WO2013016418A2 - Ion channel probes and methods of use thereof - Google Patents

Ion channel probes and methods of use thereof Download PDF

Info

Publication number
WO2013016418A2
WO2013016418A2 PCT/US2012/048135 US2012048135W WO2013016418A2 WO 2013016418 A2 WO2013016418 A2 WO 2013016418A2 US 2012048135 W US2012048135 W US 2012048135W WO 2013016418 A2 WO2013016418 A2 WO 2013016418A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
nucleic acid
acid molecule
cell
Prior art date
Application number
PCT/US2012/048135
Other languages
French (fr)
Other versions
WO2013016418A3 (en
Inventor
Jeffrey R. MARTENS
Sarah Marie BASS
Lian Zhang
Original Assignee
The Regents Of The University Of Michigan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of Michigan filed Critical The Regents Of The University Of Michigan
Publication of WO2013016418A2 publication Critical patent/WO2013016418A2/en
Publication of WO2013016418A3 publication Critical patent/WO2013016418A3/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the disclosure generally relates to ion channel probes and methods of using them. More particularly, the disclosure provides fluorescent ion channel subunit polypeptides and methods of their use.
  • Ion channels are located in the membrane of cells, which allow ions, particularly monovalent and divalent cations and anions, to pass through the cells membrane.
  • Ion channel regulators typically chemical agents, may alter the entry of certain ions into or out of cells and cellular organelles, depending on whether the intracellular or extracellular concentration of the particular ion is greater, and on the electrical potential difference that exists between the inside and the outside of the cell. The combined effect of the concentration difference and the electrical potential difference is called the electrochemical gradient.
  • the gate of an ion channel is open, the ions will flow down their electrochemical gradient unless they are prevented from doing so as, for example, by means of a chemical ion channel regulator.
  • Ion channel regulators are commonly used for treating a variety of conditions, including cardiac conditions such as atrial fibrillation, supraventricular tachycardias, hypertrophic cardiomyopathy and hypertension, as well as migraine headaches, the prevention of brain damage, and other disorders.
  • cardiac conditions such as atrial fibrillation, supraventricular tachycardias, hypertrophic cardiomyopathy and hypertension, as well as migraine headaches, the prevention of brain damage, and other disorders.
  • Understanding how ion channels are assembled and function in cells provides insight as to new ways to target ion channels with new chemical therapies for the treatment of such conditions and disorders. For example, the precise localization of ion channels to distinct subcellular compartments plays an important role in the control of neuronal excitability. This localization is
  • Kv channels are critical determinants of both presynaptic and postsynaptic membrane excitability.
  • Kv channel stiochiometry is tetrameric, with four identical (homomeric) or non- identical (heteromeric) a subunits combining to form a functional channel.
  • the multiplicity of Kv channel function is enhanced by oligomeric assembly of channel subunits, which is especially significant since Kv channels are thought to exist almost exclusively as heteromeric complexes in the brain.
  • Immunoprecipitation of Kv subunit complexes, with channel specific antibodies, clearly demonstrate the prevalence of this oligomerization in the brain while immunolabeling has demonstrated regional and subcellular variations the expression patterns of individual Kv channel subunits.
  • the disclosure addresses one or more needs in the art relating to molecules and methods for measuring the expression of ion channel proteins. Such molecules and methods are useful as tools in the study of temporal and spatial changes in channel protein expression and localization.
  • the disclosure provides nucleic acid molecules encoding an ion channel subunit polypeptide wherein the ion channel subunit polypeptide comprises a label inserted into an extracellular loop between transmembrane- spanning segments of the ion channel polypeptide.
  • the label is inserted between segments 1 and 2 of a potassium channel subunit polypeptide.
  • the label is inserted between segments 5 and 6 of the extracellular loop of domain 1 of a sodium channel subunit polypeptide.
  • the encoded ion channel subunit polypeptide is a voltage-gated channel polypeptide, an inwardly rectifying channel polypeptide, a tandem pore domain channel polypeptide, a calcium-activated channel polypeptide, or an intracellular channel polypeptide.
  • the inwardly rectifying channel polypeptide is an inwardly rectifying potassium channel polypeptide.
  • Such inwardly rectifying channel polypeptide is selected from the group consisting of: Kir1 .1 , Kir1 .2, Kir2.1 , Kir2.2, Kir2.2v, Kir2.3, Kir2.4, Kir3.1 , Kir3.2, Kir3.3, Kir3.4, Kir4.1 , Kir5.1 , Kir6.1 , and Kir7.1 .
  • the voltage-gated channel polypeptide is a potassium channel polypeptide, a sodium channel polypeptide, or a chloride channel polypeptide.
  • the voltage-gated channel polypeptide is a voltage- gated potassium channel polypeptide.
  • Such voltage-gated potassium channel polypeptide is selected from the group consisting of: Kv1 .1 , Kv1 .2, Kv1 .3, Kv1 .4, Kv1 .5, Kv1 .6, Kv1 .7, Kv1 .8, Kv2.1 , Kv2.2, Kv3.1 , Kv3.2, Kv3.3, Kv3.4, Kv4.1 , Kv4.2, Kv4.3, Kv5.1 , Kv6.1 , Kv6.2, Kv6.3, Kv6.4, Kv7.1 , Kv8.1 , Kv8.2, Kv9.1 , Kv9.2, Kv9.3, Kvl O.1 , Kv10.2, Kv1 1 .1 , Kv1 1 .2, Kv1 1 .3, Kv12.1 , Kv12.2, and Kv12.3.
  • the voltage-gated channel polypeptide is a voltage- gated sodium channel polypeptide.
  • Such voltage-gated sodium channel polypeptide is selected from the group consisting of: Nav1 .1 , Nav1 .2, Nav1 .3, Nav1 .4, Nav1 .5, Nav1 .6, Nav1 .7, Nav1 .8, Nav1 .9, and Nax.
  • the voltage-gated channel polypeptide is a voltage- gated chloride channel polypeptide.
  • Such voltage-gated chloride channel polypeptide is selected from the group consisting of: CLCN1 , CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCNKB.
  • the calcium-activated channel polypeptide is a calcium-activated chloride channel polypeptide.
  • Such calcium-activated chloride channel polypeptide is selected from the group consisting of: CLCA1 , CLCA2, CLCA3, and CLCA4.
  • the intracellular channel polypeptide is an intracellular chloride channel polypeptide.
  • Such intracellular channel polypeptide is selected from the group consisting of: CLIC1 , CLIC2, CLIC3, CLIC4, CLIC5, CLIC6, and CLNS1 A.
  • the label is a green, yellow, cyan, orange or red fluorescent protein or fragment thereof.
  • the fluorescent protein comprises amino acid residues 1 -238 of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, or a pHluorin.
  • the fluorescent protein fragment is the amino-terminal fragment comprising amino acid residues 1 -155 of the fluorescent protein.
  • the fluorescent protein fragment is the carboxy-terminal fragment comprising amino acid residues 156-238 of the fluorescent protein.
  • the fluorescent label is selected from the group consisting of: yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (mCherry), green fluorescent protein (GFP), and pHluorin.
  • the label is inserted into the extracellular loop via a linker.
  • the label comprises a linker at either end of the label.
  • the linker comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105 (AAASGGGTG) and SEQ ID NO: 106
  • the nucleotide sequence encoding the label comprising the linker is inserted in the nucleic acid molecule at a position in the nucleotide sequence selected from the group consisting of: between the nucleotide sequence encoding amino acids at positions 201 and 202 in human Kv1 .1 , between the nucleotide sequence encoding amino acids at positions 200 and 201 of rat Kv1 .2, between the nucleotide sequence encoding amino acids at positions 348 and 349 in human Kv1 .4, between positions 212 and 213 of rat Kv2.1 , between the nucleotide sequence encoding amino acids at positions 220 and 221 of rat Kv2.2.
  • the nucleic acid molecules of the disclosure comprise a nucleotide sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to a nucleic acid sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO:
  • the nucleic acid molecules of the disclosure comprise a nucleotide sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO
  • the nucleic acid molecules of the disclosure consist of a nucleotide sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to a nucleic acid sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO:
  • the nucleic acid molecules of the disclosure consist of a nucleotide sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 1 , SEQ
  • the nucleic acid molecules of the disclosure encode a polypeptide comprising an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46
  • the nucleic acid molecules of the disclosure encode a polypeptide comprising an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
  • the nucleic acid molecules of the disclosure encode a polypeptide consisting of an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
  • the nucleic acid molecules of the disclosure encode a polypeptide consisting of the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
  • the disclosure includes vectors and cells comprising the nucleic acid molecules described herein.
  • the cells comprise the vectors.
  • the cells comprise a first nucleic acid molecule which is the nucleic acid molecule encoding a polypeptide comprising an amino- terminal fragment of a fluorescent protein and a second nucleic acid molecule encoding a polypeptide comprising a carboxy-terminal fragment of a fluorescent protein, and the first nucleic acid molecule and the second nucleic acid molecule encode the same ion channel polypeptide.
  • the first nucleic acid molecule and the second nucleic acid molecule do not encode the same ion channel polypeptide.
  • such cells can further comprise one or more additional nucleic acid molecules.
  • such cells can further comprise comprising an additional heterologous nucleic acid molecule comprising a label.
  • the cell is a mammalian cell. In more particular aspects, the cell is a human cell.
  • the disclosure provides methods of expressing an ion channel subunit polypeptide in a cell comprising incubating the cells described herein and herein above under conditions that allow the cell to express the ion channel polypeptide.
  • the disclosure provides a polypeptide comprising an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
  • the disclosure provides a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:
  • the disclosure provides a polypeptide consisting of an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48
  • the disclosure provides a polypeptide consisting of an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO
  • the disclosure provides a method for identifying a modulator of ion channel polypeptide expression comprising the step of:
  • test compound measuring expression of the ion channel polypeptide in a cell as provided herein in the presence and absence of a test compound, wherein a change in ion channel polypeptide expression in the presence of the test compound indicates the test compound is a modulator of ion channel expression.
  • the disclosure provides a method for measuring ion channel biogenesis, cellular trafficking, internalization, plasma membrane recycling, membrane insertion, or degradation, the method comprising the steps of: (a) contacting the cell as provided herein with a test compound or a control compound; and (b) detecting a change in expression of the label between the cell contacted with a test compound and the cell contacted with a control compound.
  • the expression of ion channel polypeptide is measured at the surface of the cell, extracellularly, or intracellular ⁇ .
  • an increase in expression of the label indicates trafficking of the channel polypeptide to the cell's plasma membrane.
  • a decrease in expression of the label indicates internalization of the channel polypeptide in a subcellular compartment within the cell.
  • the step of detecting a change in expression of the label is measured over a period of time.
  • the period of time comprises a lifetime of the protein.
  • the period of time comprises a cell's lifetime.
  • the period of time includes a period of minutes, a period of hours, or a period of days.
  • the cell is contacted with the compound in the presence of temperatures which range between about 4 Q C to about 40 Q C. In some aspects, the cell is contacted with the compound at a pH between about pH1 to about pH14. In some aspects, the cell is contacted with the compound in the presence of calcium in a concentration from about 0.001 M to about 1 .0 M.
  • the disclosure includes a method of using bimolecular fluorescence complementation to detect association of two or more ion channel subunit polypeptides in a cell, wherein the cell comprises a first ion channel subunit polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between a transmembrane- spanning segment of the ion channel subunit polypeptide and a second ion channel subunit polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between a transmembrane-spanning segment of the ion channel subunit polypeptide, the method comprising the step of: measuring colocalization of the amino-terminal and the carboxy-terminal fragments by measuring fluorescence, wherein an increase in fluorescence indicates association of the ion channel subunit polypeptides in the cell.
  • the disclosure includes a method of using bimolecular fluorescence complementation to monitor ion channel biogenesis, cellular trafficking, internalization, plasma membrane recycling, membrane insertion, or degradation of two or more ion channel polypeptides in a cell, wherein the cell comprises a first ion channel polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between transmembrane-spanning segments 1 and 2 of the ion channel polypeptides and a second ion channel polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between transmembrane-spanning segments 1 and 2 of the ion channel polypeptides, the method comprising the steps of: (a) measuring colocalization of the amino- terminal and carboxy-terminal fragments by measuring fluorescence, wherein an increase in fluorescence indicates association of the ion channel polypeptides in the cell; and (b) detecting a change
  • the first ion channel polypeptide and the second ion channel polypeptide are homomeric. In some aspects, the first ion channel polypeptide and the second ion channel polypeptide are heteromeric. In some aspects, the label is inserted between segments 1 and 2 of the extracellular loop of a potassium channel polypeptide. In some aspects, the label is inserted between segments 5 and 6 of the extracellular loop of domain 1 of a sodium channel polypeptide. In some aspects, the label comprises a linker at either end of the label. In some aspects, the label is inserted into the extracellular loop of the polypeptide via a polypeptide linker sequence. In some aspects, the linker comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105
  • the following disclosure describes ion channel constructs and methods for using these ion channel constructs as probes for visualizing and monitoring the movement of ion channel subunits.
  • Such constructs and methods provide new discovery tools for understanding how various molecules affect ion channel assembly and function and provide means for the development of new therapies directed at the regulation of ion channels.
  • the disclosure provides novel compositions and methods for using bimolecular fluorescence complementation (BIFC) with an extracellular
  • fluorescent epitope tag to study and visualize localization, assembly, and interaction of heteromeric and homomeric ion channel and ion channel subunit complexes.
  • BIFC of split fluorescent molecules, inserted into extracellular loops of ion channel subunits, are used to study the spatial and temporal dynamics of specific heteromeric and homomeric ion channel populations.
  • PSD-95 Postsynaptic density protein 95 (membrane-associated guanylate kinase)
  • control refers to an active, positive, negative or vehicle control. As will be understood by those of skill in the art, controls are used to establish the relevance of experimental results, and provide a
  • nucleic acid or “nucleic acid sequence” or “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • Nucleic acids encoding an ion channel subunit polypeptide of the disclosure include, for example and without limitation, genes, pre-mRNAs, mRNAs, cDNAs, polymorphic variants, alleles, and synthetic and naturally-occurring mutants.
  • Nucleic acids of the disclosure also include, without limitation, those that (1 ) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence as described herein, and conservatively modified variants thereof; (2) have a nucleic acid sequence that has greater than about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater nucleotide sequence identity, over a region of at least about 25, about 50, about 100, about 150, about 200, about 250, about 500, about 1000, or more nucleotides (up to the full length sequence of nucleotides of the mature protein), to a reference nucleic acid sequence as described herein.
  • Exemplary "stringent hybridization” conditions include hybridization at 42 Q C in 50% formamide, 5X SSC, 20 mM Na-P04, pH 6.8; and washing in 1 X SSC at 55 Q C for 30 minutes. It is understood that variation in these exemplary conditions can be made based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining appropriate hybridization conditions. See Sambrook et al., Molecular Cloning: A Laboratory Manual (Second ed., Cold Spring Harbor Laboratory Press, 1989) ⁇ 9.47-9.51 .
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions are achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • an "analog,” “variant” or “derivative” is a compound substantially similar in structure and having the same biological activity, albeit in certain instances to a differing degree, to a naturally-occurring molecule.
  • a polypeptide variant refers to a polypeptide sharing substantially similar structure and having the same biological activity as a reference polypeptide.
  • Variants or analogs differ in the composition of their amino acid sequences compared to the naturally- occurring polypeptide from which the analog is derived, based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and/or one or more internal regions of the naturally-occurring polypeptide sequence (e.g., fragments), (ii) insertion or addition of one or more amino acids at one or more termini (typically an "addition” or “fusion") of the polypeptide and/or one or more internal regions (typically an "insertion") of the naturally-occurring polypeptide sequence or (iii) substitution of one or more amino acids for other amino acids in the naturally-occurring polypeptide sequence.
  • a “derivative” refers to a polypeptide sharing the same or substantially similar structure as a reference polypeptide that has been modified, e.g., chemically.
  • fragment of a polypeptide refers to any portion of the polypeptide smaller than the full-length polypeptide or protein expression product. Fragments are deletion analogs of the full-length polypeptide wherein one or more amino acid residues have been removed from the amino terminus and/or the carboxy terminus of the full-length polypeptide.
  • ion channel polypeptide and “ion channel” are used interchangeably herein to refer to pore-forming membrane proteins that enable the passive flow of inorganic ions by forming hydrated pores across biological membranes.
  • ion channel subunit polypeptide and “ion channel subunit” refer to alpha, beta, gamma and delta subunits of various ion channel
  • polypeptides Pore-forming alpha subunits determine ion permeation whereas beta, gamma and delta subunits modulate channel gating, surface expression, targeting and stability. Moreover, some of these subunits constitute binding sites for toxins as well as for therapeutic drugs.
  • the term "homomer” refers to a complex composed of only one subunit.
  • the disclosure includes homomers of ion channel subunits wherein like ion channel subunits associate with each other, e.g. a homomer of a voltage- gated potassium ion (Kv) channel subunit 1 .1 (Kv1 .1 -Kv1 .1 ).
  • the term “heteromer” refers to a complex compose of more than one type of subunit.
  • the disclosure includes heteromers of ion channel subunits wherein unlike ion channel subunits associate with each other, e.g. a heteromer of a voltage-gated potassium ion (Kv) channel subunits 1 .1 and 1 .2 (Kv1 .1 -Kv1 .2).
  • label or “fluorescent label” or “fluorescent protein” are used herein to refer to a fluorescent protein, or fragment thereof, that is attached to an ion channel subunit polypeptide to detect expression, changes in expression, and localization of the polypeptide in a cell.
  • linker or "linker polypeptide” is used herein to refer to a short peptide sequence used to connect the fluorescent label to the ion channel subunit polypeptide.
  • identity or “percent identity” or “percent sequence identity” are used herein to refer to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences.
  • Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms").
  • Substantial identity refers to sequences with at least or about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity over a specified sequence.
  • the identity exists over a region that is at least about 50-100 amino acids or nucleotides in length. In other aspects, the identity exists over a region that is at least about 100-200 amino acids or nucleotides in length. In other aspects, the identity exists over a region that is at least about 200-500 amino acids or nucleotides in length. In certain aspects, percent sequence identity is determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit and the Smith-Waterman algorithm.
  • any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as about 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1 % to 3%, etc., are expressly enumerated in this specification. The values listed above are only examples of what is specifically intended.
  • vector is used to refer to any molecule used to transfer coding information to a host cell.
  • vector includes all those known in the art, including without limitation cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant
  • the vector is inserted (e.g., via transformation or transduction) into an appropriate host cell for expression of the polynucleotide and polypeptide via transformation or transfection using techniques known in the art. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, 1989.
  • cell or "host cell” as used herein are used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing that selected nucleic acid sequence as a polypeptide.
  • the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected nucleic acid sequence, e.g., gene, is present.
  • the cell being transformed is either a prokaryotic or a eukaryotic cell.
  • the cell being transformed includes, but is not limited to, a mammalian cell.
  • the cell is a human cell.
  • agent or “test compound” describes any molecule, e.g. protein or pharmaceutical, with the capability of affecting or regulating an ion channel or ion channel subunit.
  • agent refers to ion channel blockers and/or ion channel activators, and refers to any agent that can alter the entry of certain ions into or out of cells and cellular organelles, depending on whether the intracellular or extracellular concentration of the particular ion is greater and the electrical potential difference that exists between the inside and the outside of the cell.
  • an agent affects ion channel subunit polypeptide assembly, membrane expression, biogenesis, trafficking, membrane recycling, internalization, membrane insertion, or degradation.
  • trafficking is anterograde trafficking or retrograde trafficking.
  • ion channel polypeptides ion channel polypeptides, ion channel subunit polypeptides, linker polypeptides, vectors, cells, fluorescent proteins, and bimolecular fluorescence insofar as they apply to the compositions and methods of the disclosure.
  • Ion channels are integral membrane proteins, or, more typically, an assembly of several proteins. Ion channels are present on all membranes of cell (plasma membrane) and intracellular organelles (nucleus, mitochondria, endoplasmic reticulum, golgi apparatus and the like). Such "multi-subunit" assemblies usually involve a circular arrangement of identical or homologous proteins closely packed around a water-filled pore through the plane of the membrane or lipid bilayer.
  • the pore-forming subunit(s) is called the a subunit, while the auxiliary subunits are denoted ⁇ , ⁇ , ⁇ and so on.
  • Some ion channels permit the passage of ions based solely on their charge of positive (cation) or negative (anion).
  • passage through the pore is governed by a "gate,” which may be opened or closed by chemical or electrical signals, temperature, or mechanical force, depending on the variety of channel.
  • Ion channels differ with respect to the ion they let pass (e.g., Na + , K + , and CI " ), the ways in which they may be regulated, the number of subunits of which they are composed and other aspects of structure.
  • Ion channels belonging to the largest class which includes the voltage-gated channels that underlie the nerve impulse, consists of four subunits with six transmembrane helices each. On activation, these helices move about and open the pore. Two of these six helices are separated by a loop that lines the pore and is the primary determinant of ion selectivity and conductance in this channel class and some others.
  • ion channel subunit polypeptides comprising a label inserted into an extracellular loop between transmembrane-spanning segments of the ion channel polypeptide.
  • the channel probes provided herein permit the measurement of ion channel surface levels.
  • anti-GFP antibodies are used to label surface proteins in non-permeablized cells.
  • the GFP signal can then be normalized to the GFP fluorescence to the ratio of surface channel to total channel protein.
  • PHIuorin-tagged ion channel surface levels can be assayed independent of antibody labeling by exposing cells to extracellular solutions of neutral or acidic pH.
  • these channel probes can also be a tool to study the temporal and spatial changes in the channel/protein localization. For example, channel movement can be measured, e.g., measure and visualize channel moving from the surface into the cell (endocytosis) or channel moving back to the membrane after endocytosis (recycling).
  • time, concentration (i.e., number), and location of insertion into the membrane is measured as well as the movement of channel proteins within the cell.
  • use of co-expressed split-fluorescent tagged constructs permits the selective visualization and measurement of all the trafficking events described above for homo- and heteromeric channel complexes.
  • These channel probes also permit the study of when and where within a cell, ion channel complexes are created and assembled (biogenesis) or when and where they are destroyed (degradation).
  • these fluorescent ion channel probes are useful in screening for compounds or conditions that change the surface density of channel proteins. When used in combination with high-throughput assays, ion channel probes described herein are a valuable drug-discovery or screening tool.
  • the methods of the disclosure detect trafficking of ion channel subunit polypeptides.
  • this trafficking is anterograde or retrograde.
  • Anterograde trafficking is movement of the newly synthesized channel protein to the cell surface.
  • Retrograde trafficking is movement of surface channel protein to intracellular compartments.
  • the disclosure provides compositions and methods for studying how various compounds or agents affect the biological activity of ion channels and the ion channel subunits.
  • many natural toxins target ion channels. Examples include the voltage-gated sodium channel blocker tetrodotoxin, the irreversible nicotinic acetylcholine receptor antagonist alpha- bungarotoxin, and plant-derived alkaloids, such as strychnine and d-tubocurarine.
  • Such compounds and methods are useful in the development of therapeutic drugs, which act directly or indirectly to modulate ion channel activity.
  • the ion channel polypeptide is a potassium channel.
  • Potassium channels form potassium-selective pores that span cell membranes.
  • potassium channels are found in most cell types and control a wide variety of cell functions.
  • the disclosure includes all four classes of potassium channels.
  • Potassium channels have a tetrameric structure in which, in one aspect, four identical protein subunits associate to form a four-fold symmetric complex arranged around a central ion conducting pore (i.e., a homotetramer). Alternatively, four related, but not identical protein subunits, may associate, in some aspects, to form heterotetrameric complexes. Potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selective permeability.
  • the potassium channel is a voltage-gated potassium (Kv) channel.
  • Kv channel polypeptides include, but are not limited to, Kv1 , Kv2, Kv3, Kv4, Kv5, Kv6, Kv7, Kv8, Kv9, Kv10, Kv1 1 , and Kv12.
  • Kv channel polypeptides further comprise polypeptide subunits including, but not limited to, Kv1 .1 , Kv1 .2, Kv1 .3, Kv1 .4, Kv1 .5, Kv1 .6, Kv1 .7, Kv1 .8, Kv2.1 , Kv2.2, Kv3.1 , Kv3.2, Kv3.3, Kv3.4, Kv4.1 , Kv4.2, Kv4.3, Kv5.1 , Kv6.1 , Kv6.2, Kv6.3, Kv6.4, Kv7.1 , Kv8.1 , Kv8.2, Kv9.1 , Kv9.2, Kv9.3, Kv10.1 , Kv10.2, Kv1 1 .1 , Kv1 1 .2, Kv1 1 .3, Kv12.1 , Kv12.2, Kv12.3.
  • the potassium channel is an inward-rectifying (Kir) potassium channel.
  • Kir channel polypeptides include, but are not limited to, Kir1 , Kir2, Kir3, Kir4, Kir5, Kir6, and Kir7.
  • Such Kir channel polypeptides further comprise polypeptide subunits including, but not limited to, Kir1 .1 , Kir1 .2, Kir2.1 , Kir2.2, Kir2.2v, Kir2.3, Kir2.4, Kir3.1 , Kir3.2, Kir3.3, Kir3.4, Kir4.1 , Kir5.1 , Kir6.1 , and Kir7.1 .
  • the disclosure includes sodium channel polypeptides.
  • Sodium channels are integral membrane proteins that form ion channels, conducting sodium ions (Na + ) through a cell's plasma membrane.
  • Sodium channel polypeptides are classified according to the trigger that opens the channel for such ions, i.e. either a voltage-change (voltage-gated sodium channels, i.e. Na v ) or binding of a substance (a ligand) to the channel (ligand- gated sodium channels).
  • the disclosure includes both voltage-gated sodium channels and ligand-gated sodium channels.
  • the disclosure includes voltage-gated sodium (Na v ) channel polypeptides.
  • Na v channel polypeptides include, but are not limited to, Na v channel polypeptides selected from the group consisting of:
  • the disclosure includes chloride channel
  • Chloride channels display a variety of important physiological and cellular roles that include regulation of pH, volume homeostasis, organic solute transport, cell migration, cell proliferation and differentiation. Based on sequence homology, the chloride channels are subdivided into a number of groups.
  • Chloride channels are important for setting cell resting membrane potential and maintaining proper cell volume. These channels conduct chloride as well as other anions such as HC0 3 " , , SCN “ , and N03 " . The structure of chloride channels are not like other known channels. Chloride channel subunits contain between 1 and 12 transmembrane segments. Some members of this family are activated by voltage, while others are activated by Ca 2+ , extracellular ligands, pH, and the like.
  • the disclosure includes calcium-activated chloride channel regulator proteins 1 (CLCA1 ), 2 (CLCA2), 3 (CLCA3), and 4 (CLCA4).
  • the disclosure includes the CLCN family of voltage- dependent chloride channel proteins.
  • the CLCN family comprises nine members (CLCN1 , CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCNKB) which demonstrate quite diverse functional characteristics while sharing significant sequence homology.
  • the disclosure includes chloride intracellular (CLI) channel proteins.
  • the CLI family comprises at least 6 members (CLIC1 , CLIC2, CLIC3, CLIC4, CLIC5, and CLIC6.
  • the disclosure includes chloride channel, nucleotide-sensitive, 1 A (CLNS1 A).
  • CLNS1 A is a protein channel subunit shown to interact with small nuclear ribonucleoprotein D1 , integrin alpha chain 2b (ITGA2B), small nuclear ribonucleoprotein Sm D3 (SNRPD3), and Protein arginine N-methyltransferase 5 (PRMT5).
  • the disclosure includes the following polypeptides of various ion channel subunits comprising a linker and a fluorescent label.
  • Kir2.1 -PHN (597) (SEQ ID NO: 86)
  • Kir2.2-PHN (601 ) (SEQ ID NO: 92)
  • a label is attached to an ion channel subunit polypeptide to detect expression, changes in expression, and localization of the polypeptide in a cell.
  • label includes all fluorescent proteins, including enhanced and bright versions of fluorescent proteins, as well as analogs, fragments, variants and derivatives thereof.
  • Fluorescent proteins are members of a structurally homologous class of proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of three amino acids within their own polypeptide sequence.
  • a gene (or a gene chimera) encoding an engineered fluorescent protein is introduced into a living cell or multiple living cells, which allows for the subsequent visualization of the location and dynamics of the gene product using fluorescence microscopy.
  • fluorescent proteins include fluorescent proteins as described by Shaner et al. (Nature Methods 2: 905-9, 2009), incorporated herein by reference in its entirety.
  • fluorescent proteins include, but are not limited to, green, yellow, cyan, orange and red fluorescent proteins.
  • fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), pHluorin, and mCherry.
  • N-terminal and C-terminal fragments i.e. split fluorescent protein fragments
  • BiFC bimolecular fluorescence complementation
  • split constructs are used in co-expression studies to measure and monitor hetero- or homo-meric channel proteins.
  • full-length fluorescent proteins are used to study general protein movement, location, and surface expression in the same way but will not allow for the resolution of multisubunit channels.
  • N-terminal fragment of a fluorescent protein label e.g., YFP, CFP, or pHluorin
  • the designated abbreviation for that construct is YN, CN, or PHN, respectively.
  • the designated abbreviation for that construct is YC, CC, or PHC, respectively.
  • fluorescent protein fragments are attached to components of the same macromolecular complex. More particularly, ion channel subunits that are postulated to interact are fused to unfolded complementary fragments of a fluorescent label and expressed in live cells. Interaction of these proteins brings fluorescent fragments within proximity, allowing the fluorescent label to reform in its native three-dimensional structure (i.e. amino- and carboxy-fragments of the fluorescent label are brought together) allowing the fluorescent label to emit its fluorescent signal.
  • the label is inserted into the loop of an ion channel subunit polypeptide between transmembrane segments 1 and 2. In other aspects, the label is inserted via a linker into the loop between transmembrane segments 5 and 6.
  • a label is inserted into an extracellular loop of an ion channel subunit polypeptide via a linker.
  • the linker is a peptide linker.
  • the peptide linker comprises from about 1 to about 100 amino acids, from about 5 to about 50 amino acids, from about 8 to about 20 amino acids.
  • the peptide linker comprises about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, and about 100 amino acids.
  • the peptide linker comprises about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 1 7, about 18, about 19, and about 20 amino acids.
  • an N-terminal fragment or a C-terminal fragment of a label is inserted into an extra-cellular loop of an ion channel subunit between transmembrane-spanning segments of the ion channel subunit via a peptide linker.
  • the label comprises a linker at either end of the label.
  • the label comprises a linker at the N-terminus of the label.
  • the label comprises a linker at the C-terminus of the label.
  • the linker sequence comprises the following amino acids: AAASGGTG (SEQ ID NO: 105) and VDGGSAAA (SEQ ID NO: 106). Synthesis of Ion Channel Nucleic Acid Molecules and Polypeptide Molecules
  • nucleic acid molecules encode a polypeptide comprising the amino acid sequence of an ion channel subunit polypeptide and can readily be obtained in a variety of ways including, without limitation, recombinant DNA methods and chemical synthesis.
  • Recombinant DNA methods are generally those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and/or Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994).
  • Recombinant expression techniques conducted in accordance with the descriptions set forth below, in various aspects, are followed to produce these polynucleotides and to express the encoded polypeptides. For example, by inserting a nucleic acid sequence which encodes the amino acid sequence of an ion channel subunit polypeptide into an appropriate vector, one skilled in the art can readily produce large quantities of the desired nucleotide sequence.
  • sequences can then be used to generate detection probes or amplification primers.
  • a polynucleotide encoding the amino acid sequence of an ion channel subunit polypeptide can be inserted into an expression vector.
  • the encoded ion channel subunit polypeptide or ion channel subunit polypeptides are, in some aspects, produced in large amounts.
  • nucleic acids and polypeptides are well known in the art, such as those described by Engels et al., Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include, inter alia, the
  • nucleic acid variants contain codons which have been altered for the optimal expression of an ion channel subunit
  • codon alterations depend upon the ion channel subunit polypeptide(s) and host cell(s) selected for expression.
  • Such "codon optimization” can be carried out by a variety of methods, for example, by selecting codons which are preferred for use in highly expressed genes in a given host cell.
  • Computer algorithms which incorporate codon frequency tables such as "Ecohigh.cod” for codon preference of highly expressed bacterial genes are used, in some instances, and are provided by the University of Wisconsin
  • Codon frequency tables include “Celegans_high.cod”, “Celegansjow.cod”, “Drosophila_high.cod”, “Human_high.cod”, “Maize_high.cod”, and
  • a nucleic acid molecule encoding the amino acid sequence of an ion channel subunit polypeptide is inserted into an appropriate vector using standard ligation techniques.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • Cloning vectors include all those known in the art. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, 1989.
  • Expression vectors include all those known in the art, including without limitation cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
  • the expression vector is inserted (e.g., via transformation or transduction) into an appropriate host cell for expression of the polynucleotide and polypeptide via transformation or
  • the vector is pcDNA3.1 /V5/His vector.
  • the completed vector is inserted into a suitable host cell for amplification and/or polypeptide expression.
  • the transformation of a vector encoding an ion channel subunit polypeptide into a selected host cell is, in various aspects, accomplished by well-known methods such as transfection, infection, calcium chloride-mediated transformation, electroporation, microinjection, lipofection or the DEAE-dextran method or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan and are set forth, for example, in Sambrook et al., supra.
  • Host cells are prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as yeast, insect or vertebrate cells).
  • the selection of an appropriate host cell depends upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation), ease of folding into a biologically active molecule, and cell type, i.e. physiological function of the cell so that appropriate ion channel expression and regulation is observed.
  • Such host cells include, but are not limited to, host cells of bacterial, yeast, fungal, viral, invertebrate, and mammalian sources.
  • mammalian host cells are used.
  • human host cells are used.
  • Such cells include, but are not limited to, a vascular cell, a myocyte, a cardiomyocyte, a neuron, a renal cell, a fibroblast, an endothelial cell, an epithelial cell, a myoepithelial cell, a squamous cell, a basal cell, an interstitial cell, an immune cell, a glial cell, an astrocyte, a tumor cell, an ovarian cell, a sperm cell, a stem cell, a kidney cell, a liver cell, a fat cell, a lung cell, a bladder cell, an intestinal cell, a colon cell, a parafollicular cell, a blood cell, a pigment cell, a corneal cell, a hormone-secreting cell, a nurse cell, and a bone cell.
  • the cells include, but are not limited to, cancer cells.
  • Host cells comprising an ion channel subunit polypeptide expression vector are cultured using standard media well known to the skilled artisan.
  • the media will usually contain all nutrients necessary for the growth and survival of the cells.
  • Suitable media for culturing eukaryotic cells include Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which, in some instances, are supplemented with serum and/or growth factors as indicated by the particular cell line being cultured.
  • an antibiotic or other compound useful for selective growth of transformed cells is added as a supplement to the media.
  • the compound used is dictated by the selectable marker element present on the plasmid with which the host cell was transformed.
  • the selectable marker element is kanamycin resistance
  • the compound added to the culture medium will be kanamycin.
  • Other compounds for selective growth include ampicillin, tetracycline and neomycin.
  • the amount of an ion channel subunit polypeptide produced by a host cell is evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, chromatographic separation, such as High Performance Liquid Chromatography (HPLC), immunodetection such as immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.
  • HPLC High Performance Liquid Chromatography
  • immunodetection such as immunoprecipitation
  • activity assays such as DNA binding gel shift assays.
  • the disclosure includes all cells that are amenable to transfection or infection by the ion channel subunit polypeptides.
  • Bimolecular fluorescence complementation (BiFC)
  • BiFC complementation
  • BIFC is a technology typically used to validate protein interactions.
  • BIFC is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex.
  • macromolecular complex comprises ion channel subunits. Proteins that are postulated to interact, i.e. various ion channel subunits, are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells.
  • the polypeptides are attached to split fragments of a fluorescent reporter protein by a linker. Examples of appropriate linkers are discussed in further detail herein. Interaction of the proteins of interest, i.e.
  • ion channel subunits brings fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal.
  • This fluorescent signal is detected and located within a cell using an inverted fluorescence microscope, a confocal microscope, or a total internal reflectance microscope that allows imaging of fluorescence in cells. With complementation, the signal is all or nothing. Therefore, through the visualization and analysis of the intensity and distribution of fluorescence in these cells, one of skill in the art
  • the expression of the cDNA in cells results in expression of the ion channel probes. Fluorescence measurement or imaging is used to detect the signal, measure the signal intensity, or determine signal localization within a cell. Live cell fluorescence imaging is used in various aspects to visualize trafficking effects. When using pHluorin constructs, application of solutions with acidic or basic pH is used to measure surface expression.
  • Further aspects of the disclosure include antibodies to proteins described herein.
  • immunocytochemistry can be used to validate BIFC or used in labeling and localizing surface proteins.
  • GFP antibodies which recognize pHluorin are useful in measuring channel protein endocytosis and/or recycling (Schumacher et al., Circ. Res. 104:1390-8, 2009).
  • Amino terminal residues 1 -155 of YFP (YN), CFP (CN), and pHluorin (PHN) and carboxy terminal residues 156-238 of YFP (YC), CFP (CC), and pHluorin (PHC) are inserted between transmembrane-spanning segments of an ion channel subunit.
  • DNA sequences encoding amino acid residues 1 -238, 1 -155 and 156-238 of YFP, CFP and pHluorin were inserted into extra-cellular loop between transmembrane-spanning segments 1 and 2 in human Kv1 .1 , rat 1 .2, human 1 .4, rat 2.1 and rat 2.2 between the amino acid positions of 201 /202, 200/201 , 348/349, 212/213, and 220/221 respectively with the linker sequences encoding AAASGGTG (SEQ ID NO: 105) and
  • VDGGSAAA (SEQ ID NO: 106).
  • COS-7 cells were maintained in DMEM (Gibco #1 1960) with 10% fetal bovine serum (FBS) (Gibco) and 1 x Penicillin-Streptomycin (Gibco) at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .
  • COS-7 cells grown to 60-80% confluence on glass coverslips were transiently transfected with 2 ⁇ g of DNA total combined with 3 ⁇ of Lipofectamine 2000 (Invitrogen) in serum-free Opti-Mem I (Gibco) for 3-5 hours and then changed to normal media followed by incubation for 1 -2 days before performing experiments.
  • Transfection kit (Clontech) using 3 ⁇ g total DNA according to manufacturer's protocol. After 1 hour of transfection the medium was replaced with acidified Neurobasal medium pre-equilibrated at 10% CO 2 and neurons were placed in a 10% C0 2 incubator at 37°C for 20 minutes (Jiang et al., Nat. Protoc. 1 : 695-700, 2006). Cells were then washed three times in Neurobasal medium, fed with growth medium and placed back in the 5% C0 2 incubator at 37°C for 3 days until experiments were carried out.
  • coverslips were washed briefly with ice-cold PBS and then incubated with polyclonal anti-GFP antibody in 2% goat serum for 30 minutes on ice. After three washes with PBS, cells were incubated with AlexaFluor 594 goat anti-rabbit for 30 min on ice. Cells were then washed with PBS twice, and fixed with 4% paraformaldehyde for 7 minutes. In some experiments, cells were incubated with anti-V5 antibody and AlexaFluor 647 goat anti-mouse following permeabilization with 0.1 % Triton X-100 to verify channel expression. Coverslips were mounted with Prolong Gold anti-fade reagent (Invitrogen).
  • Images were obtained by taking a series of stacks every 0.5 ⁇ through the cells and combining the images into a composite stack.
  • the following filters were used: a 405 nm laser diode with a 430-460 nm bandpass filter, a 488 nm laser with a 505-525 nm bandpass filter, a 543 nm laser with a 560 nm long- pass filter, and a 633 nm laser with a 660 nm long-pass filter.
  • Images were analyzed with ImageJ software (NIH), and statistics were carried out with Prism 5 software from Graphpad Prism Software. Adjustments of contrast and brightness were performed using Adobe Photoshop 9.0.
  • Fluorescence recovery after photobleaching was performed essentially as described previously (Jenkins et al., Curr. Biol. 16:121 1 -16, 2006). Briefly, five single confocal plane prebleach images were acquired at a resolution of 512 x 512 pixels at 5% laser intensity before bleaching a region of approximately 5 - 10 square microns at 100% laser intensity for 3 - 5 s.
  • the beam was focused on the periphery of the back focal plane of a 60x 1 .49 NA oil immersion objective (Olympus) so that the laser beam was incident on the coverslip at -70° from the normal, giving a decay constant for the evanescent field of ⁇ 1 10 nm.
  • Digital images were captured on a cooled EM CCD camera (Andor iXon; Andor
  • the bath solution contained (in mM): NaCI 1 10, KCI 4, MgCI 2 1 , CaCI 2 1 .8, HEPES 10, and glucose 1 .8; and was adjusted to pH 7.35 with NaOH. Cells were held at -80 mV resting potential and voltage steps were applied at 20 mV intervals for 1 .5 s to elicit outward potassium currents.
  • an ion channel subunit construct was created with the neuronal Kv channel, Kv1 .4, with either the amino-terminal 1 -155 amino acid fragment of yellow fluorescent protein (YFP) (Kv1 .4-YN) or the carboxyl-terminal 156-238 amino acid fragment of YFP (Kv1 .4-YC) inserted into the S1 -S2 loop of the Kv1 .4 ion channel subunit polypeptide.
  • YFP yellow fluorescent protein
  • Kv1 .4-YC carboxyl-terminal 156-238 amino acid fragment of YFP
  • TIRFM total internal reflection fluorescence microscopy
  • Kv1 .1 homomeric channels have been shown to be restricted to the endoplasmic reticulum (ER) through an ER-retention motif found in the channel pore (Manganas, et al., Proc. Natl. Acad. Sci. USA 98: 14055-9, 2001 .
  • mutation of a conserved threonine residue within the S1 -S2 linker of Kv1 .4 has also been shown to be strictly required for surface expression (McKeown et al., supra).
  • surface localization of either of these two Kv channel subunits can be rescued by coexpression with another wild-type Kv a subunit (Manganas et al., supra; McKeown et al., supra).
  • Kv1 channel family Members of the Kv1 channel family have been shown (Kim et al., Nature 378: 85-8, 1995) to interact with a membrane-associated guanylate kinase protein, PSD-95, to direct channel to cell surface clusters through interaction with channel carboxyl terminus. This compartmentalization has been shown to be dependent on cell surface expression of the channel, since channels unable to traffic to the plasma membrane are not clustered by PSD-95.
  • Kv1 .4 , Kv1 .2, and Kv1 .4 represent the predominant Kv1 channel subunits.
  • Hippocampal neurons were transfected with (1 ) Kv1 .4-PHN + Kv1 .4- PHC, (2) Kv1 .2-PHN + Kv1 .4-PHC, (3) Kv1 .1 -PHN + Kv1 .4-PHC, (4) Kv1 .2-PHN + Kv1 .1 -PHC, and (5) Kv1 .2-PHN + Kv1 .2-PHC. Neurons were fixed,
  • heteromeric channels revealed that Kv1 .4-Kv1 .2 heteromers, like Kv1 .4-Kv1 .4 homomers, were also capable of axonal localization.
  • the heteromeric channel localized to the axonal compartment exhibited a distinct punctate pattern typical of presynaptic sites, whereas the homomeric channel was diffusely localized throughout the axon.
  • heteromeric channels containing Kv1 .4 and Kv1 .1 failed to traffic to the axon, indicated by a lack of pHluorin colocalization with neurofilament immunostaining.
  • Uncomplemented channel subunits presumably tetramerized with other endogenous Kv channel subunits.
  • These uncomplemented channel subunits were able to traffic to the axon, as indicated by anti-GFP immunostaining, demonstrating that the lack of axonal trafficking of the complemented channel is not caused by unhealthy cells or by other abnormal trafficking.
  • Kv1 .4/Kv1 .1 heteromer failed to traffic to the axon, since it had been reported that Kv1 .1 is localized to the axon in several types of neurons (Rasband et al., J. Neurocytol. 28, 319-31 ; 1999; Raab-Graham et al., Science 314: 144-48, 2006). Indeed, when Kv1 .1 was coexpressed with another predominant neuronal Kv1 channel subunit, Kv1 .2, the heteromeric complex was able to localize to the axonal compartment and was clustered in similar puncta as seen in the Kv1 .4/Kv1 .2 heteromer.
  • Kv2.1 and Kv2.2 are expressed at high levels in the mammalian hippocampus. Expression of Kv2.1 in mammalian cells results in formation of large cell surface clusters, whereas Kv2.2 appears more evenly- distributed throughout the neuron. The detection of Kv2.1 -2.2 heteromeric channels has been limited by the fact that the currents produced by the two channels are nearly identical. Despite work that has demonstrated the presence of heteromeric channel populations (Blaine et al., J. Neurosci.

Abstract

The disclosure relates to ion channel constructs and methods for using these ion channel constructs as probes for visualizing and monitoring the movement of ion channel subunits. Such constructs and methods provide new discovery tools for understanding how various molecules affect ion channel assembly and function and provide means for the development of new therapies directed at the regulation of ion channels.

Description

ION CHANNEL PROBES AND METHODS OF USE THEREOF
[0001] This application claims priority of U.S. Provisional Application No.
61 /512,257, filed July 27, 201 1 , which is incorporated by reference herein in its entirety.
[0002] This invention was made with government support under HL070973 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] The disclosure generally relates to ion channel probes and methods of using them. More particularly, the disclosure provides fluorescent ion channel subunit polypeptides and methods of their use.
BACKGROUND
[0004] Ion channels are located in the membrane of cells, which allow ions, particularly monovalent and divalent cations and anions, to pass through the cells membrane. Ion channel regulators, typically chemical agents, may alter the entry of certain ions into or out of cells and cellular organelles, depending on whether the intracellular or extracellular concentration of the particular ion is greater, and on the electrical potential difference that exists between the inside and the outside of the cell. The combined effect of the concentration difference and the electrical potential difference is called the electrochemical gradient. When the gate of an ion channel is open, the ions will flow down their electrochemical gradient unless they are prevented from doing so as, for example, by means of a chemical ion channel regulator. Ion channel regulators are commonly used for treating a variety of conditions, including cardiac conditions such as atrial fibrillation, supraventricular tachycardias, hypertrophic cardiomyopathy and hypertension, as well as migraine headaches, the prevention of brain damage, and other disorders. [0005] Understanding how ion channels are assembled and function in cells provides insight as to new ways to target ion channels with new chemical therapies for the treatment of such conditions and disorders. For example, the precise localization of ion channels to distinct subcellular compartments plays an important role in the control of neuronal excitability. This localization is
dynamically regulated in response to both intrinsic and extrinsic factors. Activity- dependent changes in plasticity seen in long term depression and potentiation occur through trafficking of distinct a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptor heteromeric channels to and from the synapse (Kessels et al, Neuron 61 :340-50, 2009). Recently, the importance of dynamic channel trafficking has been demonstrated at the organismal level where ion channels were shown to rapidly and transiently localize to different subcellular compartments in response to activity, social cues, or time of day (Markham et al., PLoS. Biol. 7: e1000203, 2009; Kim et al., Neuron 54: 933-47, 2007). While these examples highlight the importance of protein dynamics in the control of neuronal function, the mechanism regulating the temporal and spatial localization of ion channels remains poorly understood.
[0006] In neurons, voltage-gated potassium (Kv) channels are critical determinants of both presynaptic and postsynaptic membrane excitability. Kv channel stiochiometry is tetrameric, with four identical (homomeric) or non- identical (heteromeric) a subunits combining to form a functional channel. The multiplicity of Kv channel function is enhanced by oligomeric assembly of channel subunits, which is especially significant since Kv channels are thought to exist almost exclusively as heteromeric complexes in the brain. Immunoprecipitation of Kv subunit complexes, with channel specific antibodies, clearly demonstrate the prevalence of this oligomerization in the brain while immunolabeling has demonstrated regional and subcellular variations the expression patterns of individual Kv channel subunits.
[0007] The study of ion channels involves many scientific techniques such as voltage clamp electrophysiology (in particular patch clamp),
immunohistochemistry, and RT-PCR. To date, however, direct visualization of heteromultimeric and homomeric channel complexes with the resolution to monitor the spatial and temporal dynamics of surface localized channels has not been reported.
SUMMARY
[0008] The disclosure addresses one or more needs in the art relating to molecules and methods for measuring the expression of ion channel proteins. Such molecules and methods are useful as tools in the study of temporal and spatial changes in channel protein expression and localization.
[0009] In one aspect, the disclosure provides nucleic acid molecules encoding an ion channel subunit polypeptide wherein the ion channel subunit polypeptide comprises a label inserted into an extracellular loop between transmembrane- spanning segments of the ion channel polypeptide. In some aspects, the label is inserted between segments 1 and 2 of a potassium channel subunit polypeptide. In some aspects, the label is inserted between segments 5 and 6 of the extracellular loop of domain 1 of a sodium channel subunit polypeptide.
[0010] In some aspects, the encoded ion channel subunit polypeptide is a voltage-gated channel polypeptide, an inwardly rectifying channel polypeptide, a tandem pore domain channel polypeptide, a calcium-activated channel polypeptide, or an intracellular channel polypeptide.
[0011] In some aspects, the inwardly rectifying channel polypeptide is an inwardly rectifying potassium channel polypeptide. Such inwardly rectifying channel polypeptide is selected from the group consisting of: Kir1 .1 , Kir1 .2, Kir2.1 , Kir2.2, Kir2.2v, Kir2.3, Kir2.4, Kir3.1 , Kir3.2, Kir3.3, Kir3.4, Kir4.1 , Kir5.1 , Kir6.1 , and Kir7.1 .
[0012] In some aspects, the voltage-gated channel polypeptide is a potassium channel polypeptide, a sodium channel polypeptide, or a chloride channel polypeptide.
[0013] In some aspects, the voltage-gated channel polypeptide is a voltage- gated potassium channel polypeptide. Such voltage-gated potassium channel polypeptide is selected from the group consisting of: Kv1 .1 , Kv1 .2, Kv1 .3, Kv1 .4, Kv1 .5, Kv1 .6, Kv1 .7, Kv1 .8, Kv2.1 , Kv2.2, Kv3.1 , Kv3.2, Kv3.3, Kv3.4, Kv4.1 , Kv4.2, Kv4.3, Kv5.1 , Kv6.1 , Kv6.2, Kv6.3, Kv6.4, Kv7.1 , Kv8.1 , Kv8.2, Kv9.1 , Kv9.2, Kv9.3, Kvl O.1 , Kv10.2, Kv1 1 .1 , Kv1 1 .2, Kv1 1 .3, Kv12.1 , Kv12.2, and Kv12.3.
[0014] In some aspects, the voltage-gated channel polypeptide is a voltage- gated sodium channel polypeptide. Such voltage-gated sodium channel polypeptide is selected from the group consisting of: Nav1 .1 , Nav1 .2, Nav1 .3, Nav1 .4, Nav1 .5, Nav1 .6, Nav1 .7, Nav1 .8, Nav1 .9, and Nax.
[0015] In some aspects, the voltage-gated channel polypeptide is a voltage- gated chloride channel polypeptide. Such voltage-gated chloride channel polypeptide is selected from the group consisting of: CLCN1 , CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCNKB.
[0016] In some aspects, the calcium-activated channel polypeptide is a calcium-activated chloride channel polypeptide. Such calcium-activated chloride channel polypeptide is selected from the group consisting of: CLCA1 , CLCA2, CLCA3, and CLCA4.
[0017] In some aspects, the intracellular channel polypeptide is an intracellular chloride channel polypeptide. Such intracellular channel polypeptide is selected from the group consisting of: CLIC1 , CLIC2, CLIC3, CLIC4, CLIC5, CLIC6, and CLNS1 A.
[0018] In some aspects, the label is a green, yellow, cyan, orange or red fluorescent protein or fragment thereof. In some aspects, the fluorescent protein comprises amino acid residues 1 -238 of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, or a pHluorin. In some aspects, the fluorescent protein fragment is the amino-terminal fragment comprising amino acid residues 1 -155 of the fluorescent protein. In some aspects, the fluorescent protein fragment is the carboxy-terminal fragment comprising amino acid residues 156-238 of the fluorescent protein. In some aspects, the fluorescent label is selected from the group consisting of: yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (mCherry), green fluorescent protein (GFP), and pHluorin.
[0019] In some aspects, the label is inserted into the extracellular loop via a linker. In various aspects, the label comprises a linker at either end of the label. In some aspects, the linker comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105 (AAASGGGTG) and SEQ ID NO: 106
(VDGGSAAA). In some aspects, the nucleotide sequence encoding the label comprising the linker is inserted in the nucleic acid molecule at a position in the nucleotide sequence selected from the group consisting of: between the nucleotide sequence encoding amino acids at positions 201 and 202 in human Kv1 .1 , between the nucleotide sequence encoding amino acids at positions 200 and 201 of rat Kv1 .2, between the nucleotide sequence encoding amino acids at positions 348 and 349 in human Kv1 .4, between positions 212 and 213 of rat Kv2.1 , between the nucleotide sequence encoding amino acids at positions 220 and 221 of rat Kv2.2.
[0020] In some aspects, the nucleic acid molecules of the disclosure comprise a nucleotide sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to a nucleic acid sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
[0021] In some aspects, the nucleic acid molecules of the disclosure comprise a nucleotide sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
[0022] In some aspects, the nucleic acid molecules of the disclosure consist of a nucleotide sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to a nucleic acid sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
[0023] In some aspects, the nucleic acid molecules of the disclosure consist of a nucleotide sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
[0024] In some aspects, the nucleic acid molecules of the disclosure encode a polypeptide comprising an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
[0025] In some aspects, the nucleic acid molecules of the disclosure encode a polypeptide comprising an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ I D NO: 102, and SEQ ID NO: 104.
[0026] In some aspects, the nucleic acid molecules of the disclosure encode a polypeptide consisting of an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
[0027] In some aspects, the nucleic acid molecules of the disclosure encode a polypeptide consisting of the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ I D NO: 102, and SEQ ID NO: 104.
[0028] In some aspects, the disclosure includes vectors and cells comprising the nucleic acid molecules described herein. In some aspects, the cells comprise the vectors. In some aspects, the cells comprise a first nucleic acid molecule which is the nucleic acid molecule encoding a polypeptide comprising an amino- terminal fragment of a fluorescent protein and a second nucleic acid molecule encoding a polypeptide comprising a carboxy-terminal fragment of a fluorescent protein, and the first nucleic acid molecule and the second nucleic acid molecule encode the same ion channel polypeptide. In other aspects, the first nucleic acid molecule and the second nucleic acid molecule do not encode the same ion channel polypeptide. In some aspects, such cells can further comprise one or more additional nucleic acid molecules. In some aspects, such cells can further comprise comprising an additional heterologous nucleic acid molecule comprising a label. In particular aspects, the cell is a mammalian cell. In more particular aspects, the cell is a human cell.
[0029] In some aspects, the disclosure provides methods of expressing an ion channel subunit polypeptide in a cell comprising incubating the cells described herein and herein above under conditions that allow the cell to express the ion channel polypeptide.
[0030] In some aspects, the disclosure provides a polypeptide comprising an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
[0031] In some aspects, the disclosure provides a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
[0032] In some aspects, the disclosure provides a polypeptide consisting of an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
[0033] In some aspects, the disclosure provides a polypeptide consisting of an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
[0034] In some aspects, the disclosure provides a method for identifying a modulator of ion channel polypeptide expression comprising the step of:
measuring expression of the ion channel polypeptide in a cell as provided herein in the presence and absence of a test compound, wherein a change in ion channel polypeptide expression in the presence of the test compound indicates the test compound is a modulator of ion channel expression.
[0035] In some aspects, the disclosure provides a method for measuring ion channel biogenesis, cellular trafficking, internalization, plasma membrane recycling, membrane insertion, or degradation, the method comprising the steps of: (a) contacting the cell as provided herein with a test compound or a control compound; and (b) detecting a change in expression of the label between the cell contacted with a test compound and the cell contacted with a control compound.
[0036] In some aspects, the expression of ion channel polypeptide is measured at the surface of the cell, extracellularly, or intracellular^. In some aspects, an increase in expression of the label indicates trafficking of the channel polypeptide to the cell's plasma membrane. In some aspects, a decrease in expression of the label indicates internalization of the channel polypeptide in a subcellular compartment within the cell. In some aspects, the step of detecting a change in expression of the label is measured over a period of time. In some aspects, the period of time comprises a lifetime of the protein. In some aspects, the period of time comprises a cell's lifetime. In some aspects, the period of time includes a period of minutes, a period of hours, or a period of days. In some aspects, the cell is contacted with the compound in the presence of temperatures which range between about 4QC to about 40QC. In some aspects, the cell is contacted with the compound at a pH between about pH1 to about pH14. In some aspects, the cell is contacted with the compound in the presence of calcium in a concentration from about 0.001 M to about 1 .0 M.
[0037] In some aspects, the disclosure includes a method of using bimolecular fluorescence complementation to detect association of two or more ion channel subunit polypeptides in a cell, wherein the cell comprises a first ion channel subunit polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between a transmembrane- spanning segment of the ion channel subunit polypeptide and a second ion channel subunit polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between a transmembrane-spanning segment of the ion channel subunit polypeptide, the method comprising the step of: measuring colocalization of the amino-terminal and the carboxy-terminal fragments by measuring fluorescence, wherein an increase in fluorescence indicates association of the ion channel subunit polypeptides in the cell.
[0038] In some aspects, the disclosure includes a method of using bimolecular fluorescence complementation to monitor ion channel biogenesis, cellular trafficking, internalization, plasma membrane recycling, membrane insertion, or degradation of two or more ion channel polypeptides in a cell, wherein the cell comprises a first ion channel polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between transmembrane-spanning segments 1 and 2 of the ion channel polypeptides and a second ion channel polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between transmembrane-spanning segments 1 and 2 of the ion channel polypeptides, the method comprising the steps of: (a) measuring colocalization of the amino- terminal and carboxy-terminal fragments by measuring fluorescence, wherein an increase in fluorescence indicates association of the ion channel polypeptides in the cell; and (b) detecting a change in fluorescence localization in the cell over time.
[0039] In some aspects, in the methods of bimolecular fluorescence
complementation, the first ion channel polypeptide and the second ion channel polypeptide are homomeric. In some aspects, the first ion channel polypeptide and the second ion channel polypeptide are heteromeric. In some aspects, the label is inserted between segments 1 and 2 of the extracellular loop of a potassium channel polypeptide. In some aspects, the label is inserted between segments 5 and 6 of the extracellular loop of domain 1 of a sodium channel polypeptide. In some aspects, the label comprises a linker at either end of the label. In some aspects, the label is inserted into the extracellular loop of the polypeptide via a polypeptide linker sequence. In some aspects, the linker comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105
(AAASGGGTG) and SEQ ID NO: 106 (VDGGSAAA).
[0040] The foregoing summary is not intended to define every aspect of the subject matter of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the subject matter of the disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
DETAILED DESCRIPTION
[0041] The following disclosure describes ion channel constructs and methods for using these ion channel constructs as probes for visualizing and monitoring the movement of ion channel subunits. Such constructs and methods provide new discovery tools for understanding how various molecules affect ion channel assembly and function and provide means for the development of new therapies directed at the regulation of ion channels.
[0042] The disclosure provides novel compositions and methods for using bimolecular fluorescence complementation (BIFC) with an extracellular
fluorescent epitope tag to study and visualize localization, assembly, and interaction of heteromeric and homomeric ion channel and ion channel subunit complexes. BIFC of split fluorescent molecules, inserted into extracellular loops of ion channel subunits, are used to study the spatial and temporal dynamics of specific heteromeric and homomeric ion channel populations. [0043] This approach allows specific detection of ion channel subunits and their association with other ion channel subunits with high spatial and temporal resolution that was previously unattainable.
[0044] Before any embodiments of the subject matter of the disclosure are explained in detail, however, it is to be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the figures and examples. Accordingly, the disclosure embraces other embodiments and is practiced or carried out in various ways.
[0045] The section headings as used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0046] Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. Definitions
[0047] The terms "including," "comprising," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter.
[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger, et al. (eds.), Springer Verlag (1991 ); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991 ).
[0049] The following abbreviations are used throughout.
AA Amino acid
CFP Cyan fluorescent protein
CC C-terminal fragment of CFP
CN N-terminal fragment of CFP DMEM Dulbecco's modified Eagle's medium
DNA Deoxyribonucleic acid
ELISA Enzyme-linked immunosorbent assay
FBS Fetal bovine serum
FRAP Fluorescence recovery after photobleaching
GFP Green fluorescent protein
HPLC High performance liquid chromatography
mCherry Red fluorescent protein mCherry μΜ Micromolar
M Molar
MEM Minimal essential medium
mM Millimolar
PBS Phosphate-buffered saline
pHluorin pH-sensitive mutant of GFP
PHC C-terminal fragment of pHluorin
PHN N-terminal fragment of pHluorin
PSD-95 Postsynaptic density protein 95 (membrane-associated guanylate kinase)
RNA Ribonucleic acid
RPMI Roswell Park Memorial Institute medium
SDS Sodium dodecyl sulfate
TIRF Total internal reflection fluorescence microscopy
YFP Yellow fluorescent protein
YC C-terminal fragment of YFP
YN N-terminal fragment of YFP
[0050] It is noted here that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. [0051] A "control," as used herein, refers to an active, positive, negative or vehicle control. As will be understood by those of skill in the art, controls are used to establish the relevance of experimental results, and provide a
comparison for the condition being tested.
[0052] The term "nucleic acid" or "nucleic acid sequence" or "nucleic acid molecule" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Nucleic acids encoding an ion channel subunit polypeptide of the disclosure include, for example and without limitation, genes, pre-mRNAs, mRNAs, cDNAs, polymorphic variants, alleles, and synthetic and naturally-occurring mutants.
[0053] Nucleic acids of the disclosure also include, without limitation, those that (1 ) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence as described herein, and conservatively modified variants thereof; (2) have a nucleic acid sequence that has greater than about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater nucleotide sequence identity, over a region of at least about 25, about 50, about 100, about 150, about 200, about 250, about 500, about 1000, or more nucleotides (up to the full length sequence of nucleotides of the mature protein), to a reference nucleic acid sequence as described herein.
[0054] Exemplary "stringent hybridization" conditions include hybridization at 42QC in 50% formamide, 5X SSC, 20 mM Na-P04, pH 6.8; and washing in 1 X SSC at 55QC for 30 minutes. It is understood that variation in these exemplary conditions can be made based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining appropriate hybridization conditions. See Sambrook et al., Molecular Cloning: A Laboratory Manual (Second ed., Cold Spring Harbor Laboratory Press, 1989) §§ 9.47-9.51 .
[0055] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions, in some aspects, are achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al., Nucleic Acid Res. 1 9:5081 (1991 ); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91 -98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
[0056] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues linked via peptide bonds. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides.
[0057] Individual substitutions, insertions, deletions, truncations or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, and generally less than 1 %) in an encoded sequence are considered "conservatively modified variations," provided alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions providing functionally similar amino acids are well known in the art, including the following eight groups, each of which contains amino acids that are considered conservative substitutes for each another:
1 ) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0058] An "analog," "variant" or "derivative" is a compound substantially similar in structure and having the same biological activity, albeit in certain instances to a differing degree, to a naturally-occurring molecule. For example, a polypeptide variant refers to a polypeptide sharing substantially similar structure and having the same biological activity as a reference polypeptide. Variants or analogs differ in the composition of their amino acid sequences compared to the naturally- occurring polypeptide from which the analog is derived, based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and/or one or more internal regions of the naturally-occurring polypeptide sequence (e.g., fragments), (ii) insertion or addition of one or more amino acids at one or more termini (typically an "addition" or "fusion") of the polypeptide and/or one or more internal regions (typically an "insertion") of the naturally-occurring polypeptide sequence or (iii) substitution of one or more amino acids for other amino acids in the naturally-occurring polypeptide sequence. By way of example, a "derivative" refers to a polypeptide sharing the same or substantially similar structure as a reference polypeptide that has been modified, e.g., chemically.
[0059] As used herein a "fragment" of a polypeptide refers to any portion of the polypeptide smaller than the full-length polypeptide or protein expression product. Fragments are deletion analogs of the full-length polypeptide wherein one or more amino acid residues have been removed from the amino terminus and/or the carboxy terminus of the full-length polypeptide.
[0060] The terms "ion channel polypeptide" and "ion channel" are used interchangeably herein to refer to pore-forming membrane proteins that enable the passive flow of inorganic ions by forming hydrated pores across biological membranes.
[0061] The terms "ion channel subunit polypeptide" and "ion channel subunit" refer to alpha, beta, gamma and delta subunits of various ion channel
polypeptides. Pore-forming alpha subunits determine ion permeation whereas beta, gamma and delta subunits modulate channel gating, surface expression, targeting and stability. Moreover, some of these subunits constitute binding sites for toxins as well as for therapeutic drugs.
[0062] The term "homomer" refers to a complex composed of only one subunit. For example, the disclosure includes homomers of ion channel subunits wherein like ion channel subunits associate with each other, e.g. a homomer of a voltage- gated potassium ion (Kv) channel subunit 1 .1 (Kv1 .1 -Kv1 .1 ). [0063] The term "heteromer" refers to a complex compose of more than one type of subunit. For example, the disclosure includes heteromers of ion channel subunits wherein unlike ion channel subunits associate with each other, e.g. a heteromer of a voltage-gated potassium ion (Kv) channel subunits 1 .1 and 1 .2 (Kv1 .1 -Kv1 .2).
[0064] The terms "label" or "fluorescent label" or "fluorescent protein" are used herein to refer to a fluorescent protein, or fragment thereof, that is attached to an ion channel subunit polypeptide to detect expression, changes in expression, and localization of the polypeptide in a cell.
[0065] The term "linker" or "linker polypeptide" is used herein to refer to a short peptide sequence used to connect the fluorescent label to the ion channel subunit polypeptide.
[0066] The terms "identity" or "percent identity" or "percent sequence identity" are used herein to refer to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). "Substantial identity" refers to sequences with at least or about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity over a specified sequence.
[0067] In some aspects, the identity exists over a region that is at least about 50-100 amino acids or nucleotides in length. In other aspects, the identity exists over a region that is at least about 100-200 amino acids or nucleotides in length. In other aspects, the identity exists over a region that is at least about 200-500 amino acids or nucleotides in length. In certain aspects, percent sequence identity is determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit and the Smith-Waterman algorithm.
[0068] It also is specifically understood that any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as about 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1 % to 3%, etc., are expressly enumerated in this specification. The values listed above are only examples of what is specifically intended.
[0069] The term "vector" is used to refer to any molecule used to transfer coding information to a host cell. As used herein, "vector" includes all those known in the art, including without limitation cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant
polynucleotide. The vector is inserted (e.g., via transformation or transduction) into an appropriate host cell for expression of the polynucleotide and polypeptide via transformation or transfection using techniques known in the art. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, 1989.
[0070] The terms "cell" or "host cell" as used herein are used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing that selected nucleic acid sequence as a polypeptide. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected nucleic acid sequence, e.g., gene, is present. In various aspects, the cell being transformed is either a prokaryotic or a eukaryotic cell. In further aspects, the cell being transformed includes, but is not limited to, a mammalian cell. In further aspects, the cell is a human cell.
[0071] The term "agent" or "test compound" describes any molecule, e.g. protein or pharmaceutical, with the capability of affecting or regulating an ion channel or ion channel subunit. Such agent, in various aspects, refers to ion channel blockers and/or ion channel activators, and refers to any agent that can alter the entry of certain ions into or out of cells and cellular organelles, depending on whether the intracellular or extracellular concentration of the particular ion is greater and the electrical potential difference that exists between the inside and the outside of the cell. In various aspects, an agent affects ion channel subunit polypeptide assembly, membrane expression, biogenesis, trafficking, membrane recycling, internalization, membrane insertion, or degradation. In some aspects, trafficking is anterograde trafficking or retrograde trafficking.
[0072] Discussed in further detail herein below are ion channel polypeptides, ion channel subunit polypeptides, linker polypeptides, vectors, cells, fluorescent proteins, and bimolecular fluorescence insofar as they apply to the compositions and methods of the disclosure.
Ion Channel Polypeptides
[0073] The disclosure provides ion channel constructs and methods for using these ion channel constructs as probes for visualizing and monitoring the movement of ion channel subunits. Ion channels are integral membrane proteins, or, more typically, an assembly of several proteins. Ion channels are present on all membranes of cell (plasma membrane) and intracellular organelles (nucleus, mitochondria, endoplasmic reticulum, golgi apparatus and the like). Such "multi-subunit" assemblies usually involve a circular arrangement of identical or homologous proteins closely packed around a water-filled pore through the plane of the membrane or lipid bilayer. For most voltage-gated ion channels, the pore-forming subunit(s) is called the a subunit, while the auxiliary subunits are denoted β, γ, δ and so on. Some ion channels permit the passage of ions based solely on their charge of positive (cation) or negative (anion). In some ion channels, passage through the pore is governed by a "gate," which may be opened or closed by chemical or electrical signals, temperature, or mechanical force, depending on the variety of channel.
[0074] Ion channels differ with respect to the ion they let pass (e.g., Na+, K+, and CI"), the ways in which they may be regulated, the number of subunits of which they are composed and other aspects of structure. Ion channels belonging to the largest class, which includes the voltage-gated channels that underlie the nerve impulse, consists of four subunits with six transmembrane helices each. On activation, these helices move about and open the pore. Two of these six helices are separated by a loop that lines the pore and is the primary determinant of ion selectivity and conductance in this channel class and some others. For potassium-selective channels, which are among the best-characterized ion channels, four homologous transmembrane subunits come together to create a tunnel, known as the conducting pore, that provides a polar pathway through the nonpolar lipid membrane. The present disclosure provides ion channel subunit polypeptides comprising a label inserted into an extracellular loop between transmembrane-spanning segments of the ion channel polypeptide.
[0075] In some aspects, the channel probes provided herein permit the measurement of ion channel surface levels. In certain aspects, anti-GFP antibodies are used to label surface proteins in non-permeablized cells. The GFP signal can then be normalized to the GFP fluorescence to the ratio of surface channel to total channel protein. In other aspects, PHIuorin-tagged ion channel surface levels can be assayed independent of antibody labeling by exposing cells to extracellular solutions of neutral or acidic pH. Importantly, these channel probes can also be a tool to study the temporal and spatial changes in the channel/protein localization. For example, channel movement can be measured, e.g., measure and visualize channel moving from the surface into the cell (endocytosis) or channel moving back to the membrane after endocytosis (recycling).
[0076] In further aspects, time, concentration (i.e., number), and location of insertion into the membrane is measured as well as the movement of channel proteins within the cell. In addition, the use of co-expressed split-fluorescent tagged constructs permits the selective visualization and measurement of all the trafficking events described above for homo- and heteromeric channel complexes. These channel probes also permit the study of when and where within a cell, ion channel complexes are created and assembled (biogenesis) or when and where they are destroyed (degradation). Importantly, these fluorescent ion channel probes are useful in screening for compounds or conditions that change the surface density of channel proteins. When used in combination with high-throughput assays, ion channel probes described herein are a valuable drug-discovery or screening tool.
[0077] In some aspects, the methods of the disclosure detect trafficking of ion channel subunit polypeptides. In various aspects, this trafficking is anterograde or retrograde. Anterograde trafficking is movement of the newly synthesized channel protein to the cell surface. Retrograde trafficking is movement of surface channel protein to intracellular compartments.
[0078] In some aspects, the disclosure provides compositions and methods for studying how various compounds or agents affect the biological activity of ion channels and the ion channel subunits. For example, many natural toxins target ion channels. Examples include the voltage-gated sodium channel blocker tetrodotoxin, the irreversible nicotinic acetylcholine receptor antagonist alpha- bungarotoxin, and plant-derived alkaloids, such as strychnine and d-tubocurarine. Such compounds and methods are useful in the development of therapeutic drugs, which act directly or indirectly to modulate ion channel activity.
[0079] In some aspects, the ion channel polypeptide is a potassium channel. Potassium channels form potassium-selective pores that span cell membranes. Furthermore, potassium channels are found in most cell types and control a wide variety of cell functions. There are four major classes of potassium channels: (1 ) calcium-activated potassium channels - open in response to the presence of calcium ions or other signaling molecules; (2) inwardly rectifying potassium channels - pass current (positive charge) more easily in the inward direction (into the cell); (3) tandem pore domain potassium channels - are constitutively open or possess high basal activation, such as the "resting potassium channels" or "leak channels" that set the negative membrane potential of neurons; and (4) voltage- gated potassium channels - open or close in response to changes in the transmembrane voltage. In various aspects, the disclosure includes all four classes of potassium channels.
[0080] Potassium channels have a tetrameric structure in which, in one aspect, four identical protein subunits associate to form a four-fold symmetric complex arranged around a central ion conducting pore (i.e., a homotetramer). Alternatively, four related, but not identical protein subunits, may associate, in some aspects, to form heterotetrameric complexes. Potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selective permeability.
[0081] In particular aspects, the potassium channel is a voltage-gated potassium (Kv) channel. Such Kv channel polypeptides include, but are not limited to, Kv1 , Kv2, Kv3, Kv4, Kv5, Kv6, Kv7, Kv8, Kv9, Kv10, Kv1 1 , and Kv12. Such Kv channel polypeptides further comprise polypeptide subunits including, but not limited to, Kv1 .1 , Kv1 .2, Kv1 .3, Kv1 .4, Kv1 .5, Kv1 .6, Kv1 .7, Kv1 .8, Kv2.1 , Kv2.2, Kv3.1 , Kv3.2, Kv3.3, Kv3.4, Kv4.1 , Kv4.2, Kv4.3, Kv5.1 , Kv6.1 , Kv6.2, Kv6.3, Kv6.4, Kv7.1 , Kv8.1 , Kv8.2, Kv9.1 , Kv9.2, Kv9.3, Kv10.1 , Kv10.2, Kv1 1 .1 , Kv1 1 .2, Kv1 1 .3, Kv12.1 , Kv12.2, Kv12.3.
[0082] In other aspects, the potassium channel is an inward-rectifying (Kir) potassium channel. Such Kir channel polypeptides include, but are not limited to, Kir1 , Kir2, Kir3, Kir4, Kir5, Kir6, and Kir7. Such Kir channel polypeptides further comprise polypeptide subunits including, but not limited to, Kir1 .1 , Kir1 .2, Kir2.1 , Kir2.2, Kir2.2v, Kir2.3, Kir2.4, Kir3.1 , Kir3.2, Kir3.3, Kir3.4, Kir4.1 , Kir5.1 , Kir6.1 , and Kir7.1 .
[0083] In some aspects, the disclosure includes sodium channel polypeptides. Sodium channels are integral membrane proteins that form ion channels, conducting sodium ions (Na+) through a cell's plasma membrane. Sodium channel polypeptides are classified according to the trigger that opens the channel for such ions, i.e. either a voltage-change (voltage-gated sodium channels, i.e. Nav) or binding of a substance (a ligand) to the channel (ligand- gated sodium channels). The disclosure includes both voltage-gated sodium channels and ligand-gated sodium channels.
[0084] In particular aspects, the disclosure includes voltage-gated sodium (Nav) channel polypeptides. Such Nav channel polypeptides include, but are not limited to, Nav channel polypeptides selected from the group consisting of:
Nav1 .1 , Nav1 .2, Nav1 .3, Nav1 .4, Nav1 .5, Nav1 .6, Nav1 .7, Nav1 .8, Nav1 .9, and Nax. [0085] In further aspects, the disclosure includes chloride channel
polypeptides. Chloride channels display a variety of important physiological and cellular roles that include regulation of pH, volume homeostasis, organic solute transport, cell migration, cell proliferation and differentiation. Based on sequence homology, the chloride channels are subdivided into a number of groups.
Chloride channels are important for setting cell resting membrane potential and maintaining proper cell volume. These channels conduct chloride as well as other anions such as HC03 ", , SCN", and N03". The structure of chloride channels are not like other known channels. Chloride channel subunits contain between 1 and 12 transmembrane segments. Some members of this family are activated by voltage, while others are activated by Ca2+, extracellular ligands, pH, and the like.
[0086] Thus, in some aspects, the disclosure includes calcium-activated chloride channel regulator proteins 1 (CLCA1 ), 2 (CLCA2), 3 (CLCA3), and 4 (CLCA4). In other aspects, the disclosure includes the CLCN family of voltage- dependent chloride channel proteins. The CLCN family comprises nine members (CLCN1 , CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCNKB) which demonstrate quite diverse functional characteristics while sharing significant sequence homology. In further aspects, the disclosure includes chloride intracellular (CLI) channel proteins. The CLI family comprises at least 6 members (CLIC1 , CLIC2, CLIC3, CLIC4, CLIC5, and CLIC6. In even further aspects, the disclosure includes chloride channel, nucleotide-sensitive, 1 A (CLNS1 A). CLNS1 A is a protein channel subunit shown to interact with small nuclear ribonucleoprotein D1 , integrin alpha chain 2b (ITGA2B), small nuclear ribonucleoprotein Sm D3 (SNRPD3), and Protein arginine N-methyltransferase 5 (PRMT5).
Polypeptides of Various Ion Channel Subunit Constructs
[0087] The disclosure includes the following polypeptides of various ion channel subunits comprising a linker and a fluorescent label.
Kv1 .1
Kv1 .1 -GFP (750) (SEQ ID NO: 2)
[0088] mtvmsgenvdeasaapghpqdgsyprqadhddheccervvinisglrfetqlktlaqfpntllgn pkkrmryfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldmfseeikfyelgeeamekfredegfikeeer plpekeyqrqvwllfeypessgparviaivsvmvilisivifcletlpelkddkdftgtaaasggtgmvskgeelftgv vpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkqhdffksamp egyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimadkqkngikvnfki rhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgmdelykvdggs aaavhridnttviynsniftdpffivetlciiwfsfelvvrffacpsktdffknimnfidivaiipyfitlgteiae qatslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaeeaeshfssipdafwwavv smttvgygdmypvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqllhvsspnlasdsdlsrrss stmskseymeieedmnnsiahyrqvnirtancttanqncvnksklltdv
Kv1 .1 -pHluorin (741 ) (SEQ ID NO: 4)
[0089] mtvmsgenvdeasaapghpqdgsyprqadhddheccervvinisglrfetqlktlaqfpntllgn pkkrmryfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldmfseeikfyelgeeamekfredegfikeeer plpekeyqrqvwllfeypessgparviaivsvmvilisivifcletlpelkddkdftgtaaaklmskgeelftgvvpilv eldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyv qertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngikanfkirhni edggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykaaavhridnt tviynsniftdpffivetlciiwfsfelvvrffacpsktdffknimnfidivaiipyfitlgteiaeqegnqkgeqatslai vrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaeeaeshfssipdafwwavvsmttvgygd mypvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqllhvsspnlasdsdlsrrssstmsksey meieedmnnsiahyrqvnirtancttanqncvnksklltdv
Kv1 .1 -PHN (665) (SEQ ID NO: 6)
[0090] mtvmsgenvdeasaapghpqdgsyprqadhddheccervvinisglrfetqlktlaqfpntllgn pkkrmryfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldmfseeikfyelgeeamekfredegfikeeer plpekeyqrqvwllfeypessgparviaivsvmvilisivifcletlpelkddkdftgtaaasggtgmskgeelftgvv pilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampe gyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggsaaavhrid nttviynsniftdpffivetlciiwfsfelvvrffacpsktdffknimnfidivaiipyfitlgteiaeqe
virlvrvfrifklsrhskglqNgqtlkasmrelgllifflfigvNfssavyfaeaeeaeshfssipdafwwavvsmttvgy gdmypvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqllhvsspnlasdsdlsrrssstmsks eymeieedmnnsiahyrqvnirtancttanqncvnksklltdv
Kv1 .1 -PHC (595) (SEQ ID NO: 8)
[0091] mtvmsgenvdeasaapghpqdgsyprqadhddheccervvinisglrfetqlktlaqfpntllgn pkkrmryfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldmfseeikfyelgeeamekfredegfikeeer plpekeyqrqvwllfeypessgparviaivsvmvilisivifcletlpelkddkdftgtaaasggtgdkqkngikanfki rhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykvdggs aaavhridnttviynsniftdpffivetlciiwfsfelvvrffacpsktdffknimnfidivaiipyfitlgteiae
qatslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaeeaeshfssipdafwwavv smttvgygdmypvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqllhvsspnlasdsdlsrrss stmskseymeieedmnnsiahyrqvnirtancttanqncvnksklltdv
Kv1 .1 -YC (595) (SEQ ID NO: 10)
[0092] mtvmsgenvdeasaapghpqdgsyprqadhddheccervvinisglrfetqlktlaqfpntllgn pkkrmryfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldmfseeikfyelgeeamekfredegfikeeer plpekeyqrqvwllfeypessgparviaivsvmvilisivifcletlpelkddkdftgtaaasggtgdkqkngikvnfki rhniedgsvqladhyqqntpigdgpvllpdnhylsyqsalskdpnekrdhmvllefvtaagitlgmdelykvdgg saaavhridnttviynsniftdpffivetlciiwfsfelvvrffacpsktdffknimnfidivaiipyfitlgteiaeqeg eqatslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaeeaeshfssipdafwwav vsmttvgygdmypvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqllhvsspnlasdsdlsrrs sstmskseymeieedmnnsiahyrqvnirtancttanqncvnksklltdv
Kv1 .1 -YN (666) (SEQ ID NO: 12)
[0093] mtvmsgenvdeasaapghpqdgsyprqadhddheccervvinisglrfetqlktlaqfpntllgn pkkrmryfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldmfseeikfyelgeeamekfredegfikeeer plpekeyqrqvwllfeypessgparviaivsvmvilisivifcletlpelkddkdftgtaaasggtgmvskgeelftgv vpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttfgyglqcfarypdhmkqhdffksam pegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimavdggsaaavh ridnttviynsniftdpffivetlciiwfsfelvvrffacpsktdffknimnfidivaiipyfitlgtei^
NrvirlvrvfrifklsrhskglqNgqtlkasmrelgllifflfigvNfssavyfaeaeeaeshfssipdafwwavvsmttvg ygdmypvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqllhvsspnlasdsdlsrrssstmsk seymeieedmnnsiahyrqvnirtancttanqncvnksklltdv
Kv1 .2
Kv1 .2-GFP (754) (SEQ ID NO: 14)
[0094] mtvatgdpvdeaaalpghpqdtydpeadheccervvinisglrfetqlktlaqfpetllgdpkkrmr yfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldifseeirfyelgeeamemfredegyikeeerplpenef qrqvwllfeypessgpariiaivsvmvilisivsfcletlpifrdenedmhgggvaaasggtgmvskgeelftgvvpi
Iveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkqhdffksampeg yvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnNghkleynynshnvyimadkqkngikvnfkirh niedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgmdelykvdggsa aatfhtysnstigyqqstsftdpffivetlciiwfsfeflvrffacpskagfftnimniidivaiipyfitlgtelaekpedaqq gqqamslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaderdsqfpsipdafww avvsmttvgygdmvpttiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqylqvtscpkipsspdl kksrsastisksdymeiqegvnnsnedfreenlktanctlantnyvnitkmltdv
Kv1 .2-PHN (669) (SEQ ID NO: 16)
[0095] mtvatgdpvdeaaalpghpqdtydpeadheccervvinisglrfetqlktlaqfpetllgdpkkrmr yfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldifseeirfyelgeeamemfredegyikeeerplpenef qrqvwllfeypessgpariiaivsvmvilisivsfcletlpifrdenedmhgggvaaasggtgmskgeelftgvvpil veldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegy vqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggsaaatfhtysn stigyqqstsftdpffivetlciiwfsfeflvrffacpskagfftnimniidivaiipyfitlgtelaekpedaqqgqqamsla ilrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaderdsqfpsipdafwwavvsmttvg ygdmvpttiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqylqvtscpkipsspdlkksrsastisk sdymeiqegvnnsnedfreenlktanctlantnyvnitkmltdv
Kv1 .2-PHC (599) (SEQ ID NO: 18)
[0096] mtvatgdpvdeaaalpghpqdtydpeadheccervvinisglrfetqlktlaqfpetllgdpkkrmr yfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldifseeirfyelgeeamemfredegyikeeerplpenef qrqvwllfeypessgpariiaivsvmvilisivsfcletlpifrdenedmhgggvaaasggtgdkqkngikanfkirh niedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykvdggsaa atfhtysnstigyqqstsftdpffivetlciiwfsfeflvrffac
qqamslaNrvirlvrvfrifklsrhskglqNgqtlkasmrelgllifflfigvilfssavyfaeaderdsqfpsipdafwwa vvsmttvgygdmvpttiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqylqvtscpkipsspdlkk srsastisksdymeiqegvnnsnedfreenlktanctlantnyvnitkmltdv
Kv1 .2-YN (670) (SEQ ID NO: 20)
[0097] mtvatgdpvdeaaalpghpqdtydpeadheccervvinisglrfetqlktlaqfpetllgdpkkrmr yfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldifseeirfyelgeeamemfredegyikeeerplpenef qrqvwllfeypessgpariiaivsvmvilisivsfcletlpifrdenedmhgggvaaasggtgmvskgeelftgvvpi
Iveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttfgyglqcfarypdhmkqhdffksampeg yvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimavdggsaaatfhtysn stigyqqstsftdpffivetlciiwfsfeflvrffacpskagfftnimniidivaiipyfitlgtelaekpedaqqgqqam
NrvirlvrvfrifklsrhskglqNgqtlkasmrelgllifflfigvilfssavyfaeaderdsqfpsipdafwwavvsmttvg ygdmvpttiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqylqvtscpkipsspdlkksrsastisk sdymeiqegvnnsnedfreenlktanctlantnyvnitkmltdv
Kv1 .2-YC (599) (SEQ ID NO: 22)
[0098] mtvatgdpvdeaaalpghpqdtydpeadheccervvinisglrfetqlktlaqfpetllgdpkkrmr yfdplrneyffdrnrpsfdailyyyqsggrlrrpvnvpldifseeirfyelgeeamemfredegyikeeerplpenef qrqvwllfeypessgpariiaivsvmvilisivsfcletlpifrdenedmhgggvaaasggtgdkqkngikvnfkirh niedgsvqladhyqqntpigdgpvllpdnhylsyqsalskdpnekrdhmvllefvtaagitlgmdelykvdggsa aatfhtysnstigyqqstsftdpffivetlciiwfsfeflvrffacpskagfftnimniidivaiipyfitlgtelaekpedaqq gqqamslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaderdsqfpsipdafww avvsmttvgygdmvpttiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqaqylqvtscpkipsspdl kksrsastisksdymeiqegvnnsnedfreenlktanctlantnyvnitkmltdv
Kv1 .3
Kv1 .3-GFP (778) (SEQ ID NO: 24)
[0099] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrfe tqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekfr edegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgm vskgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmk qhdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimad kqkngikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlg mdelykvdggsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidiv aiipyfitlgtelaerqgngqqamslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfae dptsgfssipdafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsq ymhvgscqhlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkift dv
Kv1 .3-YN (694) (SEQ ID NO: 26)
[00100] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrf etqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekf redegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgm vskgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttfgyglqcfarypdhmk qhdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimav dggsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidivaiipyfitlg telaerqgngqqamslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaddptsgfss ipdafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsqymhvgsc qhlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkiftdv
Kv1 .3-YC (623) (SEQ ID NO: 28)
[00101 ] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrf etqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekf redegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgd kqkngikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylsyqsalskdpnekrdhmvllefvtaagitlg mdelykvdggsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidiv aiipyfitlgtelaerqgngqqamslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssa dptsgfssipdafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsq ymhvgscqhlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkift dv
Kv1 .3-PHN (693) (SEQ ID NO: 30)
[00102] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrf etqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekf redegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgm skgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkr hdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavd ggsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidivaiipyfitlgt elaerqgngqqamslaNrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaddptsgfssi pdafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsqymhvgsc qhlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkiftdv
Kv1 .3-PHC (623) (SEQ ID NO: 32)
[00103] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrf etqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekf redegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgd kqkngikanfkirhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithg mdelykvdggsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidiv aiipyfitlgtelaerqgngqqamslaNrvirlvrvfrifklsrhskglqNgqtlkasmrelgllifflfigvNfssavyfae dptsgfssipdafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsq ymhvgscqhlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkift dv Kv1 .3-CN (694) (SEQ ID NO: 34)
[00104] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrf etqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekf redegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgm vskgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltwgvqcfsrypdhmk qhdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyishnvyitavdg gsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidivaiipyfitlgtel aerqgngqqamslailrvirlvrvfrifklsrhskglqilgqtlkasmrelgllifflfigvilfssavyfaeaddptsgfssip dafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsqymhvgscq hlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkiftdv
Kv1 .3-CC (623) (SEQ ID NO: 36)
[00105] mtvvpgdhllepevadgggappqggcggggcdryeplppslpaageqdccgervvinisglrf etqlktlcqfpetllgdpkrrmryfdplrneyffdrnrpsfdailyyyqsggrirrpvnvpidifseeirfyqlgeeamekf redegflreeerplprrdfqrqvwllfeypessgpargiaivsvlvilisivifcletlpefrdekdypastaaasggtgd kqkngikanfkirhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlg mdelykvdggsaaasqdsfeaagnstsgsragassfsdpffvvetlciiwfsfellvrffacpskatfsrnimnlidiv aiipyfitlgtelaerqgngqqamslaNrvirlvrvfrifklsrhskglqNgqtlkasmrelgllifflfigvNfssavyfae dptsgfssipdafwwavvtmttvgygdmhpvtiggkivgslcaiagvltialpvpvivsnfnyfyhretegeeqsq ymhvgscqhlsssaeelrkarsnstlskseymvieeggmnhsafpqtpfktgnstatcttnnnpnscvnikkift dv
Kv1 .4
Kv1 .4-GFP (908) (SEQ ID NO: 38)
[00106] mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvNisivifcletlpefrddrdlvmalsagghggllndtsaphlaaasggtgmv skgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkq hdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimadk qkngikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgm delykvdggsaaaensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqq gggngqqqqamsfaNriirlvrvfrifklsrhskglqNghtlrasmrelgllifflfigvilfssavyfaeadeptthfqsipd afwwavvtmttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylp snllkkfrsstssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-CFP (908) (SEQ ID NO: 40)
[00107] mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgmvskgeelftgvv pilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltwgvqcfsrypdhmkqhdffksamp egyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyishnvyitadkqkngikanfkirh niedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgmdelykvdggsa aagllndtsaphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqgg gngqqqqamsfailriirlvrvfrifklsrhskglqilghtlrasmrelgllifflfigvilfssavyfaeadeptthfqsip wwavvtmttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylps nllkkfrsstssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-CN (824) (SEQ ID NO: 42)
[00108] mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgmvskgeelftgvv pNveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltwgvqcfsrypdhmkqhdffksamp egyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyishnvyitavdggsaaagllndt saphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqgggngqqq qamsfaNriirlvrvfrifklsrhskglqNghtlrasmrelgllifflfigvNfssavyfaeadeptthfqsipdafwwavvt mttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylpsnllkkfrss tssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-CC (753) (SEQ ID NO: 44)
[00109] Mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgdkqkngikanfki rhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgmdelykvdggs aaagllndtsaphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqg ggngqqqqamsfailriirlvrvfrifklsrhskglqilghtlrasmrelgllifflfigvilfssavyfaeadeptthfqsip fwwavvtmttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylps nllkkfrsstssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-pHluorin (899) (SEQ ID NO: 46)
[00110] Mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaaklmskgeelftgvvpilve
Idgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyvq ertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngikanfkirhnie dggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykaaagllndtsa phlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqgggngqqqqa msfailriirlvrvfrifklsrhskglqilghtlrasmrelgllifflfigvilfssavyfaeadeptthfqsipdafwwavvtmtt vgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylpsnllkkfrsstss slgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-PHN (823) (SEQ ID NO: 48)
[00111] Mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgmskgeelftgvv pilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampe gyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggsaaagllnd tsaphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqgggngqqq qamsfaNriirlvrvfrifklsrhskglqNghtlrasmrelgllifflfigvNfssavyfaeadeptthfqsipdafwwavvt mttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylpsnllkkfrss tssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv Kv1 .4-PHC (753) (SEQ ID NO: 50)
[00112] Mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgdkqkngikanfki rhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykvdggs aaagllndtsaphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqg ggngqqqqamsfailriirlvrvfrifklsrhskglqilghtlrasmrelgllifflfigvilfssavyfaeadeptthfqsip fwwavvtmttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylps nllkkfrsstssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-YN (824) (SEQ ID NO: 52)
[00113] Mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgmvskgeelftgvv pilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttfgyglqcfarypdhmkqhdffksamp egyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimavdggsaaaglln dtsaphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqgggngqq qqamsfaNriirlvrvfrifklsrhskglqNghtlrasmrelgllifflfigvNfssavyfaeadeptthfqsipdafwwavv tmttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylpsnllkkfrs stssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .4-YC (753) (SEQ ID NO: 54)
[00114] Mevamvsaessgcnshmpygyaaqararererlahsraaaaaavaaataavegsggsgg gshhhhqsrgactshdpqssrgsrrrrrqrsekkkahyrqssfphcsdlmpsgseekilrelseeeedeeeee eeeeegrfyyseddhgdecsytdllpqdeggggyssvrysdccervvinvsglrfetqmktlaqfpetllgdpekr tqyfdplrneyffdrnrpsfdailyyyqsggrlkrpvnvpfdifteevkfyqlgeeallkfredegfvreeedralpenef kkqiwllfeypessspargiaivsvlvilisivifcletlpefrddrdlvmalsagghgaaasggtgdkqkngikvnfkir hniedgsvqladhyqqntpigdgpvllpdnhylsyqsalskdpnekrdhmvllefvtaagitlgmdelykvdggs aaagllndtsaphlensghtifndpffivetvcivwfsfefvvrcfacpsqalffknimniidivsilpyfitlgtdlaqqqg ggngqqqqamsfaNriirlvrvfrifklsrhskglqNghtlrasmrelgllifflfigvilfssavyfaeadeptthfqsipda fwwavvtmttvgygdmkpitvggkivgslcaiagvltialpvpvivsnfnyfyhreteneeqtqltqnavscpylps nllkkfrsstssslgdkseylemeegvkeslcakeekcqgkgddsetdknncsnakavetdv
Kv1 .5
Kv1 .5-GFP (865) (SEQ ID NO: 56)
[00115] Meialvplenggamtvrggdearagcgqatggelqcpptaglsdgpkepapkgrgaqrdads gvrplpplpdpgvrplpplpeelprprrpppedeeeegdpglgtvedqalgtaslhhqrvhinisglrfetqlgtlaqf pntllgdpakrlryfdplrneyffdrnrpsfdgilyyyqsggrlrrpvnvsldvfadeirfyqlgdeamerfredegfike eekplprnefqrqvwlifeypessgsaraiaivsvlvilisiitfcletlpefrderellrhppaphqppaaasggtgmv skgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkq hdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimadk qkngikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgm delykvdggsaaapgangsgvmappsgptvapllprtladpffivettcviwftfellvrffacpskagfsrnimniid vvaifpyfitlgtelaeqqpggggggqngqqamslailrvirlvrvfrifklsrhskglqilgktlqasmrelgllifflfigvilf ssavyfaeadnqgthfssipdafwwavvtmttvgygdmrpitvggkivgslcaiagvltialpvpvivsnfnyfyhr etdheepavlkeeqgtqsqgpgldrgvqrkvsgsrgsfckaggtlenadsarrgscplekcnvkaksnvdlrrsl yalcldtsretdl
Kv1 .5-pHluorin (856) (SEQ ID NO: 58)
[00116] Meialvplenggamtvrggdearagcgqatggelqcpptaglsdgpkepapkgrgaqrdads gvrplpplpdpgvrplpplpeelprprrpppedeeeegdpglgtvedqalgtaslhhqrvhinisglrfetqlgtlaqf pntllgdpakrlryfdplrneyffdrnrpsfdgilyyyqsggrlrrpvnvsldvfadeirfyqlgdeamerfredegfike eekplprnefqrqvwlifeypessgsaraiaivsvlvilisiitfcletlpefrderellrhppaphqppaaaklmskge elftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffk sampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngi kanfkirhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelyk aaapgangsgvmappsgptvapllprtladpffivettcviwftfellvrffacpskagfsrnimniidvvaifpyfitlgt elaeqqpggggggqngqqamslaNrvirlvrvfrifklsrhskglqilgktlqasmrelgllifflfigvilfssavyfaea dnqgthfssipdafwwavvtmttvgygdmrpitvggkivgslcaiagvltialpvpvivsnfnyfyhretdheepa vlkeeqgtqsqgpgldrgvqrkvsgsrgsfckaggtlenadsarrgscplekcnvkaksnvdlrrslyalcldtsre tdl
Kv1 .5-YN (781 ) (SEQ ID NO: 60)
[00117] Meialvplenggamtvrggdearagcgqatggelqcpptaglsdgpkepapkgrgaqrdads gvrplpplpdpgvrplpplpeelprprrpppedeeeegdpglgtvedqalgtaslhhqrvhinisglrfetqlgtlaqf pntllgdpakrlryfdplrneyffdrnrpsfdgilyyyqsggrlrrpvnvsldvfadeirfyqlgdeamerfredegfike eekplprnefqrqvwlifeypessgsaraiaivsvlvilisiitfcletlpefrderellrhppaphqppaaasggtgmv skgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttfgyglqcfarypdhmkq hdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimavd ggsaaapgangsgvmappsgptvapllprtladpffivettcviwftfellvrffacpskagfsrnimniidvvaifpyf itlgtelaeqqpggggggqngqqamslailrvirlvrvfrifklsrhskglqilgktlqasmrelgllifflfigvilfssavyfa eadnqgthfssipdafwwavvtmttvgygdmrpitvggkivgslcaiagvltialpvpvivsnfnyfyhretdhee pavlkeeqgtqsqgpgldrgvqrkvsgsrgsfckaggtlenadsarrgscplekcnvkaksnvdlrrslyalcldts retdl
Kv1 .5-YC (710) (SEQ ID NO: 62)
[00118] Meialvplenggamtvrggdearagcgqatggelqcpptaglsdgpkepapkgrgaqrdads gvrplpplpdpgvrplpplpeelprprrpppedeeeegdpglgtvedqalgtaslhhqrvhinisglrfetqlgtlaqf pntllgdpakrlryfdplrneyffdrnrpsfdgilyyyqsggrlrrpvnvsldvfadeirfyqlgdeamerfredegfike eekplprnefqrqvwlifeypessgsaraiaivsvlvilisiitfcletlpefrderellrhppaphqppaaasggtgdk qkngikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylsyqsalskdpnekrdhmvllefvtaagitlg mdelykvdggsaaapgangsgvmappsgptvapllprtladpffivettcviwftfellvrffacpskagfsrnimn iidvvaifpyfitlgtelaeqqpggggggqngqqamslailrvirlvrvfrifklsrhskglqilgktlqasmrelgllifflfig vilfssavyfaeadnqgthfssipdafwwavvtmttvgygdmrpitvggkivgslcaiagvltialpvpvivsnfnyf yhretdheepavlkeeqgtqsqgpgldrgvqrkvsgsrgsfckaggtlenadsarrgscplekcnvkaksnvdl rrslyalcldtsretdl
Kv1 .5-PHN (780) (SEQ ID NO: 64)
[00119] Meialvplenggamtvrggdearagcgqatggelqcpptaglsdgpkepapkgrgaqrdads gvrplpplpdpgvrplpplpeelprprrpppedeeeegdpglgtvedqalgtaslhhqrvhinisglrfetqlgtlaqf pntllgdpakrlryfdplrneyffdrnrpsfdgilyyyqsggrlrrpvnvsldvfadeirfyqlgdeamerfredegfike eekplprnefqrqvwlifeypessgsaraiaivsvlvilisiitfcletlpefrderellrhppaphqppaaasggtgms kgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrh dffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdg gsaaapgangsgvmappsgptvapllprtladpffivettcviwftfellvrffacpskagfsrnimniidvvaifpyfit
Igtelaeqqpggggggqngqqamslailrvi rlvrvfrifklsrhskglqilgktlqasmrelgllifflfigvilfssavyfa eadnqgthfssipdafwwavvtmttvgygdmrpitvggkivgslcaiagvltialpvpvivsnfnyfyhretdhee pavlkeeqgtqsqgpgldrgvqrkvsgsrgsfckaggtlenadsarrgscplekcnvkaksnvdlrrslyalcldts retdl Kv1 .5-PHC (710) (SEQ ID NO: 66)
[00120] Meialvplenggamtvrggdearagcgqatggelqcpptaglsdgpkepapkgrgaqrdads gvrplpplpdpgvrplpplpeelprprrpppedeeeegdpglgtvedqalgtaslhhqrvhinisglrfetqlgtlaqf pntllgdpakrlryfdplrneyffdrnrpsfdgilyyyqsggrlrrpvnvsldvfadeirfyqlgdeamerfredegfike eekplprnefqrqvwlifeypessgsaraiaivsvlvilisiitfcletlpefrderellrhppaphqppaaasggtgdk qkngikanfkirhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgm delykvdggsaaapgangsgvmappsgptvapllprtladpffivettcviwftfellvrffacpskagfsrnimniid vvaifpyfitlgtelaeqqpggggggqngqqamslailrvirlvrvfrifklsrhskglqilgktlqasmrelgllifflfigvilf ssavyfaeadnqgthfssipdafwwavvtmttvgygdmrpitvggkivgslcaiagvltialpvpvivsnfnyfyhr etdheepavlkeeqgtqsqgpgldrgvqrkvsgsrgsfckaggtlenadsarrgscplekcnvkaksnvdlrrsl yalcldtsretdl
Kv2.1
Kv2.1 -GFP (1 108) (SEQ ID NO: 68)
[00121 ] Mtkhgsrstsslppepmeivrskacsrrvrlnvgglahevlwrtldrlprtrlgklrdcnthdsllqvc ddysledneyffdrhpgaftsNnfyrtgrlhmmeemcalsfsqeldywgideiylesccqaryhqkkeqmneel kreaetlreregeefdntccaekrkklwdllekpnssvaakNaiisimfivlstialslntlpelqsaaasggtgmvsk geelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkqhd ffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynynshnvyimadkqkn gikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgmdel ykvdggsaaaldefgqstdnpqlahveavciawftmeyllrflsspkkwkffkgplnaidllailpyyvtifltesnks vlqfqnvrrvvqifrimrNrNklarhstglqslgftlrrsynelgllNflamgimifsslvffaekdeddtkfksipasfwwa titmttvgygdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnmk dafarsiemmdivvekngesiakkdkvqdnhlspnkwkwtkralsetsssksfetkeqgspekarsssspqhl nvqqledmyskmaktqsqpNntkemapqskppeelemssmpspvaplpartegvidmrsmssidsfisca tdfpeatrfshsplaslsskagsstapevgwrgalgasggrltetnpipetsrsgffvesprssmktnnplklralkv nfvegdptpllpslglyhdplrnrggaaaavaglecaslldkpvlspessiyttasartpprspekhtaiafnfeagv hhyidtdtddegqllysvdssppkslhgstspkfstgarteknhfessplptspkflrpncvyssegltgkgpgaqe kcklenhtppdvhmlpgggahgstrdqsi
Kv2.1 -PHN (1023) (SEQ ID NO: 70)
[00122] Mtkhgsrstsslppepmeivrskacsrrvrlnvgglahevlwrtldrlprtrlgklrdcnthdsllqvc ddysledneyffdrhpgaftsNnfyrtgrlhmmeemcalsfsqeldywgideiylesccqaryhqkkeqmneel kreaetlreregeefdntccaekrkklwdllekpnssvaakNaiisimfivlstialslntlpelqsaaasggtgmskg eelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdff ksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggs aaaldefgqstdnpqlahveavciawftmeyllrflsspkkwkffkgplnaidllailpyyvtifltesnksvlqfqnvrr vvqifrimrilrilklarhstglqslgftlrrsynelgllilflamgimifsslvffaekdeddtkfksipasfwwat gdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnmkdafarsie mmdivvekngesiakkdkvqdnhlspnkwkwtkralsetsssksfetkeqgspekarsssspqhlnvqqled myskmaktqsqpilntkemapqskppeelemssmpspvaplpartegvidmrsmssidsfiscatdfpeatr fshsplaslsskagsstapevgwrgalgasggrltetnpipetsrsgffvesprssmktnnplklralkvnfvegdpt pllpslglyhdplrnrggaaaavaglecaslldkpvlspessiyttasartpprspekhtaiafnfeagvhhyidtdtd degqllysvdssppkslhgstspkfstgarteknhfessplptspkflrpncvyssegltgkgpgaqekcklenht ppdvhmlpgggahgstrdqsi
Kv2.1 -PHC (953) (SEQ ID NO: 72)
[00123] Mtkhgsrstsslppepmeivrskacsrrvrlnvgglahevlwrtldrlprtrlgklrdcnthdsllqvc ddysledneyffdrhpgaftsilnfyrtgrlhmmeemcalsfsqeldywgideiylesccqaryhqkkeqmneel kreaetlreregeefdntccaekrkklwdllekpnssvaakilaiisimfivlstialslntlpelqsaaasggtgdkqk ngikanfkirhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdel ykvdggsaaaldefgqstdnpqlahveavciawftmeyllrflsspkkwkffkgplnaidllailpyyvtifltesnks vlqfqnvrrvvqifrimrilrilklarhstglqslgftlrrsynelgllilflamgimifsslvffaekdeddtkfksipasfwwa titmttvgygdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnmk dafarsiemmdivvekngesiakkdkvqdnhlspnkwkwtkralsetsssksfetkeqgspekarsssspqhl nvqqledmyskmaktqsqpilntkemapqskppeelemssmpspvaplpartegvidmrsmssidsfisca tdfpeatrfshsplaslsskagsstapevgwrgalgasggrltetnpipetsrsgffvesprssmktnnplklralkv nfvegdptpllpslglyhdplrnrggaaaavaglecaslldkpvlspessiyttasartpprspekhtaiafnfeagv hhyidtdtddegqllysvdssppkslhgstspkfstgarteknhfessplptspkflrpncvyssegltgkgpgaqe kcklenhtppdvhmlpgggahgstrdqsi
Kv2.1 -YN (1023) (SEQ ID NO: 74)
[00124] Mtkhgsrstsslppepmeivrskacsrrvrlnvgglahevlwrtldrlprtrlgklrdcnthdsllqvc ddysledneyffdrhpgaftsNnfyrtgrlhmmeemcalsfsqeldywgideiylesccqaryhqkkeqmneel kreaetlreregeefdntccaekrkklwdllekpnssvaakNaiisimfivlstialslntlpelqsaaasggtgmskg eelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdff ksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggs aaaldefgqstdnpqlahveavciawftmeyllrflsspkkwkffkgplnaidllailpyyvtifltesnksvlqfqnvrr vvqifrimrilrilklarhstglqslgftlrrsynelgllilflamgimifsslvffaekdeddtkfksipasfwwatitmt^^ gdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnmkdafarsie mmdivvekngesiakkdkvqdnhlspnkwkwtkralsetsssksfetkeqgspekarsssspqhlnvqqled myskmaktqsqpilntkemapqskppeelemssmpspvaplpartegvidmrsmssidsfiscatdfpeatr fshsplaslsskagsstapevgwrgalgasggrltetnpipetsrsgffvesprssmktnnplklralkvnfvegdpt pllpslglyhdplrnrggaaaavaglecaslldkpvlspessiyttasartpprspekhtaiafnfeagvhhyidtdtd degqllysvdssppkslhgstspkfstgarteknhfessplptspkflrpncvyssegltgkgpgaqekcklenht ppdvhmlpgggahgstrdqsi
Kv2.1 -YC (953) (SEQ ID NO: 76)
[00125] Mtkhgsrstsslppepmeivrskacsrrvrlnvgglahevlwrtldrlprtrlgklrdcnthdsllqvc ddysledneyffdrhpgaftsilnfyrtgrlhmmeemcalsfsqeldywgideiylesccqaryhqkkeqmneel kreaetlreregeefdntccaekrkklwdllekpnssvaakilaiisimfivlstialslntlpelqsaaasggtgdkqk ngikvnfkirhniedgsvqladhyqqntpigdgpvllpdnhylsyqsalskdpnekrdhmvllefvtaagitlgmd elykvdggsaaaldefgqstdnpqlahveavciawftmeyllrflsspkkwkffkgplnaidllailpyyvtifltesnk svlqfqnvrrvvqifrimrilrilklarhstglqslgftlrrsynelgllilflamgimifsslvffaekdeddtkfksi atitmttvgygdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnm kdafarsiemmdivvekngesiakkdkvqdnhlspnkwkwtkralsetsssksfetkeqgspekarsssspq hlnvqqledmyskmaktqsqpilntkemapqskppeelemssmpspvaplpartegvidmrsmssidsfis catdfpeatrfshsplaslsskagsstapevgwrgalgasggrltetnpipetsrsgffvesprssmktnnplklral kvnfvegdptpllpslglyhdplrnrggaaaavaglecaslldkpvlspessiyttasartpprspekhtaiafnfea gvhhyidtdtddegqllysvdssppkslhgstspkfstgarteknhfessplptspkflrpncvyssegltgkgpga qekcklenhtppdvhmlpgggahgstrdqsi
Kv2.2
Kv2.2-pHluorin (1 153) (SEQ ID NO: 78)
[00126] Maekappglnrktsrstlslppepvdiirsktcsrrvkinvgglnhevlwrtldrlprtrlgklrdcnthe sllevcddynlneneyffdrhpgaftsNnfyrtgklhmmeemcalsfgqeldywgideiylesccqaryhqkkeq mneelrreaetmreregeefdntccpekrkklwdllekpnssvaakilaivsilfivlstialslntlpelqeaaaklms kgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrh dffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkq kngikanfkirhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgm elykaaandefgqpsdnrklahveavciawftmeyllrflsspnkwkffkgplnvidllailpyyvtifltesnksvlqf qnvrrvvqifrimrilrilklarhstglqslgftlrrsynelgllilflamgimifsslvffaekdedatkftsipasfwwati tvgygdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnlkdafars melidvavekagesanikdsvddnhlspsrwkwarkalsetssnksyenkyqevsqkdsheqlnntssssp qhlsaqklemlyneitktqthshpnpdcqeqperpsayeeeiemeevvcpqeqlavaqtevivdmkstssids ftscatdftetersplpppsashlqmkfptdlpgmdehqrvrappfltlsrdkgpaareaaldyapiditvnldagas hgplqpdsasdspksslkgsnplksrslkvnfqenrgsapqtppstarplpvttadfplttpqhmstilleealpqg qrplpgadvsahcqgpskglsprvpkqklfpfssrerrsfteidtgededfldlqrprpdkqadpspncladkpge ardplreegcvgssspqntdhncrqdiyqavgevkkdssqegykmenhlfapeihsnpgdtgycptretsm
Kv2.2-PHN (1077) (SEQ ID NO: 80)
[00127] Maekappglnrktsrstlslppepvdiirsktcsrrvkinvgglnhevlwrtldrlprtrlgklrdcnthe sllevcddynlneneyffdrhpgaftsilnfyrtgklhmmeemcalsfgqeldywgideiylesccqaryhqkkeq mneelrreaetmreregeefdntccpekrkklwdllekpnssvaakilaivsilfivlstialslntlpelqeaaasggt gmskgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdh mkrhdffksampegyvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyim avdggsaaandefgqpsdnrklahveavciawftmeyllrflsspnkwkffkgplnvidllailpyyvtifltesnksv
Iqfqnvrrvvqifrimrilrilklarhstglqslgftlrrsynelgllilflamgimifsslvffaekdedatkftsi mttvgygdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsivsmnlkdaf arsmelidvavekagesanikdsvddnhlspsrwkwarkalsetssnksyenkyqevsqkdsheqlnntsss spqhlsaqklemlyneitktqthshpnpdcqeqperpsayeeeiemeevvcpqeqlavaqtevivdmkstssi dsftscatdftetersplpppsashlqmkfptdlpgmdehqrvrappfltlsrdkgpaareaaldyapiditvnldag ashgplqpdsasdspksslkgsnplksrslkvnfqenrgsapqtppstarplpvttadfplttpqhmstilleealp qgqrplpgadvsahcqgpskglsprvpkqklfpfssrerrsfteidtgededfldlqrprpdkqadpspncladkp geardplreegcvgssspqntdhncrqdiyqavgevkkdssqegykmenhlfapeihsnpgdtgycptrets m
Kv2.2-PHC (1007) (SEQ ID NO: 82)
[00128] Maekappglnrktsrstlslppepvdiirsktcsrrvkinvgglnhevlwrtldrlprtrlgklrdcnthe sllevcddynlneneyffdrhpgaftsNnfyrtgklhmmeemcalsfgqeldywgideiylesccqaryhqkkeq mneelrreaetmreregeefdntccpekrkklwdllekpnssvaakilaivsilfivlstialslntlpelqeaaasggt gdkqkngikanfkirhniedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagith gmdelykvdggsaaandefgqpsdnrklahveavciawftmeyllrflsspnkwkffkgplnvidllailpyyvtifl tesnksvlqfqnvrrvvqifrimrNrNklarhstglqslgftlrrsynelgllNflamgimifsslvffaekdedatkftsi^ sfwwatitmttvgygdiypktllgkivgglcciagvlvialpipiivnnfsefykeqkrqekaikrrealerakrngsi mnlkdafarsmelidvavekagesanikdsvddnhlspsrwkwarkalsetssnksyenkyqevsqkdshe qlnntsssspqhlsaqklemlyneitktqthshpnpdcqeqperpsayeeeiemeevvcpqeqlavaqteviv dmkstssidsftscatdftetersplpppsashlqmkfptdlpgmdehqrvrappfltlsrdkgpaareaaldyapi ditvnldagashgplqpdsasdspksslkgsnplksrslkvnfqenrgsapqtppstarplpvttadfplttpqhm stilleealpqgqrplpgadvsahcqgpskglsprvpkqklfpfssrerrsfteidtgededfldlqrprpdkqadps pncladkpgeardplreegcvgssspqntdhncrqdiyqavgevkkdssqegykmenhlfapeihsnpgdt gycptretsm
Kir2
Kir2.1 -pHluorin (673) (SEQ ID NO: 84)
[00129] Mgsvrtnrysivsseedgmklatmavangfgngkskvhtrqqcrsrfvkkdghcnvqfinvge kgqryladifttcvdinA rwmlvifclafvlswlffgcvfwliallhgdldaskeaaaklmskgeelftgvvpilveldgd vnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyvqertiff kddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngikanfkirhniedggv qladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykaaaskacvsevnsft aaflfsietqttigygfrcvtdecpiavfmvvfqsivgciidafiigavmakmakpkkrnetlvfshnaviamrdgklcl mwrvgnlrkshlveahvraqllksritsegeyipldqidinvgfdsgidriflvspitivheidedsplydlskqdidna dfeivvilegmveatamttqcrssylaneilwghryepvlfeekhyykvdysrfhktyevpntplcsardlaekkyil snansfcyenevaltskeeddsengvpeststdtppdidlhnqasvpleprplrresei
Kir2.1 -PHN (597) (SEQ ID NO: 86)
[00130] Mgsvrtnrysivsseedgmklatmavangfgngkskvhtrqqcrsrfvkkdghcnvqfinvge kgqryladifttcvdinA rwmlvifclafvlswlffgcvfwliallhgdldaskeaaasggtgmskgeelftgvvpNvel dgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyvq ertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggsaaaskacvsev nsftaaflfsietqttigygfrcvtdecpiavfmvvfqsivgciidafiigavmakmakpkkrnetlvfshnaviamr^ gklclmwrvgnlrkshlveahvraqllksritsegeyipldqidinvgfdsgidriflvspitivheidedsplydlskqdi dnadfeivvNegmveatamttqcrssylaneilwghryepvlfeekhyykvdysrfhktyevpntplcsardlae kkyilsnansfcyenevaltskeeddsengvpeststdtppdidlhnqasvpleprplrresei
Kir2.1 -PHC (527) (SEQ ID NO: 88)
[00131] Mgsvrtnrysivsseedgmklatmavangfgngkskvhtrqqcrsrfvkkdghcnvqfinvge kgq ry I ad if ttcvd i iwwm I vif cl af vl swlf f g ^
dggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykvdggsaaas kacvsevnsftaaflfsietqttigygfrcvtdecpiavfmvvfqsivgciidafiigavmakmakpkkrnetlvfshn aviamrdgklclmwrvgnlrkshlveahvraqllksritsegeyipldqidinvgfdsgidriflvspitivheidedspl ydlskqdidnadfeivvilegmveatamttqcrssylaneilwghryepvlfeekhyykvdysrfhktyevpntplc sardlaekkyilsnansfcyenevaltskeeddsengvpeststdtppdidlhnqasvpleprplrresei
Kir2.2-pHluorin (677) (SEQ ID NO: 90)
[00132] Mtaasranpysivsseedglhlvtmsgangfgngkvhtrrrcrnrfvkkngqcniefanmdeks qryladmfttcvdirwrymllifslaflaswllfgvifwviavahgdlepaenrgaaaklmskgeelftgvvpilveldg dvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyvqerti ffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngikanfkirhniedgg vqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykaaapcvmqvhgf maaflfsietqttigyglrcvteecpvavfmvvaqsivgciidsfmigaimakmarpkkraqtllfdhnavvalrdgk lclmwrvgnlrkshiveahvraqlikprvteegeyipldqididvgfdkgldriflvspitilheideasplfgisrqdletd dfeivvilegmveatamttqarssylaneilwghrfepvlfeeknqykidyshfhktyevpstprcsakdlvenkfll psansfcyenelaflsrdeedevegdqdgcsrdglspqprhdfdrlqaggaaleqrpyrresei
Kir2.2-PHN (601 ) (SEQ ID NO: 92)
[00133] Mtaasranpysivsseedglhlvtmsgangfgngkvhtrrrcrnrfvkkngqcniefanmdeks qryladmfttcvdirwrymllifslaflaswllfgvifwviavahgdlepaenrgaaasggtgmskgeelftgvvpilv eldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyv qertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimavdggsaaapcvmqv hgfmaaflfsietqttigyglrcvteecpvavfmvvaqsivgciidsfmigaimakmarpkkraqtllfdhnavvalr dgklclmwrvgnlrkshiveahvraqlikprvteegeyipldqididvgfdkgldriflvspitNheideasplfgisrqd letddfeivvNegmveatamttqarssylaneilwghrfepvlfeeknqykidyshfhktyevpstprcsakdlve nkfllpsansfcyenelaflsrdeedevegdqdgcsrdglspqprhdfdrlqaggaaleqrpyrresei
Kir2.2-PHC (531 ) (SEQ ID NO: 94)
[00134] Mtaasranpysivsseedglhlvtmsgangfgngkvhtrrrcrnrfvkkngqcniefanmdeks qryladmfttcvdirwrymllifslaflaswllfgvifwviavahgdlepaenrgaaasggtgdkqkngikanfkirhn iedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykvdggsaaa pcvmqvhgfmaaflfsietqttigyglrcvteecpvavfmvvaqsivgciidsfmigaimakmarpkkraqtllfdh navvalrdgklclmwrvgnlrkshiveahvraqlikprvteegeyipldqididvgfdkgldriflvspitilheideasp IfgisrqdletddfeivvNegmveatamttqarssylaneilwghrfepvlfeeknqykidyshfhktyevpstprcs akdlvenkfllpsansfcyenelaflsrdeedevegdqdgcsrdglspqprhdfdrlqaggaaleqrpyrresei Kir2.3-pHluorin (685) (SEQ ID NO: 96)
[00135] Mhghsrngqahvprrkrrnrfvkkngqcnvyfanlsnksqrymadifttcvdtrwrymlmifsaa flvswlffgllfwciaffhgdleypydvpdyaasaavpapaaaklmskgeelftgvvpilveldgdvnghkfsvsg egegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyvqertiffkddgnyktra evkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngikanfkirhniedggvqladhyqqnt pigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykaaapkpcimhvngflgaflfsvetqtt igygfrcvteecplaviavvvqsivgcvidsfmigtimakmarpkkraqtllfshhavisvrdgklclmwrvgnlrks hiveahvraqlikpymtqegeylpldqrdlglgydigldriflvspiiivheidedsplygmgkeelesedfeivvileg mveatamttqarssylaseilwghrfepvvfeekshykvdysrfhktyevagtpccsarelqeskitvlpapppp psafcyenelalmsqeeeemeeeaaaaavaaglgleagskeeagiirmlefgshldlermqatlpldnisyrre sai
Kir2.3-PHN (609) (SEQ ID NO: 98)
[00136] Mhghsrngqahvprrkrrnrfvkkngqcnvyfanlsnksqrymadifttcvdtrwrymlmifsaa flvswlffgllfwciaffhgdleypydvpdyaasaavpapaaasggtgmskgeelftgvvpilveldgdvnghkfs vsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampegyvqertiffkddgny ktraevkfegdtlvnrielkgidfkedgnNghkleynyndhqvyimavdggsaaapkpcimhvngflgaflfsvet qttigygfrcvteecplaviavvvqsivgcvidsfmigtimakmarpkkraqtllfshhavisvrdgklclmwrvgnlr kshiveahvraqlikpymtqegeylpldqrdlglgydigldriflvspiiivheidedsplygmgkeelesedfeivvil egmveatamttqarssylaseilwghrfepvvfeekshykvdysrfhktyevagtpccsarelqeskitvlpapp pppsafcyenelalmsqeeeemeeeaaaaavaaglgleagskeeagiirmlefgshldlermqatlpldnisy rresai
Kir2.3-PHC (539) (SEQ ID NO: 100)
[00137] Mhghsrngqahvprrkrrnrfvkkngqcnvyfanlsnksqrymadifttcvdtrwrymlmifsaa flvswlffgllfwciaffhgdleypydvpdyaasaavpapaaasggtgdkqkngikanfkirhniedggvqladhy qqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykvdggsaaapkpcimhvngfl gaflfsvetqttigygfrcvteecplaviavvvqsivgcvidsfmigtimakmarpkkraqtllfshhavisvrdgklcl mwrvgnlrkshiveahvraqlikpymtqegeylpldqrdlglgydigldriflvspiiivheidedsplygmgkeele sedfeivvilegmveatamttqarssylaseilwghrfepvvfeekshykvdysrfhktyevagtpccsarelqes kitvlpapppppsafcyenelalmsqeeeemeeeaaaaavaaglgleagskeeagiirmlefgshldlermq atlpldnisyrresai SCN5A
SCN5A-pHluorin (2208) (SEQ ID NO: 102)
[00138] manfllprgtssfrrftreslaaiekrmaekqargsttlqesreglpeeeaprpqldlqaskklpdlyg nppqeligepledldpfystqktfivlnkgktifrfsatnalyvlspfhpirraavkilvhslfnmlimctiltncvfmaqhd pppwtkyveytftaiytfeslvkilargfclhaftflrdpwnwldfsviimayvseniklgnlsalrtfrvlralktisvipglk tivgaliqsvkkladvmvltvfclsvfaliglqlfmgnlrhkcvrnftalngtngsveadglaaaklmskgeelftgvvp ilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwptlvttltygvqcfsrypdhmkrhdffksampeg yvqertiffkddgnyktraevkfegdtlvnrielkgidfkedgnilghkleynyndhqvyimadkqkngikanfkirh niedggvqladhyqqntpigdgpvllpdnhylfttstlskdpnekrdhmvllefvtaagithgmdelykaaavwes
Idlylsdpenyllkngtsdvllcgnssdagtcpegyrclkagenpdhgytsfdsfawaflalfrlmtqdcwerlyqqtl rsagkiymiffmlviflgsfylvnlilavvamayeeqnqatiaeteekekrfqeamemlkkehealtirgvdtvsrss lemsplapvnsherrskrrkrmssgteecgedrlpksdsedgpramnhlsltrglsrtsmkprssrgsiftfrrrdlg seadfaddenstageseshhtsllvpwplrrtsaqgqpspgtsapghalhgkknstvdcngvvsllgagdpeat spgshllrpvmlehppdtttpseepggpqmltsqapcvdgfeepgarqralsavsvltsaleeleesrhkcppc wnrlaqryliweccplwmsikqgvklvvmdpftdltitmcivlnW
mtfkiialdpyyyfqqgwnifdsiivilslmelglsrmsnlsvlrsfrllrvfklakswptlntlikiignsvgalgn fifavvgmqlfgknyselrdsdsgllprwhmmdffhafliifrilcgewietmwdcmevsgqslcllvfllvmvign vlnlflalllssfsadnltapdedremnnlqlalariqrglrfvkrttwdfccgllrqrpqkpaalaaqgqlpsciatpysp pppetekvpptrketrfeegeqpgqgtpgdpepvcvpiavaesdtddqeedeenslgteeesskqtpedscs egstadmtntaelleqipdlgqdvkdpedcftegcvrrcpccavdttqapgkvwwrlrktcyhivehswfetfiifm illssgalafediyleerktikvlleyadkmftyvfvlemllkwvaygfkkyftnawcwldflivdvslvslvantlgfae mgpikslrtlralrplralsrfegmrvvvnalvgaipsimnvllvclifwlifsimgvnlfagkfgrcinqtegdl^ nnksqceslnltgelywtkvkvnfdnvgagylallqvatfkgwmdimyaavdsrgyeeqpqweynlymyiyfvi fiifgsfftlnlfigviidnfnqqkkklggqdifmteeqkkyynamkklgskkpqkpiprplnkyqgfifdivtkqafdvti mfliclnmvtmmvetddqspekinilakinllfvaiftgecivklaalrhyyftnswnifdfvvvilsivgtvlsdi sptlfrvirlarigrilrlirgakgirtllfalmmslpalfniglllflvmfiysifgmanfayvkweagiddmfn
Ifqittsagwdgllspilntgppycdptlpnsngsrgdcgspavgilffttyiiisflivvnmyiaiilenfsvateest^ eddfdmfyeiwekfdpeatqfieysvlsdfadalseplriakpnqislinmdlpmvsgdrihcmdilfaftkrvlges gemdalkiqmeekfmaanpskisyepitttlrrkheevsamviqrafrrhllqrslkhasflfrqqagsglseedap eregliayvmsenfsrplgppssssisstsfppsydsvtratsdnlqvrgsdyshsedladfppspdrdresiv
SCN5A-mCherry (2214) (SEQ ID NO: 104)
[00139] manfllprgtssfrrftreslaaiekrmaekqargsttlqesreglpeeeaprpqldlqaskklpdlyg nppqeligepledldpfystqktfivlnkgktifrfsatnalyvlspfhpirraavkNvhslfnmlimctNtncvfmaqhd pppwtkyveytftaiytfeslvkilargfclhaftflrdpwn
tivgaliqsvkkladvmvltvfclsvfaliglqlfmgnlrhkcvrnftalngtngsveadglaaasggtgmvskgeed nmaiikefmrfkvhmegsvnghefeiegegegrpyegtqtaklkvtkggplpfawdilspqfmygskayvkhp adipdylklsfpegfkwervmnfedggvvtvtqdsslqdgefiykvklrgtnfpsdgpvmqkktmgweasser mypedgalkgeikqrlklkdgghydaevkttykakkpvqlpgaynvniklditshnedytiveqyeraegrhstg gmdelykvdggsaaavwesldlylsdpenyllkngtsdvllcgnssdagtcpegyrclkagenpdhgytsfdsf awaflalfrlmtqdcwerlyqqtlrsagkiymiffmlviflgsfylvnlilavvamayeeqnqatiaeteekekrfqea memlkkehealtirgvdtvsrsslemsplapvnsherrskrrkrmssgteecgedrlpksdsedgpramnhlsl trglsrtsmkprssrgsiftfrrrdlgseadfaddenstageseshhtsllvpwplrrtsaqgqpspgtsapghalhg kknstvdcngvvsllgagdpeatspgshllrpvmlehppdtttpseepggpqmltsqapcvdgfeepgarqral savsvltsaleeleesrhkcppcwnrlaqryliweccplwmsikqgvklvvmdpftdltitmcivlntlfmalehyn mtsefeemlqvgnlvftgiftaemtfkiialdpyyyfqqgwnifdsiivilslmelglsrmsnlsvlrsfrllrvfklaksw ptlntlikiignsvgalgnltlvlaiivfifavvgmqlfgknyselrdsdsgllprwhmmdffhafliifrilcgew cmevsgqslcllvfllvmvignlvvlnlflalllssfsadnltapdedremnnlqlalariqrglrfvkrttwdfccgllrqrp qkpaalaaqgqlpsciatpysppppetekvpptrketrfeegeqpgqgtpgdpepvcvpiavaesdtddqee deenslgteeesskqtpedscsegstadmtntaelleqipdlgqdvkdpedcftegcvrrcpccavdttqapgk vwwrlrktcyhivehswfetfiifmillssgalafediyleerktikvlleyadkmftyvfvlemllkwvaygfkkyftna wcwldflivdvslvslvantlgfaemgpikslrtlralrplralsrfegmrvvvnalvgaipsimnvllvclifwlifsi nlfagkfgrcinqtegdlplnytivnnksqceslnltgelywtkvkvnfdnvgagylallqvatfkgwmdimyaavd srgyeeqpqweynlymyiyfvifiifgsfftlnlfigviidnfnqqkkklggqdifmteeqkkyynamkklgskkpqk piprplnkyqgfifdivtkqafdvtimfliclnmvtmmvetddqspekinilakinllfvaiftgecivklaalrhyyftn wnifdfvvvilsivgtvlsdiiqkyffsptlfrvirlarigrilrlirgakgirtllfalmmslpalfnigIN
vkweagiddmfnfqtfansmlclfqittsagwdgllspilntgppycdptlpnsngsrgdcgspavgilffttyiiisfli vvnmyiaiilenfsvateesteplseddfdmfyeiwekfdpeatqfieysvlsdfadalseplriakpnqislinmdl pmvsgdrihcmdilfaftkrvlgesgemdalkiqmeekfmaanpskisyepitttlrrkheevsamviqrafrrhll qrslkhasflfrqqagsglseedaperegliayvmsenfsrplgppssssisstsfppsydsvtratsdnlqvrgsd yshsedladfppspdrdresiv
Fluorescent Proteins and Labeling of Ion Channel Subunits
[00140] In various aspects of the disclosure, a label is attached to an ion channel subunit polypeptide to detect expression, changes in expression, and localization of the polypeptide in a cell. As used herein, such label includes all fluorescent proteins, including enhanced and bright versions of fluorescent proteins, as well as analogs, fragments, variants and derivatives thereof. Fluorescent proteins are members of a structurally homologous class of proteins that share the unique property of being self-sufficient to form a visible wavelength chromophore from a sequence of three amino acids within their own polypeptide sequence. In various aspects, a gene (or a gene chimera) encoding an engineered fluorescent protein is introduced into a living cell or multiple living cells, which allows for the subsequent visualization of the location and dynamics of the gene product using fluorescence microscopy.
[00141] In multiple aspects, fluorescent proteins include fluorescent proteins as described by Shaner et al. (Nature Methods 2: 905-9, 2009), incorporated herein by reference in its entirety. In various aspects, fluorescent proteins include, but are not limited to, green, yellow, cyan, orange and red fluorescent proteins. In particular aspects, fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), pHluorin, and mCherry.
[00142] In aspects of the disclosure, N-terminal and C-terminal fragments (i.e. split fluorescent protein fragments) of various fluorescent proteins are used to label ion channel subunits to utilize bimolecular fluorescence complementation (BiFC) to detect ion channel subunit interactions. In some aspects, split constructs are used in co-expression studies to measure and monitor hetero- or homo-meric channel proteins. In some aspects, full-length fluorescent proteins are used to study general protein movement, location, and surface expression in the same way but will not allow for the resolution of multisubunit channels.
[00143] More particularly, when an N-terminal fragment of a fluorescent protein label, e.g., YFP, CFP, or pHluorin, is inserted into an ion channel subunit protein, the designated abbreviation for that construct is YN, CN, or PHN, respectively. Likewise, when the C-terminal fragment of a fluorescent protein label is inserted into an ion channel subunit protein, the designated abbreviation for that construct is YC, CC, or PHC, respectively.
[00144] Thus, fluorescent protein fragments are attached to components of the same macromolecular complex. More particularly, ion channel subunits that are postulated to interact are fused to unfolded complementary fragments of a fluorescent label and expressed in live cells. Interaction of these proteins brings fluorescent fragments within proximity, allowing the fluorescent label to reform in its native three-dimensional structure (i.e. amino- and carboxy-fragments of the fluorescent label are brought together) allowing the fluorescent label to emit its fluorescent signal.
[00145] In some aspects, the label is inserted into the loop of an ion channel subunit polypeptide between transmembrane segments 1 and 2. In other aspects, the label is inserted via a linker into the loop between transmembrane segments 5 and 6.
Linker Polypeptide Sequences
[00146] In aspects of the disclosure, a label is inserted into an extracellular loop of an ion channel subunit polypeptide via a linker. In various aspects, the linker is a peptide linker. In some aspects, the peptide linker comprises from about 1 to about 100 amino acids, from about 5 to about 50 amino acids, from about 8 to about 20 amino acids. In more particular aspects, the peptide linker comprises about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, and about 100 amino acids. In even more particular aspects, the peptide linker comprises about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 1 7, about 18, about 19, and about 20 amino acids.
[00147] In further aspects, an N-terminal fragment or a C-terminal fragment of a label is inserted into an extra-cellular loop of an ion channel subunit between transmembrane-spanning segments of the ion channel subunit via a peptide linker. In some aspects, the label comprises a linker at either end of the label. In further aspects, the label comprises a linker at the N-terminus of the label. In additional aspects, the label comprises a linker at the C-terminus of the label.
[00148] In more particular aspects, the linker sequence comprises the following amino acids: AAASGGTG (SEQ ID NO: 105) and VDGGSAAA (SEQ ID NO: 106). Synthesis of Ion Channel Nucleic Acid Molecules and Polypeptide Molecules
[00149] The nucleic acid molecules encode a polypeptide comprising the amino acid sequence of an ion channel subunit polypeptide and can readily be obtained in a variety of ways including, without limitation, recombinant DNA methods and chemical synthesis.
[00150] Recombinant DNA methods are generally those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and/or Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994). Recombinant expression techniques conducted in accordance with the descriptions set forth below, in various aspects, are followed to produce these polynucleotides and to express the encoded polypeptides. For example, by inserting a nucleic acid sequence which encodes the amino acid sequence of an ion channel subunit polypeptide into an appropriate vector, one skilled in the art can readily produce large quantities of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding the amino acid sequence of an ion channel subunit polypeptide can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the encoded ion channel subunit polypeptide or ion channel subunit polypeptides are, in some aspects, produced in large amounts.
[00151] Likewise, chemical synthesis of nucleic acids and polypeptides are well known in the art, such as those described by Engels et al., Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include, inter alia, the
phosphotriester, phosphoramidite and H-phosphonate methods for nucleic acid synthesis. In one aspect, a method for such chemical synthesis is polymer- supported synthesis using standard phosphoramidite chemistry. Typically, the DNA encoding the amino acid sequence of an Ion channel subunit polypeptide will be several hundred nucleotides in length. Nucleic acids larger than about 100 nucleotides are synthesized as several fragments using these methods. The fragments are then ligated together to form the full-length nucleotide sequences of the disclosure. In particular aspects, the DNA fragment encoding the amino terminus of the polypeptide has an ATG, which encodes a methionine residue. [00152] In certain embodiments, nucleic acid variants contain codons which have been altered for the optimal expression of an ion channel subunit
polypeptide in a given host cell. Particular codon alterations depend upon the ion channel subunit polypeptide(s) and host cell(s) selected for expression. Such "codon optimization" can be carried out by a variety of methods, for example, by selecting codons which are preferred for use in highly expressed genes in a given host cell. Computer algorithms which incorporate codon frequency tables such as "Ecohigh.cod" for codon preference of highly expressed bacterial genes are used, in some instances, and are provided by the University of Wisconsin
Package Version 9.0, Genetics Computer Group, Madison, Wl. Other useful codon frequency tables include "Celegans_high.cod", "Celegansjow.cod", "Drosophila_high.cod", "Human_high.cod", "Maize_high.cod", and
"Yeast_high.cod."
[00153] A nucleic acid molecule encoding the amino acid sequence of an ion channel subunit polypeptide, in certain aspects, is inserted into an appropriate vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur). Cloning vectors include all those known in the art. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, 1989.
[00154] Expression vectors include all those known in the art, including without limitation cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide. The expression vector is inserted (e.g., via transformation or transduction) into an appropriate host cell for expression of the polynucleotide and polypeptide via transformation or
transfection using techniques known in the art. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory Press, 1989. Also, for a review of expression vectors, see Meth. Enz., vol.185, D.V. Goeddel, ed., Academic Press Inc., San Diego, CA (1990). In particular aspect, the vector is pcDNA3.1 /V5/His vector. [00155] After the vector has been constructed and a nucleic acid molecule encoding an ion channel subunit polypeptide is inserted into the proper site of the vector, the completed vector is inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of a vector encoding an ion channel subunit polypeptide into a selected host cell is, in various aspects, accomplished by well-known methods such as transfection, infection, calcium chloride-mediated transformation, electroporation, microinjection, lipofection or the DEAE-dextran method or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan and are set forth, for example, in Sambrook et al., supra.
[00156] Host cells, in some aspects, are prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as yeast, insect or vertebrate cells). The selection of an appropriate host cell depends upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation), ease of folding into a biologically active molecule, and cell type, i.e. physiological function of the cell so that appropriate ion channel expression and regulation is observed. Such host cells include, but are not limited to, host cells of bacterial, yeast, fungal, viral, invertebrate, and mammalian sources. For examples of such host cells, see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). In additional aspects, host cells used in the art since the publication of the Maniatis (supra) manual are also used in the disclosure.
[00157] In particular aspects, mammalian host cells are used. In more particular aspects, human host cells are used. Such cells include, but are not limited to, a vascular cell, a myocyte, a cardiomyocyte, a neuron, a renal cell, a fibroblast, an endothelial cell, an epithelial cell, a myoepithelial cell, a squamous cell, a basal cell, an interstitial cell, an immune cell, a glial cell, an astrocyte, a tumor cell, an ovarian cell, a sperm cell, a stem cell, a kidney cell, a liver cell, a fat cell, a lung cell, a bladder cell, an intestinal cell, a colon cell, a parafollicular cell, a blood cell, a pigment cell, a corneal cell, a hormone-secreting cell, a nurse cell, and a bone cell. In further aspects, the cells include, but are not limited to, cancer cells.
[00158] Host cells comprising an ion channel subunit polypeptide expression vector are cultured using standard media well known to the skilled artisan. The media will usually contain all nutrients necessary for the growth and survival of the cells. Suitable media for culturing eukaryotic cells include Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which, in some instances, are supplemented with serum and/or growth factors as indicated by the particular cell line being cultured.
[00159] In some aspects, an antibiotic or other compound useful for selective growth of transformed cells is added as a supplement to the media. The compound used is dictated by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. Other compounds for selective growth include ampicillin, tetracycline and neomycin.
[00160] The amount of an ion channel subunit polypeptide produced by a host cell is evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, chromatographic separation, such as High Performance Liquid Chromatography (HPLC), immunodetection such as immunoprecipitation, and/or activity assays such as DNA binding gel shift assays. In various aspects, the disclosure includes all cells that are amenable to transfection or infection by the ion channel subunit polypeptides.
Bimolecular fluorescence complementation (BiFC)
[00161] In aspects of the disclosure, bimolecular fluorescence
complementation (also known as BiFC) is used. BIFC is a technology typically used to validate protein interactions. BIFC is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. In various aspects, such macromolecular complex comprises ion channel subunits. Proteins that are postulated to interact, i.e. various ion channel subunits, are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. In various aspects, the polypeptides are attached to split fragments of a fluorescent reporter protein by a linker. Examples of appropriate linkers are discussed in further detail herein. Interaction of the proteins of interest, i.e. ion channel subunits, brings fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal is detected and located within a cell using an inverted fluorescence microscope, a confocal microscope, or a total internal reflectance microscope that allows imaging of fluorescence in cells. With complementation, the signal is all or nothing. Therefore, through the visualization and analysis of the intensity and distribution of fluorescence in these cells, one of skill in the art
detects/identifies both the location and interaction partners of proteins of interest.
[00162] The expression of the cDNA in cells results in expression of the ion channel probes. Fluorescence measurement or imaging is used to detect the signal, measure the signal intensity, or determine signal localization within a cell. Live cell fluorescence imaging is used in various aspects to visualize trafficking effects. When using pHluorin constructs, application of solutions with acidic or basic pH is used to measure surface expression.
Antibodies
[00163] Further aspects of the disclosure include antibodies to proteins described herein. In some aspects, immunocytochemistry can be used to validate BIFC or used in labeling and localizing surface proteins. For example, in particular aspects, GFP antibodies which recognize pHluorin are useful in measuring channel protein endocytosis and/or recycling (Schumacher et al., Circ. Res. 104:1390-8, 2009).
[00164] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure.
[00165] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
[00166] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.
EXAMPLE 1 :
GENERAL METHODS
Constructs
[00167] Amino terminal residues 1 -155 of YFP (YN), CFP (CN), and pHluorin (PHN) and carboxy terminal residues 156-238 of YFP (YC), CFP (CC), and pHluorin (PHC) are inserted between transmembrane-spanning segments of an ion channel subunit. More particularly, DNA sequences encoding amino acid residues 1 -238, 1 -155 and 156-238 of YFP, CFP and pHluorin (see Table 1 ) were inserted into extra-cellular loop between transmembrane-spanning segments 1 and 2 in human Kv1 .1 , rat 1 .2, human 1 .4, rat 2.1 and rat 2.2 between the amino acid positions of 201 /202, 200/201 , 348/349, 212/213, and 220/221 respectively with the linker sequences encoding AAASGGTG (SEQ ID NO: 105) and
VDGGSAAA (SEQ ID NO: 106).
[00168] Chimeric coding regions then were cloned into pcDNA3.1 /V5/His vector for mammalian expression. The Quick Change Site-directed Mutagenesis kit (Clontech) was used for the mutation of Kv1 .4 replacing the threonine at amino acid position 328 with alanine (Kv1 .4 T328A) according to manufacturer's protocol. Table 1 - DNA and Amino Acid Sequences of YFP, CFP and pHluorin
Figure imgf000055_0001
Antibodies
[00169] Mouse anti-V5 (1 :500), rabbit anti-GFP (1 :500), and AlexaFluor (405, 594, or 647)-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies (1 :250-1 :500)(lnvitrogen); mouse pan anti-neurofilament clone SMI- 312 (Covance); and mouse anti-bassoon clone SAP7F407 (1 :500) (Stressgen) antibodies were used in experiments described herein.
Cell Culture and Transfection
[00170] COS-7 cells were maintained in DMEM (Gibco #1 1960) with 10% fetal bovine serum (FBS) (Gibco) and 1 x Penicillin-Streptomycin (Gibco) at 37°C in a humidified atmosphere of 95% air and 5% CO2. COS-7 cells grown to 60-80% confluence on glass coverslips were transiently transfected with 2 μg of DNA total combined with 3 μΙ of Lipofectamine 2000 (Invitrogen) in serum-free Opti-Mem I (Gibco) for 3-5 hours and then changed to normal media followed by incubation for 1 -2 days before performing experiments.
[00171] Dissociated postnatal (P1 - P2) rat hippocampal neurons were plated at 50,000 cells/dish in poly-D-lysine-coated glass-bottom 35mm petri dishes (Mattek), after dissociation as previously described (Aakalu et al., Neuron 30: 489-502, 2001 ). Cells were maintained for 3-4 DIV at 37°C in growth medium (Neurobasal A supplemented with B27 and Glutamax-1 (Invitrogen)) prior to transfection. Transfection was performed using a CalPhos Mammalian
Transfection kit (Clontech) using 3 μg total DNA according to manufacturer's protocol. After 1 hour of transfection the medium was replaced with acidified Neurobasal medium pre-equilibrated at 10% CO2 and neurons were placed in a 10% C02 incubator at 37°C for 20 minutes (Jiang et al., Nat. Protoc. 1 : 695-700, 2006). Cells were then washed three times in Neurobasal medium, fed with growth medium and placed back in the 5% C02 incubator at 37°C for 3 days until experiments were carried out.
Immunocytochemistry
[00172] For live cell staining and cell surface labeling of COS-7 cells, coverslips were washed briefly with ice-cold PBS and then incubated with polyclonal anti-GFP antibody in 2% goat serum for 30 minutes on ice. After three washes with PBS, cells were incubated with AlexaFluor 594 goat anti-rabbit for 30 min on ice. Cells were then washed with PBS twice, and fixed with 4% paraformaldehyde for 7 minutes. In some experiments, cells were incubated with anti-V5 antibody and AlexaFluor 647 goat anti-mouse following permeabilization with 0.1 % Triton X-100 to verify channel expression. Coverslips were mounted with Prolong Gold anti-fade reagent (Invitrogen).
Confocal Microscopy and Fluorescence Recovery After Photobleaching (FRAP)
[00173] Confocal imaging was carried out as described previously (Jenkins et al., J. Neurosci. 29:10541 -51 , 2009). Images of transfected cells displaying fluorescent signals were acquired on an Olympus Fluoview 500 confocal microscope with a 60 x 1 .40 numerical aperture (N.A.) oil objective. Exposures were adjusted so that maximal pixel intensities were at least half saturation.
Images were obtained by taking a series of stacks every 0.5 μιτι through the cells and combining the images into a composite stack. For imaging, the following filters were used: a 405 nm laser diode with a 430-460 nm bandpass filter, a 488 nm laser with a 505-525 nm bandpass filter, a 543 nm laser with a 560 nm long- pass filter, and a 633 nm laser with a 660 nm long-pass filter. Images were analyzed with ImageJ software (NIH), and statistics were carried out with Prism 5 software from Graphpad Prism Software. Adjustments of contrast and brightness were performed using Adobe Photoshop 9.0.
[00174] Fluorescence recovery after photobleaching (FRAP) was performed essentially as described previously (Jenkins et al., Curr. Biol. 16:121 1 -16, 2006). Briefly, five single confocal plane prebleach images were acquired at a resolution of 512 x 512 pixels at 5% laser intensity before bleaching a region of approximately 5 - 10 square microns at 100% laser intensity for 3 - 5 s.
Recovery was measured by obtaining 512 χ 512 images at 5% laser intensity every 5 s for four minutes. Recovery of the bleached region was background subtracted and normalized to both the initial region intensity relative to whole-cell intensity and for photobleaching of sample during recovery. Total photobleaching of sample was less than 10% over entire period of recovery. Recovery kinetics were determined with a single exponential fit of the average data: y = A(1 e ("ΐχτ)) + c, in which A is amplitude, t is time, τ is the time constant, and c is the constant representing the relative fluorescence immediately postbleach.
Total Internal Reflection Fluorescence Microscopy (TIRF)
[00175] Prism-less (through the objective) TIRF microscopy was carried out as described previously (Anantharam et al., J. Cell Biol. 188, 415-28, 2010). Briefly, an Argon ion (488 nm) laser (CVI Melles Griot) was directed through a custom side port to a side-facing dichroic mirror Q495LPw/AR and a HQ500 LP emission filter (Chroma Technology) on an Inverted microscope (IX70, Olympus) with the 1 .5x internal magnifying lens in the emission path. The beam was focused on the periphery of the back focal plane of a 60x 1 .49 NA oil immersion objective (Olympus) so that the laser beam was incident on the coverslip at -70° from the normal, giving a decay constant for the evanescent field of ~1 10 nm. Digital images were captured on a cooled EM CCD camera (Andor iXon; Andor
Technology).
Electrophysiology
[00176] For electrophysiological studies, whole-cell patch clamp recordings were performed on COS-7 cells transiently-expressing Kv1 .4, Kv1 .4 T328A, Kv1 .1 or Kv1 .4 T328A and Kv1 .1 . The intracellular pipette solution contained (in mM): Potassium aspartate 1 10, KCI 20, NaCI 8, HEPES 10, K2ATP 4, CaCI2 1 , and MgCI2 1 , K2BAPTA 10; and was adjusted to pH 7.2 with KOH. The bath solution contained (in mM): NaCI 1 10, KCI 4, MgCI2 1 , CaCI2 1 .8, HEPES 10, and glucose 1 .8; and was adjusted to pH 7.35 with NaOH. Cells were held at -80 mV resting potential and voltage steps were applied at 20 mV intervals for 1 .5 s to elicit outward potassium currents. Statistical Analysis
[00177] Statistics for comparing τ values in FRAP experiment were carried out using a two-tailed t test with the confidence interval set to 95% using Graphpad Prism software. Data are shown as mean ± SEM.
EXAMPLE 2:
BIFC ALLOWS SELECTIVE VISUALIZATION OF KV CHANNEL TETRAMERS
[00178] In order to study specific channel homo- and heteromers, an ion channel subunit construct was created with the neuronal Kv channel, Kv1 .4, with either the amino-terminal 1 -155 amino acid fragment of yellow fluorescent protein (YFP) (Kv1 .4-YN) or the carboxyl-terminal 156-238 amino acid fragment of YFP (Kv1 .4-YC) inserted into the S1 -S2 loop of the Kv1 .4 ion channel subunit polypeptide. Channels were expressed in COS-7 cells. When either channel construct alone was expressed, no fluorescence could be detected. However, cell surface protein could be detected by live-cell labeling with anti-GFP antibodies. In addition, immunostaining directed against an intracellular carboxyl terminal V5 epitope revealed robust channel expression. Interestingly, the anti- GFP antibodies used in this study were able to detect either fragment of YFP. Importantly, expression of both Kv1 .4-YN and Kv1 .4-YC in the same cell resulted in robust fluorescence, since the fragments of YFP are close enough to allow assembly of the complete fluorophore. However, when Kv1 .4 was expressed with the mutation of a conserved threonine that has been shown to prevent tetramerization of the channel (T189D in Kv1 .4 which corresponds to Kv1 .3 T65D1 1 ), no fluorescent complementation could be detected despite efficient channel expression as demonstrated by anti-V5 immunoreactivity. These data demonstrate that BiFC is useful for monitoring channel assembly without forcing aberrant tetramerization. EXAMPLE 3:
SE OF PH-SENSITIVE PHLUORIN IN BIFC ALLOWS DETECTION OF CELL
SURFACE OLIGOMERIC CHANNELS IN LIVE CELLS
[00179] When Kv1 .4 homomers with a complemented YFP on the extracellular epitope were expressed, only a very small fraction of the complemented fluorescent signal colocalized with the cell surface anti-GFP labeling. These results indicate that channel structure is indeed allowing BiFC, but that
complemented channels are unable to traffic properly to the cell surface. To test this hypothesis, channel constructs where the pH-sensitive variant of GFP, pHluorin, was used in place of YFP were created. Transfection of COS-7 cells with Kv1 .4 tagged on the S1 -S2 loop with either the amino-terminal (Kv1 .4-PHN) or carboxyl-terminal (Kv1 .4-PHC) fragments of pHluorin resulted in efficient complementation and robust pHluorin fluorescence. However, as discussed herein above, cell surface labeling with anti-GFP antibodies allows recognition of either half of the fluorescent protein, thus precluding the affirmative detection of cell surface, complemented fluorophores.
[00180] Because fluorescence from pHluorin is rapidly and efficiently quenched by the addition of acidic solution, placement of pHluorin in the extracellular space by insertion in an extracellular loop of an ion channel protein, followed by perfusion with an acidic bath solution, specifically quenches the fluorescence from cell surface protein. In addition, because fluorescence demonstrated in the experiments is the result of BiFC from two known channel subunits, a novel tool is now available to monitor specific cell surface populations of Kv channels.
[00181] In order to test the cell surface localization of specific channel complexes, total internal reflection fluorescence microscopy (TIRFM) was carried out as described previously (Anantharam et al., J. Cell Biol. 188: 415-28, 2010). TIRFM imaging of COS-7 cells transfected with Kv1 .4-PHN and Kv1 .4-PHC allowed the detection of cell surface fluorescence, which was efficiently quenched by the addition of pH 5.5 bath solution, and recovered with the return of the bath to pH of 7.4. Importantly, the complemented pHluorin molecule displayed pH- sensitivity nearly identical to the full-length pHluorin molecule. As a control, pHluorin was inserted into Kv1 .4 subunit that fails to traffic to the cell surface (Kv1 .4 T328A) (McKeown et al., J. Biol. Chem. 283: 30421 -32, 2008). No change in pHluorin fluorescence was detected upon the addition of acidic bath solution to COS-7 cells expressing Kv1 .4 T328A, indicating that the loss of fluorescence signal is due to the quenching of cell surface protein only. These results demonstrate that by using BiFC of extracellular pHluorin, cell surface channels of known subunit composition can be monitored.
EXAMPLE 4:
BIMOLECULAR FLUORESCENT COMPLEMENTATION CAN BE USED TO MONITOR HETEROMERIC CHANNEL POPULATIONS
[00182] Because the majority of Kv channels in the nervous system are thought to exist as heterotetramers, BiFC was used with the highly-expressed Kv1 family subunits Kv1 .1 , Kv1 .2 and Kv1 .4 in COS-7 cells. Expression of either Kv1 .2-PHN + Kv1 .4-PHC or Kv1 .1 -PHN + Kv1 .4-PHC resulted in robust pHluorin fluorescence and cell surface anti-GFP labeling. Because Kv channels do not assemble with members of other Kv subfamilies because of family-specific interactions of the T1 domains, Kv1 .4-PHN and Kv2.1 -PHC were co-expressed as controls. As expected, no coassembly was detected indicating that BiFC cannot force interfamily tetramerization. However, when the T1 domain within Kv2.1 was replaced with the T1 domain from Kv1 .4 (Kv2.1 (1 .4T1 )-PHC) and expressed, efficient coassembly of the two subunits of the new construct with Kv1 .4-PHN were detected by pHluorin fluorescence. These results demonstrate that BiFC can be used to monitor assembly and localization of heteromeric Kv channel populations, and that this assembly is dependent on the T1
tetramerization domain.
EXAMPLE 5:
BIFC CAN MEASURE THE TRAFFICKING OF MUTANT CHANNEL SUBUNITS TO DISCRIMINATE BETWEEN ASSEMBLY AND LOCALIZATION DEFECTS
[00183] Because the anti-GFP antibodies used in these experiments recognize either half of the split BiFC fluorophore, a complementary method was used to show that heteromeric channel was trafficking to the plasma membrane. For these experiments, two different Kv1 subunits which have been shown to be restricted to the cytoplasm , thus preventing surface expression, were used.
First, Kv1 .1 homomeric channels have been shown to be restricted to the endoplasmic reticulum (ER) through an ER-retention motif found in the channel pore (Manganas, et al., Proc. Natl. Acad. Sci. USA 98: 14055-9, 2001 . In addition, mutation of a conserved threonine residue within the S1 -S2 linker of Kv1 .4 (Kv1 .4 T328A) has also been shown to be strictly required for surface expression (McKeown et al., supra). Interestingly, surface localization of either of these two Kv channel subunits can be rescued by coexpression with another wild-type Kv a subunit (Manganas et al., supra; McKeown et al., supra).
[00184] When homomeric Kv1 .4 T328A or Kv1 .1 with split pHluorin inserted into the S1 -S2 loop (Kv1 .4 T328A-PHN + Kv1 .4 T328A-PHC or Kv1 .1 -PHN + Kv1 .1 -PHC, respectively) was expressed, no surface localization was detected, as predicted. However, both homomeric channels were able to assemble within the cell as shown by pHluorin fluorescence. When Kv1 .1 -PHN was coexpressed with Kv1 .4 T328A, cell surface localization was detected by anti-GFP live cell labeling. These results were confirmed using whole-cell patch clamp recordings of COS-7 cells transiently-expressing different Kv channel combinations. Cells expressing wild-type Kv1 .4 exhibited a rapidly-inactivating A-type outward potassium current typical of Kv1 .4 (McKeown et al., supra). Expression of either Kv1 .1 or Kv1 .4 T328A alone resulted in negligible outward current, as expected from anti-GFP immunolabeling as previously described (Manganas et al., supra; McKeown et al., supra). Interestingly, coexpression of Kv1 .1 -PHN with Kv1 .4 T328A-PHC rescued surface expression of the two channel subunits, resulting in a current trace showing a combination of non-inactivating Kv1 .1 and rapidly- inactivating Kv1 .4 current.
EXAMPLE 6:
MONITORING THE ASSOCIATION OF DIFFERENT HETEROMERIC CHANNELS
INTO DISTINCT PROTEIN COMPLEXES
[00185] Members of the Kv1 channel family have been shown (Kim et al., Nature 378: 85-8, 1995) to interact with a membrane-associated guanylate kinase protein, PSD-95, to direct channel to cell surface clusters through interaction with channel carboxyl terminus. This compartmentalization has been shown to be dependent on cell surface expression of the channel, since channels unable to traffic to the plasma membrane are not clustered by PSD-95.
[00186] As expected, experiments showed that wild-type complemented homomeric Kv1 .4 channels are efficiently clustered by PSD-95. In contrast, no clustering by PSD-95 is detected in cells expressing either complemented Kv1 .1 or Kv1 .4 T328A. However, coexpression of Kv1 .1 -PHN and Kv1 .4 T328A-PHC, which rescues surface localization, is able to restore channel surface clustering through PSD-95. These results demonstrate that BiFC can be used to monitor subcellular localization and compartmentalization of homo- and heteromeric channel populations within a cell.
EXAMPLE 7:
BIMOLECULAR FLUORESCENT COMPLEMENTATION CAN BE USED TO MONITOR HETEROMERIC CHANNEL POLARIZED TRAFFICKING IN
HIPPOCAMPAL NEURONS
[00187] With tools to monitor subcellular localization of cell surface channels, trafficking of heteromeric Kv channels in hippocampal neurons, where Kv1 .1 , Kv1 .2, and Kv1 .4 represent the predominant Kv1 channel subunits, was then examined. Hippocampal neurons were transfected with (1 ) Kv1 .4-PHN + Kv1 .4- PHC, (2) Kv1 .2-PHN + Kv1 .4-PHC, (3) Kv1 .1 -PHN + Kv1 .4-PHC, (4) Kv1 .2-PHN + Kv1 .1 -PHC, and (5) Kv1 .2-PHN + Kv1 .2-PHC. Neurons were fixed,
permeabilized and immunostained using antibodies against GFP to mark transfected cells (red) and neurofilament to mark the axonal compartment (cyan).
[00188] Transfection of dissociated hippocampal neuronal cultures with Kv1 .4- PHN + Kv1 .4-PHC resulted in pHluorin fluorescence in the soma and along the axon, as indicated by colocalization with an axonal marker, neurofilament.
Examination of heteromeric channels revealed that Kv1 .4-Kv1 .2 heteromers, like Kv1 .4-Kv1 .4 homomers, were also capable of axonal localization. However, the heteromeric channel localized to the axonal compartment exhibited a distinct punctate pattern typical of presynaptic sites, whereas the homomeric channel was diffusely localized throughout the axon. Interestingly, heteromeric channels containing Kv1 .4 and Kv1 .1 failed to traffic to the axon, indicated by a lack of pHluorin colocalization with neurofilament immunostaining. Uncomplemented channel subunits presumably tetramerized with other endogenous Kv channel subunits. These uncomplemented channel subunits were able to traffic to the axon, as indicated by anti-GFP immunostaining, demonstrating that the lack of axonal trafficking of the complemented channel is not caused by unhealthy cells or by other abnormal trafficking.
[00189] It was surprising that the Kv1 .4/Kv1 .1 heteromer failed to traffic to the axon, since it had been reported that Kv1 .1 is localized to the axon in several types of neurons (Rasband et al., J. Neurocytol. 28, 319-31 ; 1999; Raab-Graham et al., Science 314: 144-48, 2006). Indeed, when Kv1 .1 was coexpressed with another predominant neuronal Kv1 channel subunit, Kv1 .2, the heteromeric complex was able to localize to the axonal compartment and was clustered in similar puncta as seen in the Kv1 .4/Kv1 .2 heteromer. This axonal localization and clustering phenotype was also seen with coexpression of Kv1 .2-PHN and Kv1 .2-PHC. These results demonstrate that Kv channel heteromeric complexes display surprisingly diverse trafficking and axonal localization.
EXAMPLE 8:
CELL SURFACE KV CHANNEL COMPLEXES COMPOSED OF DIFFERENT ALPHA SUBUNITS EXHIBIT UNIQUE LOCALIZATION AND DYNAMICS
[00190] To test whether complemented Kv channels are localized to the plasma membrane in hippocampal neurons, neuronal cultures were
cotransfected with Kv1 .4-PHN + Kv1 .4-PHC or Kv1 .2-PHN + Kv1 .4-PHC and surface localization was examined using an acid quenching assay. When neurons were perfused with a pH 5.5 solution, there was a significant reduction in fluorescence from both the homomer Kv1 .4-1 .4 and the heteromer Kv1 .2-1 .4, which returned to baseline upon restoration of the extracellular solution to pH 7.4. These results demonstrate that homomeric and heteromeric Kv channels are on the plasma membrane in hippocampal neurons. However, as mentioned herein above, the Kv1 .4-1 .2 heteromeric channel appeared in punctate clusters along the length of the axon.
[00191] To determine if the heteromeric Kv1 .4-Kv1 .2 channel was localized to presynaptic sites, transiently-transfected hippocampal neuron cultures were immunostained with antibodies against the presynaptic marker bassoon. Kv1 .4- 1 .2- complemented pHluorin fluorescence colocalized with anti-bassoon immunostaining, suggesting that channels of this subunit configuration are localized to sites of synaptic vesicle release. Interestingly, when mobility of the Kv1 .4-1 .2 heteromer was compared with mobility of the homomeric channel using fluorescence recovery after photobleaching (FRAP), it was found that the heteromer Kv1 .4-1 .2 had significantly slower recovery than Kv1 .4-1 .4 homomers. These results indicate that there is a possible stabilization in the plasma membrane. Results also demonstrate that cell surface pHluorin and BiFC can be utilized to distinguish heteromeric channel populations and homomeric channel populations and discover unique properties of different channel subunit configurations.
[00192] In addition to the Kv1 family of channels, the Kv2 family is highly represented in the central nervous system. It previously has been shown that the delayed-rectifier channels Kv2.1 and Kv2.2 are expressed at high levels in the mammalian hippocampus. Expression of Kv2.1 in mammalian cells results in formation of large cell surface clusters, whereas Kv2.2 appears more evenly- distributed throughout the neuron. The detection of Kv2.1 -2.2 heteromeric channels has been limited by the fact that the currents produced by the two channels are nearly identical. Despite work that has demonstrated the presence of heteromeric channel populations (Blaine et al., J. Neurosci. 18: 9585-93, 1998), the presence of true heteromeric channels remained controversial (Malin et al., J. Neurosci. 22: 10094-105, 2002; Chung et al., FEBS J. 272: 3743-55, 2005; Guan et al., J. Physiol. 581 : 941 -960, 2007).
[00193] Expression of Kv2.1 or Kv2.2 homomeric channels in hippocampal neurons resulted in a typical large somatic cluster or homogeneous expression pattern, respectively. Coexpression of Kv2.1 -PHN with Kv2.2-PHC resulted in complemented pHluorin fluorescence, indicating the formation of heteromeric channels. Interestingly, localization of the heteromeric population more closely resembled the homogeneous pattern seen with Kv2.2, indicating that Kv2.2 may act dominantly to control subcellular localization of the heteromeric channel. These results highlight the ability of cell surface pHluorin and BiFC to elucidate the subcellular localization and properties of distinct heteromeric channel populations. [00194] The disclosure has been described in terms of particular embodiments found or proposed to comprise specific modes for the practice of the disclosure. Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the subject matter of the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure would be apparent to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

What is Claimed is:
1 . A nucleic acid molecule encoding an ion channel subunit polypeptide wherein the ion channel subunit polypeptide comprises a label inserted into an extracellular loop between transmembrane-spanning segments of the ion channel polypeptide.
2. The nucleic acid molecule of claim 1 wherein the label is inserted between segments 1 and 2 of a potassium channel subunit polypeptide.
3. The nucleic acid molecule of claim 1 wherein the label is inserted between segments 5 and 6 of the extracellular loop of domain 1 of a sodium channel subunit polypeptide.
4. The nucleic acid molecule of claim 1 or 2 wherein the encoded ion channel subunit polypeptide is a voltage-gated channel polypeptide, an inwardly rectifying channel polypeptide, a tandem pore domain channel polypeptide, a calcium- activated channel polypeptide, or an intracellular channel polypeptide.
5. The nucleic acid molecule of claim 4 wherein the inwardly rectifying channel polypeptide is selected from the group consisting of: Kir1 .1 , Kir1 .2, Kir2.1 , Kir2.2, Kir2.2v, Kir2.3, Kir2.4, Kir3.1 , Kir3.2, Kir3.3, Kir3.4, Kir4.1 , Kir5.1 , Kir6.1 , and Kir7.1 .
6. The nucleic acid molecule of claim 1 , 2, or 4 wherein the voltage-gated channel polypeptide is a potassium channel polypeptide, a sodium channel polypeptide, or a chloride channel polypeptide.
7. The nucleic acid molecule of claim 6 wherein the voltage-gated channel polypeptide is a voltage-gated potassium channel polypeptide.
8. The nucleic acid molecule of claim 6 wherein the voltage-gated potassium channel polypeptide is selected from the group consisting of: Kv1 .1 , Kv1 .2, Kv1 .3, Kv1 .4, Kv1 .5, Kv1 .6, Kv1 .7, Kv1 .8, Kv2.1 , Kv2.2, Kv3.1 , Kv3.2, Kv3.3, Kv3.4, Kv4.1 , Kv4.2, Kv4.3, Kv5.1 , Kv6.1 , Kv6.2, Kv6.3, Kv6.4, Kv7.1 , Kv8.1 , Kv8.2, Kv9.1 , Kv9.2, Kv9.3, Kv10.1 , Kv10.2, Kv1 1 .1 , Kv1 1 .2, Kv1 1 .3, Kv12.1 , Kv12.2, and Kv12.3.
9. The nucleic acid molecule of claim 4 wherein the voltage-gated channel polypeptide is a voltage-gated sodium channel polypeptide.
10. The nucleic acid molecule of claim 9 wherein the voltage-gated sodium channel polypeptide is selected from the group consisting of: Nav1 .1 , Nav1 .2, Nav1 .3, Nav1 .4, Nav1 .5, Nav1 .6, Nav1 .7, Nav1 .8, Nav1 .9, and Nax.
1 1 . The nucleic acid molecule of claim 6 wherein the voltage-gated channel polypeptide is a voltage-gated chloride channel polypeptide.
12. The nucleic acid molecule of claim 1 1 wherein the voltage-gated chloride channel polypeptide is selected from the group consisting of: CLCN1 , CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCNKB.
13. The nucleic acid molecule of claim 6 wherein the calcium-activated channel polypeptide is a calcium-activated chloride channel polypeptide.
14. The nucleic acid molecule of claim 13 wherein the calcium-activated chloride channel polypeptide is selected from the group consisting of: CLCA1 , CLCA2, CLCA3, and CLCA4.
15. The nucleic acid molecule of claim 6 wherein the intracellular channel polypeptide is an intracellular chloride channel polypeptide.
16. The nucleic acid molecule of claim 15 wherein the intracellular channel polypeptide is selected from the group consisting of: CLIC1 , CLIC2, CLIC3, CLIC4, CLIC5, CLIC6, and CLNS1 A.
17. The nucleic acid molecule of any one of claims 1 -16 wherein the label is a green, yellow, cyan, orange or red fluorescent protein or fragment thereof.
18. The nucleic acid molecule of claim 17 wherein the fluorescent protein comprises amino acid residues 1 -238 of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, or a pHluorin.
19. The nucleic acid molecule of claim 17 wherein the fluorescent protein fragment is the amino-terminal fragment comprising amino acid residues 1 -155 of the fluorescent protein.
20. The nucleic acid molecule of claim 17 wherein the fluorescent protein fragment is the carboxy-terminal fragment comprising amino acid residues 156-238 of the fluorescent protein.
21 . The nucleic acid molecule of claim 17 wherein the fluorescent label is selected from the group consisting of: yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (mCherry), green fluorescent protein (GFP), and pHluorin.
22. The nucleic acid molecule of any one of claims 1 -21 wherein the label is inserted into the extracellular loop via a linker.
23. The nucleic acid molecule of any one of claims 1 -22 wherein the label comprises a linker at either end of the label.
24. The nucleic acid molecule of claim 22 or 23 wherein the linker comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105 (AAASGGGTG) and SEQ ID NO: 106 (VDGGSAAA).
25. The nucleic acid molecule of claim 23 or 24 wherein the nucleotide sequence encoding the label comprising the linker is inserted in the nucleic acid molecule at a position in the nucleotide sequence selected from the group consisting of: between the nucleotide sequence encoding amino acids at positions 201 and 202 in human Kv1 .1 , between the nucleotide sequence encoding amino acids at positions 200 and 201 of rat Kv1 .2, between the nucleotide sequence encoding amino acids at positions 348 and 349 in human Kv1 .4, between positions 212 and 213 of rat Kv2.1 , between the nucleotide sequence encoding amino acids at positions 220 and 221 of rat Kv2.2.
26. The nucleic acid molecule of claim 1 comprising a nucleotide sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to a nucleic acid sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
27. The nucleic acid molecule of claim 1 comprising a nucleotide sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ I D NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
28. The nucleic acid molecule of claim 1 consisting of a nucleotide sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to a nucleic acid sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
29. The nucleic acid molecule of claim 1 consisting of a nucleotide sequence selected from the group consisting of : SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ I D NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101 , and SEQ ID NO: 103.
30. The nucleic acid molecule of claim 1 encoding a polypeptide comprising an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ I D NO: 102, and SEQ ID NO: 104.
31 . The nucleic acid molecule of claim 1 encoding a polypeptide comprising an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
32. The nucleic acid molecule of claim 1 encoding a polypeptide consisting of an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ I D NO: 102, and SEQ ID NO: 104.
33. The nucleic acid molecule of claim 1 encoding a polypeptide consisting of the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:
34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
34. A vector comprising the nucleic acid molecule of any one of claims 1 -33.
35. A cell comprising the vector of claim 34.
36. A cell comprising a nucleic acid molecule of any one of claims 1 -33.
37. A cell comprising a first nucleic acid molecule which is the nucleic acid molecule of claim 19 and a second nucleic acid molecule which is the nucleic acid molecule of claim 20, and the first nucleic acid molecule and the second nucleic acid molecule encode the same ion channel polypeptide.
38. A cell comprising a first nucleic acid molecule which is the nucleic acid molecule of claim 19 and a second nucleic acid molecule which is the nucleic acid molecule of claim 20, and the first nucleic acid molecule and the second nucleic acid molecule do not encode the same ion channel polypeptide.
39. The cell of claim 37 or 38 further comprising one or more additional nucleic acid molecules.
40. The cell of claim 37, 38, or 39 further comprising an additional heterologous nucleic acid molecule comprising a label.
41 . The cell of any one of claims 35-40 wherein the cell is a human cell.
42. A method of expressing an ion channel subunit polypeptide in a cell comprising incubating the cell of any one of claims 35-41 under conditions that allow the cell to express the ion channel polypeptide.
43. A polypeptide comprising an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
44. A polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
45. A polypeptide consisting of an amino acid sequence having at least or about 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
46. A polypeptide consisting of an amino acid sequence selected from the group consisting of : SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, and SEQ ID NO: 104.
47. A method for identifying a modulator of ion channel polypeptide expression comprising the step of:
measuring expression of the ion channel polypeptide in the cell of any one of claims 35-41 in the presence and absence of a test compound, wherein a change in ion channel polypeptide expression in the presence of the test compound indicates the test compound is a modulator of ion channel expression.
48. A method for measuring ion channel biogenesis, cellular trafficking, internalization, plasma membrane recycling, membrane insertion, or degradation, the method comprising the steps of:
(a) contacting the cell of any one of claims 35-41 with a test compound or a control compound; and
(b) detecting a change in expression of the label between the cell contacted with a test compound and the cell contacted with a control compound.
49. The method of claim 47 or 48 wherein the expression is measured at the surface of the cell, extracellularly, or intracellular^.
50. The method of any one of claims 47-49 wherein an increase in expression of the label indicates trafficking of the channel polypeptide to the cell's plasma membrane.
51 . The method of any one of claims 47-49 wherein a decrease in expression of the label indicates internalization of the channel polypeptide in a subcellular compartment within the cell.
52. The method of any one of claims 47-49 wherein the step of detecting a change in expression of the label is measured over a period of time.
53. The method of claim 52 wherein the period of time comprises a lifetime of the protein.
54. The method of claim 52 wherein the period of time comprises a cell's lifetime.
55. The method of claim 52 wherein the period of time includes a period of minutes, a period of hours, or a period of days.
56. The method of any one of claims 47-49 wherein the cell is contacted with the compound in the presence of temperatures which range between about 4QC to about 40QC.
57. The method of any one of claims 47-49 wherein the cell is contacted with the compound at a pH between about pH1 to about pH14.
58. The method of any one of claims 47-49 wherein the cell is contacted with the compound in the presence of calcium in a concentration from about 0.001 M to about 1 .0 M.
59. A method of using bimolecular fluorescence complementation to detect association of two or more ion channel subunit polypeptides in a cell, wherein the cell comprises a first ion channel subunit polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between a transmembrane-spanning segment of the ion channel subunit polypeptide and a second ion channel subunit polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between a transmembrane-spanning segment of the ion channel subunit
polypeptide, the method comprising the step of:
measuring colocalization of the amino-terminal and the carboxy-terminal fragments by measuring fluorescence, wherein an increase in fluorescence indicates
association of the ion channel subunit polypeptides in the cell.
60. A method of using bimolecular fluorescence complementation to monitor ion channel biogenesis, cellular trafficking, internalization, plasma membrane recycling, membrane insertion, or degradation of two or more ion channel polypeptides in a cell, wherein the cell comprises a first ion channel polypeptide comprising an amino- terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between transmembrane-spanning segments 1 and 2 of the ion channel polypeptides and a second ion channel polypeptide comprising an amino-terminal fragment of a split fluorescent polypeptide label inserted into an extracellular loop between transmembrane-spanning segments 1 and 2 of the ion channel
polypeptides, the method comprising the steps of:
(a) measuring colocalization of the amino-terminal and carboxy-terminal fragments by measuring fluorescence, wherein an increase in fluorescence indicates association of the ion channel polypeptides in the cell; and
(b) detecting a change in fluorescence localization in the cell over time.
61 . The method of claim 59 or 60 wherein the first ion channel polypeptide and the second ion channel polypeptide are homomeric.
62. The method of claim 59 or 60 wherein the first ion channel polypeptide and the second ion channel polypeptide are heteromeric.
63. The method of claim 59 or 60 wherein the label is inserted between segments 1 and 2 of the extracellular loop of a potassium channel polypeptide.
64. The method of claim 59 or 60 wherein the label is inserted between segments 5 and 6 of the extracellular loop of domain 1 of a sodium channel polypeptide.
65. The method of claim 63 or 64 wherein the label comprises a linker at either end of the label.
66. The method of any one of claims 63-65 wherein the label is inserted into the extracellular loop of the polypeptide via a polypeptide linker sequence.
67. The method of any one of claims 63-66 wherein the linker comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 105 (AAASGGGTG) and SEQ ID NO: 106 (VDGGSAAA).
PCT/US2012/048135 2011-07-27 2012-07-25 Ion channel probes and methods of use thereof WO2013016418A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161512257P 2011-07-27 2011-07-27
US61/512,257 2011-07-27

Publications (2)

Publication Number Publication Date
WO2013016418A2 true WO2013016418A2 (en) 2013-01-31
WO2013016418A3 WO2013016418A3 (en) 2013-06-13

Family

ID=46614639

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/048135 WO2013016418A2 (en) 2011-07-27 2012-07-25 Ion channel probes and methods of use thereof

Country Status (1)

Country Link
WO (1) WO2013016418A2 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014145554A2 (en) * 2013-03-15 2014-09-18 Tota Michael R Novel green fluorescent protein (gfp) peptides
WO2018191322A1 (en) * 2017-04-12 2018-10-18 Tempo Bioscience, Inc. Biosensors for cellular osmolarity
US10640766B2 (en) 2017-04-12 2020-05-05 Tempo Bioscience, Inc. Biosensors for chloride ions
CN111538727A (en) * 2020-04-16 2020-08-14 四川轻化工大学 Multi-path fast searching and classifying method and system based on Bloom Filter
WO2022246089A1 (en) * 2021-05-20 2022-11-24 Artema Therapeutics, Inc. Kcnv2 variants and their use
WO2022256251A1 (en) * 2021-05-30 2022-12-08 Genentech, Inc. Mutant human nax proteins and screening methods

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7211407B2 (en) * 2003-07-15 2007-05-01 Chan Test, Inc. High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins
AU2003904237A0 (en) * 2003-08-08 2003-08-21 Garvan Institute Of Medical Research Novel translocation assay
US20050063989A1 (en) * 2003-09-22 2005-03-24 Commissariat A L'energie Atomique Ion channel hybrid proteins as electrical sensors and use thereof
US20050191710A1 (en) * 2004-03-01 2005-09-01 Hanrahan John W. Method for labeling a membrane-localized protein
US20060148104A1 (en) * 2004-10-29 2006-07-06 Massachusetts Institute Of Technology Detection of ion channel or receptor activity
EP1828232A2 (en) * 2004-12-22 2007-09-05 The Salk Institute For Biological Studies Compositions and methods for producing recombinant proteins
EP2288620A2 (en) * 2008-06-12 2011-03-02 Université Joseph Fourier Hybrid proteins comprising membrane receptor and ion channel, and their use as biosensors

Non-Patent Citations (34)

* Cited by examiner, † Cited by third party
Title
AAKALU ET AL., NEURON, vol. 30, 2001, pages 489 - 502
ANANTHARAM ET AL., J. CELL BIOL., vol. 188, 2010, pages 415 - 28
AUSUBEL ET AL.,: "Current Protocols in Molecular Biology", 1994, GREEN PUBLISHERS INC. AND WILEY AND SONS
BATZER ET AL., NUCLEIC ACID RES., vol. 19, 1991, pages 5081
BLAINE ET AL., J. NEUROSCI., vol. 18, 1998, pages 9585 - 93
CHUNG ET AL., FEBS J., vol. 272, 2005, pages 3743 - 55
CREIGHTON, PROTEINS, 1984
D.V. GOEDDEL,: "Meth. Enz.", vol. 185, 1990, ACADEMIC PRESS INC.
ENGELS ET AL., ANGEW. CHEM. INTL. ED., vol. 28, 1989, pages 716 - 734
GUAN ET AL., J. PHYSIOL., vol. 581, 2007, pages 941 - 960
HALE; MARHAM: "THE HARPER COLLINS DICTIONARY OF BIOLOGY", 1991
JENKINS ET AL., CURR. BIOL., vol. 16, 2006, pages 1211 - 16
JENKINS ET AL., J. NEUROSCI., vol. 29, 2009, pages 10541 - 51
JIANG ET AL., NAT. PROTOC., vol. 1, 2006, pages 695 - 700
KESSELS ET AL., NEURON, vol. 61, 2009, pages 340 - 50
KIM ET AL., NATURE, vol. 378, 1995, pages 85 - 8
KIM ET AL., NEURON, vol. 54, 2007, pages 933 - 47
MALIN ET AL., J. NEUROSCI., vol. 22, 2002, pages 10094 - 105
MANGANAS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 14055 - 9
MANIATIS ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
MARKHAM ET AL., PLOS., vol. 7, 2009, pages E1000203
MCKEOWN ET AL., J. BIOL. CHEM., vol. 283, 2008, pages 30421 - 32
OHTSUKA ET AL., J. BIOL. CHEM., vol. 260, 1985, pages 2605 - 2608
R. RIEGER ET AL.: "THE GLOSSARY OF GENETICS, 5TH ED.", 1991, SPRINGER VERLAG
RAAB-GRAHAM ET AL., SCIENCE, vol. 314, 2006, pages 144 - 48
RASBAND ET AL., J. NEUROCYTOL, vol. 28, 1999, pages 319 - 31
ROSSOLINI ET AL., MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual(Second ed., )", 1989, COLD SPRING HARBOR LABORATORY PRESS, pages: 9.47 - 9.51
SAMBROOK; FRITSCH; MANIATIS: "Molecular Cloning: A Laboratory Manual, Second Edition.", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHUMACHER ET AL., CIRC. RES., vol. 104, 2009, pages 1390 - 8
SHANER ET AL., NATURE METHODS, vol. 2, 2009, pages 905 - 9
SINGLETON ET AL.: "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY(2d ed.", 1994
WALKER: "THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY", 1988

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014145554A2 (en) * 2013-03-15 2014-09-18 Tota Michael R Novel green fluorescent protein (gfp) peptides
WO2014145554A3 (en) * 2013-03-15 2014-10-30 Tota Michael R Green fluorescent protein (gfp) peptides from rhacostoma
WO2018191322A1 (en) * 2017-04-12 2018-10-18 Tempo Bioscience, Inc. Biosensors for cellular osmolarity
US10640766B2 (en) 2017-04-12 2020-05-05 Tempo Bioscience, Inc. Biosensors for chloride ions
CN111538727A (en) * 2020-04-16 2020-08-14 四川轻化工大学 Multi-path fast searching and classifying method and system based on Bloom Filter
CN111538727B (en) * 2020-04-16 2023-01-13 四川轻化工大学 Multi-path fast searching and classifying method and system based on Bloom Filter
WO2022246089A1 (en) * 2021-05-20 2022-11-24 Artema Therapeutics, Inc. Kcnv2 variants and their use
WO2022256251A1 (en) * 2021-05-30 2022-12-08 Genentech, Inc. Mutant human nax proteins and screening methods

Also Published As

Publication number Publication date
WO2013016418A3 (en) 2013-06-13

Similar Documents

Publication Publication Date Title
Zeng et al. Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity
Koval et al. Mix and match: investigating heteromeric and heterotypic gap junction channels in model systems and native tissues
Ma et al. Golgi export of the Kir2. 1 channel is driven by a trafficking signal located within its tertiary structure
WO2013016418A2 (en) Ion channel probes and methods of use thereof
Berbari et al. Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors
Ma et al. Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart
Segretain et al. Regulation of connexin biosynthesis, assembly, gap junction formation, and removal
Chu et al. A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions
Hojo et al. μ-Opioid receptor forms a functional heterodimer with cannabinoid CB1 receptor: electrophysiological and FRET assay analysis
Gianulis et al. Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels
Valentine et al. Confinement of β1-and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae
Kowalczyk et al. Structure and function of desmosomal transmembrane core and plaque molecules
Trudeau et al. Dynamics of Ca2+-calmodulin–dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry
Kuzmenkov et al. Apamin structure and pharmacology revisited
US7060794B2 (en) Protein that enhances expression of potassium channels on cell surfaces and nucleic acids that encode the same
Kleist et al. Shaping up: Recent advances in the study of plant calcium channels
Sakamaki et al. Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage
Schlingmann et al. The role of the C-terminus in functional expression and internalization of rat connexin46 (rCx46)
Prinsen et al. The retinal rod and cone Na+/Ca 2+-K+ exchangers
Naso et al. The role of the C-terminus for functional heteromerization of the plant channel KDC1
US9034599B2 (en) Polynucleotides encoding monomeric variants of the tetrameric eqFP611
US7713713B2 (en) Polypeptide having intracellular calcium ion indicator function
Nivillac et al. Disrupted plasma membrane localization and loss of function reveal regions of human equilibrative nucleoside transporter 1 involved in structural integrity and activity
WO2013163681A1 (en) Fluorescent proteins and uses thereof
JP2001518295A (en) Methods for selectively controlling membrane protein display and protein secretion in eukaryotic cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12743614

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 12743614

Country of ref document: EP

Kind code of ref document: A2