EP1097166A2 - Potassium channel polypeptide and polynucleotide compositions - Google Patents
Potassium channel polypeptide and polynucleotide compositionsInfo
- Publication number
- EP1097166A2 EP1097166A2 EP99944996A EP99944996A EP1097166A2 EP 1097166 A2 EP1097166 A2 EP 1097166A2 EP 99944996 A EP99944996 A EP 99944996A EP 99944996 A EP99944996 A EP 99944996A EP 1097166 A2 EP1097166 A2 EP 1097166A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- sequence
- seq
- polynucleotide
- potassium channel
- Prior art date
- Legal status (The legal status 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 status listed.)
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Classifications
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- 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
Definitions
- the present invention relates to novel human potassium channel polypeptide and polynucleotide compositions, to the production of these compositions, and to the use of the compositions in the diagnosis, prevention, and treatment of disease states, as well as in screening for therapeutics for treating such disease states.
- Potassium channels are a heterogeneous group of ion channels that allow selective permeation of potassium ions across the plasma membrane, but differ in details of activation mechanism, voltage range of activity, and kinetic properties.
- They contribute to numerous physiological functions, for example, action potential repola ⁇ zation, cardiac pacemakmg, neuron bursting, muscle contraction, hormone secretion, vascular tone regulation, renal ion reabsorption, learning and memory, and cell growth and differentiation.
- Tokl is an outward rectifying channel, in that it preferentially passes outward K+ currents which is coupled to changes in the external K+ concentration, and is regulated by protein kmase C and mtracellular pH.
- d-ORKl from o Drosoph ⁇ a is expressed in neuromuscular tissues and, unlike Tokl, appears to contain only four transmembrane segments (2P/4TM topology).
- d-ORKl behaves as an open rectifier (or leak) K+ channel when expressed m Xenopus oocytes, showing similar current rectification properties to those of an unidentified leak K+ current associated with resting membrane potential in myelmated vertebrate nerves (Baker M. et al (1993) J. Physiol. 383:45-67). 5
- h-TPKCl is a 2P/4TM channel which exhibits conductance properties similar to the 2P/8TM Tokl channel, is expressed m bram, skeletal muscle, small intestine and colon, but was not detected m lung, kidney, and heart (Goldstein et al. (1998), supra).
- the mouse homolog , mTREKl is 85% identical to h-TPKCl and shows common biophysical properties, and is expressed in brain, and, in contrast to h-TPKCl, in lung, kidney and heart (Fink M. et al. (1996) EMBO J. 15:6854-6862).
- the fourth known family of 2PDs is represented by HOHOl from human brain, a 337 amino 5 acid protein with probable 2P/4TM topology, the identical TWIK1 from human kidney, and the mouse homolog mTWIKl.
- the TWIKs reportedly behave as inwardly-rectifying K+ channels when expressed in Xenopus oocytes (Lesage F. et al. (1996) EMBO J. 15:1004-1011; Lesage F. et al. (1997) FEBS Lett 402:28-32), but it has been suggested that HOHOl/TWIKl may need to associate with other channel subunits and/or are subjected to other regulatory influences (Goldstein 0 et al. (1998), supra).
- rTASK TWIK-related acid-sensitive K+ channel
- TWIK family isolated from rat is most abundantly expressed in heart, lung and brain, lacks voltage sensitivity and exhibits open-rectifier channel properties, and is inhibited by mtracellular protein kinase-A activation and by extracellular acidity (Leonoudakis D. et al. (1998) 18:868-877).
- the homologous human hTASK channel is likewise voltage-insensitive and susceptible to variations in 5 extracellular pH but, unlike rTASK, is most abundantly expressed in pancreas and placenta, with lower levels observed in brain, lung, prostate, and heart (Duprat, F. et al. (1997) EMBO J. 16:5464- 5471).
- Potassium channels are associated with a variety of disease states. In some diseases and disorders, abnormal ion channels are believed to be causative factors, while other diseases appear to o arise from inappropriate regulation of otherwise normal ion channels. Diseases believed to have a particular association with potassium channels include neurological disorders such as epilepsy, cardiovascular, and proliferative disorders such as cancers.
- the discovery of new channel proteins of the emerging 2P-domain (2PD) potassium channel family, and the polynucleotides which encode them, satisfies a need in the art by providing new compositions which are useful in treatment of 5 various diseases associated with ion channel dysfunction.
- the invention is directed to the discovery of novel potassium channel subunits characterized by the presence of two pore forming domains, collectively referred to as TWIK family two P domain (2PD) potassium channel polypeptides and exemplified herein by KT4 and o KT5.
- TWIK family two P domain (2PD) potassium channel polypeptides characterized by the presence of two pore forming domains
- 2PD two pore forming domains
- the invention provides a composite amino acid sequence for such TWIK family polypeptide subunits, defined by an alignment of the sequences SEQ ID NO: 2 and SEQ ID NO: 6.
- residues that are conserved between SEQ ID NO: 2 and SEQ ID NO: 6 are conserved or "fixed” in the composite sequence, and residues that are different between the two sequences are varied between the residue found in SEQ ID NO: 2 and the residue found in 5 SEQ ID NO: 6.
- the invention is not construed to include the specific sequence hTWIK-1 (SEQ ID NO: 3)
- the invention is exemplified by two novel TWIK 2PD potassium channel polypeptides, KT4 (SEQ ID NO: 2) and KT5 (SEQ ID NO: 6).
- the invention includes these polypeptides, and variants thereof, as well as polypeptides having at least 80%, and more preferably 90%, sequence identity to SEQ ID NO: 2 or SEQ ID NO: 6.
- the polypeptide is purified and crystallized in a composition suitable for 5 performing X-ray crystallographic studies, which are useful for determining ligand binding site coordinates and optimal binding structures.
- a composition may further include an agonist or antagonist compound.
- the invention also includes antibodies that binds to a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6.
- the invention includes an isolated polynucleotide which encodes any of the polypeptides described above, or sequences which are complementary to such coding sequences.
- isolated polynucleotides that hybridize under high-stringency conditions to the following exemplified polynucleotides: SEQ ID NO: 1, the complement of SEQ ID NO: 1, SEQ ID NO: 5 and the complement of SEQ ID NO: 5.
- the 5 invention also includes polynucleotides having the sequence SEQ ID NO: 1 or SEQ ID NO: 5, or sequences substantially identical thereto.
- the invention includes recombinant expression vectors.
- such vectors include (a) a polynucleotide as described above, and (b) operably linked to the polynucleotide, a regulatory sequence effective to facilitate expression of the o polynucleotide in a selected host.
- the invention also encompasses host cells transfected with such vectors, where the host cell expresses a heterologous potassium channel polypeptide, such as described above, on the surface of the cell.
- the invention also includes a process for producing such a cell by transforming or transfecting the cell with an expression vector, as described above, and culturing the cell under appropriate culture conditions.
- the invention includes methods for detecting a polynucleotide that encodes a TWIK family potassium channel in a biological sample.
- the method includes: (a) hybridizing to nucleic acid material of a biological sample a polynucleotide fragment which encodes the sequence identified as SEQ ID NO: 2 or SEQ ID NO: 6, where the fragment has a length of at least 12 nucleotides; and(b) detecting a o hybridization complex formed thereby.
- the presence of a hybridization complex correlates with the presence of a polynucleotide encoding the TWIK family protein in said biological sample.
- the invention includes a method for detecting the presence of a TWIK family 2PD potassium channel in a biological sample.
- the method 5 includes: (a) contacting the biological sample with an antibody which binds to a polypeptide of the invention, such as KT4 or KT5, thereby forming an antibody-antigen complex; and (b) detecting the presence of the antibody-antigen complex.
- the presence of such a complex correlates with the presence of a TWIK family 2PD potassium channel, such as KT4 or KT5 in the biological sample.
- the invention includes a method of identifying a candidate compound capable of modulating potassium channel activity.
- This screening method 5 includes the steps of: (a) contacting a test compound with a potassium channel which contains a polypeptide subunit of the invention (as described above); (b) measuring the effect of the test compound on the activity of the potassium channel; and (c) selecting the test compound as a candidate compound if its effect on the activity of the potassium channel is above a selected threshold level.
- the invention further includes potassium channel agonist and antagonist 0 compounds selected according to this method.
- FIGS. 1 A-1C show the nucleic acid sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO:2) of KT4.
- FIGS. 2A-2B show the amino acid sequence alignment between KT4 (SEQ ID NO:2) and human TWIK-1 (GenBank PID gl086491; SEQ ID NO:3) produced using the CLUSTAL-W alignment program of MacVectorTM software (ver. 6.01; Oxford Molecular Ltd, Oxford, UK) using o the default pairwise parameters.
- FIGS. 3A-3C show a nucleic acid sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO: 6) of a KT5 polypeptide.
- FIGS. 4A-4B show an amino acid sequence alignment between KT5 (SEQ ID NO: 6) and human TWIK-1 (GenBank PID gl086491; SEQ ID NO:3) produced using the CLUSTAL-W 5 alignment program of MacVectorTM software (ver. 6.01; Oxford Molecular Ltd, Oxford, UK) using the default pairwise parameters.
- FIGS. 5A-5B show an amino acid sequence alignment between KT4 (SEQ ID NO: 2) and KT5 (SEQ ID NO: 6) produced using the CLUSTAL-W alignment program, using the PAM250 residue weight table and LaserGene analysis (DNAStar; Madison, WI). o Brief Description of the Sequences
- SEQ ID NO: 1 is the nucleic acid sequence of KT4.
- SEQ ID NO:2 is the deduced amino acid sequence of KT4.
- SEQ ID NO:3 is the amino acid sequence of hTWIK-1 (GenBank PID gl086491).
- SEQ ID NO:4 is the sequence of human EST AA604914 (GenBank NID g2445778; Accession No. 5 AA604914).
- SEQ ID NO: 5 is a nucleic acid sequence encoding KT5.
- SEQ ID NO:6 is a deduced amino acid sequence of KT5.
- SEQ ID NO:7 is the sequence of human EST AA533124 (GenBank NID g2277220; Accession No. AA533124).
- polypeptide refers to a compound made up of a single chain of ammo acid residues linked by peptide bonds
- protein as used herein may be synonymous with the term “polypeptide” or may refer, m addition, to a complex of two or more 5 polypeptides.
- a “channel” or “channel protein” as used herein refers to a protein comprising one or more P-domam-contammg polypeptide subunits, and may be formed of multimers of the same polypeptide (a “homome ⁇ c” channel) or of different polypeptides (a “heterome ⁇ c” channel). Channel proteins may also contain "accessory subunits” which modulate the activity of the channel.
- TWIK family 2PD polypeptide is a polypeptide which contains two potential P- domams, eight or preferably four predicted transmembrane domains, and has at least 80% sequence identity to a corresponding aligned region of a 2PD potassium channel polypeptide, such as KT4 and KT5, or preferably, has a sequence corresponding to a composite sequence defined by alignment of KT4 and KT5, as descnbed herein 5 "KT4" refers to a TWIK family 2PD polypeptide comprising a sequence having at least 70 percent, preferably at least 80 percent, more preferably at least 90 percent, and most preferably at least 95 percent sequence identity to the polypeptide identified as SEQ ID NO: 2.
- KT4 is meant to include the full-length polypeptide and fragments thereof unless the context indicates otherwise.
- KT5 refers to a TWIK family 2PD polypeptide compnsmg a sequence having at least 70 percent, preferably at least 80 percent, more preferably at least 90 percent, and most preferably at least 95 percent sequence identity to the polypeptide identified as SEQ ID NO: 6.
- reference to KT5 is meant to include the full-length polypeptide and fragments thereof unless the context indicates otherwise. 5
- a "composite" of KT4 and KT5, as used herein, refers to a polypeptide sequence that is defined by alignment of the two sequences, using methods such as CLUSTAL analysis.
- the composite retains such ammo acid as a fixed or conserved amino acid; where the two sequences have different amino acids at a given position, the composite has a variable amino acid at that position, where the variable can take the identity of either of the two amino acids found at the position.
- KT4 channel refers to a multimeric potassium channel comprising at least one 5 KT4 polypeptide
- KT5 channel refers to a multimeric potassium channel comprising at least one KT5 polypeptide
- mature polypeptide refers to a polypeptide as it exists in the cell after post- translational processing; for example, after removal of any signal sequence.
- modified when referring to a polypeptide of the invention, means a polypeptide o which is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art.
- modifications include, but are not limited to, acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, 5 ubiqutination, or any similar process.
- biologically active refers to a TWIK family 2PD polypeptide having structural, regulatory or biochemical functions similar to those of the naturally occurring KT4 or KT5 polypeptides including, but not limited to, the ability to support potassium ion conductance, when it forms a channel, such as a self-associated homomeric channel, or when associated with other o channel polypeptides into a heteromeric channel.
- immunologically active defines the capability of a natural, recombinant or synthetic polypeptide according to the invention, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
- fragment when referring to polypeptides of the invention, means a polypeptide 5 which has an amino acid sequence which is the same as part of but not all of the amino acid sequence of KT4, KT5 or a composite thereof, which retains at least one of the functions or activities of the channel protein, or which is capable of interacting with KT4, KT5, other proteins, peptides, or other molecules, to alter a function or activity or the cellular/subcellular localization of a channel formed in accordance with the present invention. Fragments contemplated include, but o are not limited to, a KT4 or KT5 fragment which retains the ability to bind a ligand of a either a
- KT4 or KT5 channel a fragment which blocks the binding of a ligand to a KT4 or KT5 channel, or a fragment which retains immunological activity of KT4 or KT5.
- the fragment preferably includes at least 20, more preferably at least 50, contiguous amino acid residues of KT4, KT5 or a fragment thereof. 5
- fragment when referring to a KT4 or KT5 coding sequence, means a polynucleotide which has a nucleic acid sequence which is the same as part of but not all of the nucleic acid sequence of the KT4 or KT5 coding sequence.
- the fragment preferably includes at least 12 contiguous bases such coding sequence.
- portion when referring to a polypeptide of the invention, means a polypeptide which has an amino acid sequence which is the same as part of the amino acid sequence of the present invention or a variant thereof, which does not necessarily retain any biological function or activity.
- a “conservative substitution” refers to the substitution of an amino acid in one class by an amino acid in the same class, where a class is defined by common physicochemical amino acid sidechain properties and high substitution frequencies in homologous proteins found in nature (as determined, e.g., by a standard Dayhoff frequency exchange matrix or BLOSUM matrix).
- Class II Ser, Thr, Pro, Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala, Gly
- Class III Ala
- non-conservative substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.
- Optimal alignment is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using a ktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program in
- MacVector operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix.
- Percent sequence identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two or more optimally aligned polypeptide sequences are identical. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the "gap" of the first sequence).
- a first polypeptide region is said to "correspond" to a second polypeptide region when the regions are essentially co-extensive when the sequences containing the regions are aligned using a sequence alignment program, as above.
- Corresponding polypeptide regions typically contain a similar, if not identical, number of residues. It will be understood, however, that corresponding regions may contain insertions or deletions of residues with respect to one another, as well as some differences in their sequences.
- Corresponding polynucleotide or polypeptide fragments typically contain a similar, if not identical, number of residues. It will be understood, however, that corresponding fragments may contain insertions or deletions of residues with respect to one another, as well as some differences in their sequences.
- sequence identity means nucleic acid or amino acid sequence identity in two or 5 more aligned sequences, aligned as defined above.
- Sequence similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
- 80% protein sequence similarity means that 80% of the amino acid residues in two or more aligned protein sequences are conserved amino acid residues, i.e. are 0 conservative substitutions.
- Hybridization includes any process by which a strand of a nucleic acid joins with a complementary nucleic acid strand through base-pairing. Thus, strictly speaking, the term refers to the ability of the complement of the target sequence to bind to the test sequence, or vice-versa.
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid 5 binding complex or probe and are typically classified by degree of “stringency” of the conditions under which hybridization is measured. For example, “maximum stringency” typically occurs at about Tm-5°C (5° below the Tm of the probe); “high stringency” at about 5-10° below the Tm; “intermediate stringency” at about 10-20° below the Tm of the probe; and “low stringency” at about 20-25° below the Tm. Functionally, maximum stringency conditions may be used to identify o nucleic acid sequences having strict identity or near-strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe.
- gene means the segment of DNA involved in producing a polypeptide chain; it may include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as o intervening sequences (introns) between individual coding segments (exons).
- 5' UTR 5' untranslated
- leader leader
- 3' UTR or “trailer” sequences as well as o intervening sequences (introns) between individual coding segments (exons).
- a "polynucleotide having a sequence which encodes a TWIK family 2PD polypeptide” is a polynucleotide which contains the coding sequence of a polypeptide of the invention (i) in combination with additional coding sequences, such as fusion protein or signal peptide, in which the polypeptide (e.g., KT4, KT5, or composite thereof) coding sequence is the dominant coding 5 sequence, (ii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment in which the polypeptide coding sequence is a heterologous gene
- heterologous DNA and “heterologous RNA” refer to nucleotides that are not endogenous to the cell or part of the genome in which they are present; generally such nucleotides have been added to the cell, by transfection, micromjection, electroporation, or the like. Such 5 nucleotides generally include at least one coding sequence, but this coding sequence need not be expressed.
- isolated means that the mate ⁇ al is removed from its ongmal environment (e.g., the natural environment if it is naturally occurring).
- a naturally-occur ⁇ ng polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide 0 or polypeptide, separated from some or all of the coexisting mate ⁇ als in the natural system, is isolated.
- Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment Further, such isolated materials are still considered to be isolated when they are added to a cell system, such as for heterologous or non-natural expression, 5 including augmentation of natural expression.
- expression vector refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell
- Many prokaryotic and eukaryotic expression vectors are commercially available Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
- substantially purified refers to molecules, either polynucleotides or polypeptides, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
- a “va ⁇ ant" polynucleotide sequence may encode a "vanant” ammo acid sequence which is 5 altered by one or more amino acids from the reference polypeptide sequence.
- the va ⁇ ant polynucleotide sequence may encode a variant ammo acid sequence which contains "conservative" substitutions, wherein the substituted ammo acid has structural or chemical properties similar to the ammo acid which it replaces.
- va ⁇ ant polynucleotide sequence may encode a variant ammo acid sequence which contains "non-conservative" substitutions, wherein the o substituted ammo acid has dissimilar structural or chemical properties to the ammo acid which it replaces
- Variant polynucleotides may also encode variant ammo acid sequences which contain ammo acid insertions or deletions, or both.
- a va ⁇ ant polynucleotide may encode the same polypeptide as the reference polynucleotide sequence but, due to the degeneracy of the genetic code, has a polynucleotide sequence which is altered by one or more bases from the 5 reference polynucleotide sequence.
- An "allehc variant” is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
- “Alternative splicing” is a process whereby multiple polypeptide isoforms are generated from a single gene, and involves the splicing together of nonconsecutive exons during the processing of some, but not all, transcripts of the gene. Thus a particular exon may be connected to 5 any one of several alternative exons to form messenger RNAs.
- the alternatively-spliced mRNAs produce polypeptides ("splice variants”) in which some parts are common while other parts are different.
- splice variants of KT4 or KT5 when referred to in the context of an rriRNA transcript, are mRNAs produced by alternative splicing of coding regions, i.e., exons, from the KT4 or KT5 0 gene, respectively.
- KT4 or KT5 when referred to in the context of the protein itself, are KT4 or KT5 translation products which are encoded by alternatively-spliced KT4 or KT5 mRNA transcripts, respectively.
- a “mutant" amino acid or polynucleotide sequence is a variant amino acid sequence, or a 5 variant polynucleotide sequence that encodes a variant amino acid sequence, which has significantly altered biological activity from that of the naturally occurring protein.
- a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
- insertion or “addition” is that change in a nucleotide or amino acid sequence which o has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
- substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
- modulate refers to the change in activity of the polypeptide of 5 the invention. Modulation may relate to an increase or a decrease in biological activity, binding characteristics, or any other biological, functional, or immunological property of the molecule.
- agonist refers to a molecule which, when bound to the channel of the present invention, modulates the activity of the channel by inducing, increasing, or prolonging the duration of the biological activity mediated by the channel.
- Agonists may o themselves be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other ligand which binds to and modulates the activity of the channel.
- Antagonist refers to a molecule which, when bound to the channel of the present invention, modulates the activity of the channel by blocking, decreasing, or shortening the duration of the biological activity mediated by the channel.
- Antagonists may 5 themselves be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other ligand which binds to and modulates the activity of the channel.
- humanized antibody refers to antibody molecule in which one or more amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding activity of the antibody.
- Treating a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
- the invention provides isolated TWTK Family 2PD polypeptides and isolated polynucleotides encoding such polypeptides. As defined more fully in Section III below, KT4 and KT5 are exemplary
- TWTK family 2PD polypeptides that (i) comprise an amino acid sequence having at least 70%, 0 preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, sequence identity to the amino acid sequence identified as SEQ ID NO: 2 and SEQ ED NO: 6, respectively.
- SEQ ID NO: 1 is a 2671 base nucleic acid sequence which contains an open reading frame that encodes a 313 amino acid polypeptide identified herein as KT4 and having the sequence SEQ ID NO: 2.
- SEQ ID NO: 5 is a 2872 base nucleic acid sequence which contains an open reading frame encoding a 449 amino acid polypeptide identified herein as a KT5 polypeptide having the sequence SEQ ID NO: 6.
- Polynucleotides encoding related TWIK 2PD family members in accordance with the present invention can be isolated from selected cDNA libraries, such as a derived from human o brain, as exemplified in Example 1. Briefly, a biotin-labeled nucleotide probe is used to capture target cDNA molecules from a cDNA library by solution hybridization, using probe sequences derived from KT4 or KT5, exemplified herein.
- TWIK 2PD family 5 member nucleic acid sequences such as those identified herein as SEQ ID NO: 1 and SEQ ID NO: 5.
- Northern analysis performed as described in Example 2 showed expression of KT4 transcripts in high abundance in pancreas, somewhat lower abundance in heart and placenta, and still lower abundance in liver, lung and brain, and of KT5 transcripts in tissues including, but not o limited to, liver, kidney, pancreas, placenta, and lung.
- the polynucleotides of the invention include sequences which encode KT4, KT5, composites thereof and sequences complementary to such coding sequences, as well as novel fragments of such polynucleotides.
- the polynucleotides may be in the form of RNA or in the form 5 of DNA, and include mRNA, cRNA, synthetic RNA and DNA and analogs thereof, cDNA, peptide nucleic acid, and genomic DNA.
- the polynucleotides may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.
- the polynucleotide hybridizes under stringent conditions, preferably high-stringency conditions, to the sequence identified as SEQ ID NO: 1, SEQ ID NO: 5 or the complement of either of such sequences. Exemplary hybridization conditions are described in Section IIB below.
- the polynucleotide of the invention has at least 70%, preferably 80% or 90% sequence identity with the sequence identified as SEQ ID NO: 1, SEQ ID NO: 5 or the complement of either of such sequences.
- the polynucleotide has a sequence substantially identical to the sequence identified as SEQ ID NO: 1 or substantially identical to the sequence identified as SEQ ID NO: 5.
- the polynucleotides may include the coding sequence of KT4 or KT5 (i) in combination with additional coding sequences, such as fusion protein or signal peptide, in which the KT4 or KT5 coding sequence is the dominant coding sequence, (ii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or (iii) in a vector or host environment in which the KT5 coding sequence is a heterologous gene.
- the polynucleotide encodes a composite polypeptide of KT4 and KT5, as described below.
- KT4 and KT5 as described below.
- persons skilled in the art will be able to select appropriate codons and prepare a coding sequence encoding such composite polypeptides.
- the polynucleotide may encode a polypeptide fragment of KT4 or KT5, for example, an extracellular fragment or an intracellular fragment which has been cleaved from a transmembrane domain of KT4 or KT5.
- the polynucleotides of the present invention may also have the protein coding sequence fused in- frame to a marker sequence which allows for purification of polypeptides of the invention.
- the marker sequence may be, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
- the HA tag co ⁇ esponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. (1984) Cell 37:767).
- polynucleotides also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 20 or 30 bases, corresponding to a region of the coding-sequence polynucleotide or the complement thereof.
- the polynucleotides may be used as probes, primers, antisense agents, and the like, according to known methods.
- the polynucleotides may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amphfy polynucleotides which encode the KT5 and fragments disclosed above.
- cDNA hbra ⁇ es prepared from a va ⁇ ety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described m, for example, Sambrook et al (1989) Molecular 5 Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plamview, N.Y. and
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe and are typically classified by degree of " stringency” of the conditions 0 under which hybridization is measured.
- Tm melting temperature
- degree of “ stringency” typically occurs at about Tm-5°C (5° below the Tm of the probe), “high stringency” at about 5-10° below the Tm; “intermediate stringency” at about 10-20° below the Tm of the probe; and “low st ⁇ ngency” at about 20-25° below the Tm.
- maximum st ⁇ ngency conditions may be used to identify nucleic acid sequences having st ⁇ ct identity or near-st ⁇ ct identity with the hyb ⁇ dization probe; 5 while high st ⁇ ngency conditions are used to identify nucleic acid sequences having about 80% or more sequence identity with the probe.
- the polynucleotides may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3' untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerous methods known to those of skill in the art, such as PCR or p ⁇ mer extension (Sambrook et al., supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # Kl 802-1) 5 Alternatively, the technique of "restriction-site" PCR (Gobmda et al. ( 1993) PCR Methods
- Applic. 2:318-22 which uses universal p ⁇ mers to ret ⁇ eve flanking sequence adjacent a known locus, may be employed.
- genomic DNA is amplified in the presence of p ⁇ mer to a linker sequence and a primer specific to the known region.
- the amplified sequences are subjected to a second round of PCR with the same linker p ⁇ mer and another specific primer internal to the first o one Products of each round of PCR are transc ⁇ bed with an approp ⁇ ate RNA polymerase and sequenced using reverse transc ⁇ ptase
- Inverse PCR can be used to amplify or extend sequences using divergent p ⁇ mers based on a known region (T ⁇ glia T et al (1988) Nucleic Acids Res 16:8186)
- the primers may be designed using OLIGO(R) 4.06 P ⁇ mer Analysis Software (1992; National Biosciences Inc, Madison, 5 Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of
- the method uses several restriction enzymes to generate a suitable fragment m the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
- Capture PCR (Lagerstrom M et al. (1991) PCR Methods Applic 1:111-19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR.
- flanking sequences Another method which may be used to retrieve flanking sequences is that of Parker, JD et al. (1991; Nucleic Acids Res 19:3055-60). Additionally, one can use PCR, nested primers and PromoterFinderTM libraries to "walk in" genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
- Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs.
- random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes. A randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA. Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
- polynucleotides and oligonucleotides of the invention can also be prepared by solid- phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to form continuous sequences up to several hundred bases.
- polynucleotide coding sequences and novel oligonucleotides of the invention have a variety of uses in (1) synthesis of TWIK family 2PD polypeptides, (2) diagnostics, (3) gene mapping, and (4) therapeutics.
- TWIK family 2PD polypeptides Synthesis of TWIK family 2PD polypeptides.
- polynucleotide sequences which encode KT4, KT5, composites of KT4 and KT5, splice variants, fragments of the polypeptide, fusion proteins, or functional equivalents thereof may be used in recombinant DNA molecules that direct the expression of KT5 in appropriate host cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express polypeptides of the invention.
- Codons prefe ⁇ ed by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of KT5 polypeptide expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
- polynucleotide sequences of the present invention can be engineered in order to alter the naturally occuring coding sequences, such as the KT4 or KT5 human sequences exemplified herein, for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
- alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction 5 sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc.
- the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
- the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
- the construct further comprises regulatory 0 sequences, including, for example, a promoter, operably linked to the sequence.
- regulatory 0 sequences including, for example, a promoter, operably linked to the sequence.
- suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, et al., (supra).
- the present invention also relates to host cells which are genetically engineered with 5 vectors of the invention, and the production of proteins and polypeptides of the invention by recombinant techniques.
- Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
- the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
- the engineered host cells can be cultured in conventional nutrient media modified as o appropriate for activating promoters, selecting transformants or amplifying the gene of interest.
- the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
- the polynucleotides of the present invention may be included in any one of a variety of expression vectors for expressing a polypeptide.
- Such vectors include chromosomal, 5 nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
- any other vector may be used as long as it is replicable and viable in the host.
- the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA o sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art.
- the DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis.
- promoters 5 include: LTR or SV40 promoter, the E. coli lac or tip promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
- the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression.
- the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin 5 resistance in E. coli.
- the vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
- appropriate expression hosts include: bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells, such as yeast; insect 0 cells such as Drosophila and Spodoptera Sf9; mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma; adenoviruses; plant cells, etc.
- a number of expression vectors may be selected depending upon the use intended for the polypeptide. For example, when large quantities of the polypeptide or fragments thereof are needed for the induction of antibodies, vectors which direct high level o expression of fusion proteins that are readily purified may be desirable.
- Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J. Biol. Chem 5 264:5503-5509); pET vectors (Novagen, Madison WI); and the like.
- yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
- the expression of a coding sequence may 0 be driven by any of a number of promoters.
- viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al. (1984) Nature 310:511-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al. (1987) EMBO J 3:1671-1680; Broglie et al.
- Polypeptides of the invention may also be expressed m an insect system.
- Autographa cahfornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda Sf9 cells or in Trichoplusia larvae.
- the coding sequence is cloned into a nonessential region of the virus, such as the polyhed ⁇ n gene, and placed under control of the polyhed ⁇ n promoter. Successful insertion of coding sequence will render the polyhed ⁇ n gene inactive and produce recombinant virus lacking coat protein coat.
- the recombinant viruses are then used to mfect S frugiperda cells or Trichoplusia larvae in which KT5 is expressed (Smith et al. (1983) J Virol 46:584; Engelhard EK et al (1994) Proc Nat Acad Sci 91 :3224-3227).
- a number of viral-based expression systems may be utilized.
- a coding sequence may be hgated into an adenovirus transc ⁇ ption/translation complex consisting of the late promoter and t ⁇ partite leader sequence Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing polypeptide m infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81.3655-3659).
- transcription enhancers such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- RSV rous sarcoma virus
- Specific initiation signals may also be required for efficient translation of a functional TWIK 2PD family polypeptide coding sequence These signals include the ATG initiation codon and adjacent sequences In cases where the polypeptide coding sequence, its initiation codon and upstream sequences are inserted into the approp ⁇ ate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transc ⁇ ptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be m the correct reading frame to ensure transcription of the entire insert.
- Exogenous transc ⁇ ptional elements and initiation codons can be of various origins, both natural and synthetic The efficiency of expression may be enhanced by the inclusion of enhancers approp ⁇ ate to the cell system m use (Scharf D et al. (1994) Results Probl Cell Differ 20.125-62; Bittner et al (1987) Methods in Enzymol 153:516-544).
- the present invention relates to host cells containing the above- described constructs.
- the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacte ⁇ al cell.
- Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology).
- KT5 cRNA may be micromjected into cells, such as Xenopus laevis oocytes, for production of KT5 for electrophysiological measurements or other assays.
- a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
- modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, 5 lipidation and acylation
- Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function.
- Different host cells such as CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
- the host cell lack endogenous functionally expressed potassium channels having cu ⁇ ent characte ⁇ stics similar to those exhibited or modulated by the KT5 channel desc ⁇ bed herein.
- cell lines which stably express KT4 or KT5 may be transformed using expression vectors which contain viral o ⁇ gms of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1 -2 days in an en ⁇ ched media before they are switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and o recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques approp ⁇ ate to the cell type.
- Host cells transformed with a nucleotide sequence encoding KT4, KT5 or composites thereof may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
- the protein or fragment thereof produced by a recombinant cell may be 5 secreted, membrane-bound, or contained mtracellularly, depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides encoding polypeptides of the invention can be designed with signal sequences which direct secretion of the protein through a prokaryotic or eukaryotic cell membrane.
- Polypeptides of the invention may also be expressed as a recombinant protein with one or o more additional polypeptide domains added to facilitate protein pu ⁇ fication.
- purification facilitating domains include, but are not limited to, metal chelatmg peptides such as histidme- tryptophan modules that allow pu ⁇ fication on immobilized metals, protein A domains that allow purification on immobilized lmmunoglobuhn, and the domain utilized m the FLAGS extension/affinity pu ⁇ fication system (Immunex Corp, Seattle, Wash.).
- the inclusion of a 5 protease-cleavable polypeptide linker sequence between the pu ⁇ fication domain and KT5 is useful to facilitate pu ⁇ fication.
- One such expression vector provides for expression of a fusion protein comprising KT5 (e g , a soluble KT5 fragment) fused to a polyhistidme region separated by an enterokinase cleavage site.
- KT5 e g , a soluble KT5 fragment
- the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al. (1992) Protein Expression and Purification 3:263-281) while the enterokinase cleavage site provides a means for isolating KT5 from the fusion protein.
- pGEX vectors may also be used to express 5 foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand- agarose beads (e.g., glutathione-agarose in the case of GST- fusions) followed by elution in the presence of free ligand.
- ligand- agarose beads e.g., glutathione-agarose in the case of GST- fusions
- the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
- Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
- Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical 5 disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
- Polypeptides of the invention can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose o chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- the polynucleotides of the present invention may be used 5 for a variety of diagnostic purposes.
- the polynucleotides may be used to detect and quantitate expression of TWIK family 2PD potassium channels, and particularly, aberrant forms thereof, in a patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for KT4, KT5 or composites thereof.
- This assay typically involves obtaining total mRNA from the tissue and contacting the mRNA with a nucleic acid probe.
- the probe is a nucleic acid molecule of at least 12 o nucleotides, preferably at least 20-30 nucleotides, capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding polypeptides of the invention under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of such potassium channel polypeptides.
- This assay can be used to distinguish between absence, presence, and excess expression of KT4, KT5, including 5 composites or variants thereof , and to monitor such levels of expression during therapeutic intervention.
- the invention also contemplates the use of the polynucleotides as a diagnostic for diseases resulting from inherited defective TWIK family polypeptide genes.
- These genes can be detected by compa ⁇ ng the sequences of the defective (i.e., mutant) gene with that of a normal one. Association of a mutant gene with abnormal KT4 or KT5 activity (for example, abnormal channel activity) may be verified.
- mutant KT4 or KT5 genes can be inserted into a suitable vector for expression in a functional assay system as yet another means to ve ⁇ fy or identify mutations. Once mutant genes have been identified, one can then screen populations of interest for carriers of the mutant gene.
- Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, u ⁇ ne, saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al. (1986) Nature 324:163-166) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
- Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et al. (1985) Proc. Natl. Acad. Sci. USA 85:4397-4401), or by differences in melting temperatures. "Molecular beacons" (Kost ⁇ kis L.G. et al.
- hairpm-shaped, single-stranded synthetic oligonucleotides containing probe sequences which are complementary to the nucleic acid of the present invention may also be used to detect point mutations or other sequence changes as well as monitor expression levels of polypeptides of the invention. Such diagnostics would be particularly useful for, e g., prenatal testing.
- Another method for detecting mutations uses two DNA probes which are designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutat ⁇ on(s) is at or near the abutting bases.
- the two probes may be joined at the abutting bases, e.g., in the presence of a hgase enzyme, but only if both probes are correctly base paired in the region of probe junction.
- the presence or absence of mutations is then detectable by the presence or absence of ligated probe.
- the TWIK family 2PD polypeptide coding sequence are oligonucleotide array methods based on sequencing by hyb ⁇ dization (SBH), as described, for example, in U.S. Patent No. 5,547,839.
- SBH sequencing by hyb ⁇ dization
- the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip.
- the sequence of the target can then be "read” from the pattern of target binding to the array C3.
- Gene mapping Gene mapping.
- the sequences of the present invention are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hyb ⁇ dize with a particular location on an individual human chromosome.
- chromosome marking reagents based on actual 5 sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
- the mapping of DNAs to chromosomes according to the present invention is an important first step m co ⁇ elatmg those sequences with genes associated with disease.
- sequences can be mapped to chromosomes by prepa ⁇ ng PCR primers (preferably 12-30 bp) from cDNA sequences de ⁇ ved from nucleotides of the invention. Computer analysis of 0 the 3' untranslated region is used to rapidly select primers that do not span more than one exon m the genomic DNA, which would complicate the amplification process. These p ⁇ mers are then used for PCR screening of somatic cell hyb ⁇ ds containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the p ⁇ mer will yield an amplified fragment.
- mapping of somatic cell hyb ⁇ ds is a rapid procedure for assigning a particular DNA 5 to a particular chromosome
- sublocahzation can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
- Other mapping strategies that can similarly be used to map to its chromosome include in situ hyb ⁇ dization, prescreenmg with labeled flow-sorted chromosomes and preselection by hyb ⁇ dization to construct chromosome specific-cDNA libra ⁇ es.
- FISH Fluorescence m situ hybridization
- the physical position 5 of the sequence on the chromosome can be correlated with genetic map data.
- genetic map data are found, for example, in the OMIM database (Center for Medical Genetics, Johns Hopkins University, Baltimore, MD and National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD).
- the OMIM gene map presents the cytogenetic map location of disease genes and other expressed genes
- the OMIM database provides information on diseases associated with the o chromosomal location Such associations include the results of linkage analysis mapped to this interval, and the correlation of translocations and other chromosomal aberrations m this area with the advent of polygenic diseases, such as cancer.
- Polynucleotides which encode polypeptides of the invention, or complements of such polynucleotides, may also be used for therapeutic purposes.
- 5 Polypeptide expression of may be modulated through antisense technology, which controls gene expression through complementary polynucleotides, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene.
- the 5' coding portion of the polynucleotide sequence which codes for the protein of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs m length. Oligonucleotides de ⁇ ved from the transcription start site, e.g.
- An antisense DNA oligonucleotide is designed to be complementary to a region of the gene involved in 5 transcription (Lee et al. (1979) Nucl. Acids Res. 3:173; Cooney et al (1988) Science 241:456; and Dervan et al. (1991) Science 251: 1360), thereby preventing transcription and the production of polypeptides of the invention.
- An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule (Okano (1991) J Neurochem. 56:560).
- the antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or 0 DNA may be expressed in vivo.
- compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable earner or excipient
- a pharmaceutically acceptable earner or excipient includes but is not limited to salme, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. 5
- the formulation should suit the mode of administration
- polypeptides, and agonist and antagonist compounds which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often refe ⁇ ed to as "gene therapy"
- Cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then o being provided to a patient to be treated with the polypeptide
- DNA or RNA polynucleotide
- RNA encoding a polypeptide ex vivo
- Such methods are well-known in the art
- cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
- cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known m the art.
- a producer cell for producing a retroviral particle 5 containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
- the expression vehicle for engineering cells may be, for example, an adenovirus which may be used to engineer cells in o vivo after combination with a suitable delivery vehicle (Yeh P., et al (1997) FASEB J 11:615-623)
- Retroviruses from which the retroviral plasmid vectors mentioned above may be de ⁇ ved include, but are not limited to, Moloney Mu ⁇ ne Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, adenovirus, Myeloprohferative Sarcoma Virus, and mammary tumor virus. 5
- the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
- packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ps ⁇ -2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller (1990; Human Gene Therapy, Vol. 1, pg. 5-14).
- the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
- the retroviral plasmid vector may be encapsulated into a liposome, 5 or coupled to a lipid, and then administered to a host.
- the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
- retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
- the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
- Eukaryotic cells which may be o transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, f ⁇ broblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
- the genes introduced into cells may be placed under the control of inducible promoters, such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et al. (1996) Cancer Res 5 56(19):4311), to stimulate polypeptide production or antisense inhibition in response to radiation, e.g., radiation therapy for treating tumors.
- inducible promoters such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et al. (1996) Cancer Res 5 56(19):4311
- the invention also includes TWIK family 2PD polypeptides, exemplified by KT4 and o KT5, described herein, and by composites of KT4 and KT5, discussed below.
- the KT4 polypeptide SEQ ID NO: 2 has 313 amino acid residues and an estimated molecular weight of approximately 34 kDal.
- KT4 contains four potential transmembrane domains, spanning approximately residues 6 to 28, 118 to 142, 170 to 190, and 234 to 238, potential N- linked glycosylation sites at residues Asn79 and Asn85, and potential phosphorylation sites at 5 residues Thr42, Serl58, Ser200, Thr255, and Ser304.
- the KT4 sequence contains two potential pore-forming P-domains spanning approximately residues 93 to 118 (PI) and 210 to 227 (P2), which are highly conserved within the family of 2P domain potassium channel polypeptides (Goldstein et al.
- KT4 SEQ ID NO:2
- hTWIK-1 SEQ ID NO:3
- the proteins have an overall amino acid sequence identity of 40%, and have several regions of closer sequence identity, particularly in the conserved PI and P2 domains, which share about 65% and 82% identity, respectively.
- the KT5 polypeptide SEQ ID NO: 6 has 499 residues and a calculated molecular weight of approximately 55 kDal.
- KT5 contains four potential transmembrane domains, spanning 5 approximately residues 5 to 26, 114 to 134, 160 to 184, and 225 to 250, a potential N-linked glycosylation site at residue Asn77, and numerous potential phosphorylation sites including Thr40, Thrl49, Serl54, Ser273, Ser295, Thr300, Thr354, Thr361, Ser371, Ser430, Ser437, Ser438, Thr434, Ser466, Thr473, Thr475, and Ser495 .
- the KT5 sequence contains two potential pore-forming P-domains, spanning approximately residues 85 to 106 (PI) and 188 to 212 (P2).
- the KT5 amino acid sequence SEQ ID NO: 6 and the hTWTK-1 sequence SEQ ID NO:3 were aligned using the CLUSTAL-W 5 pairwise alignment program of MacVectorTM software, with the results shown in FIG. 4.
- the proteins have an overall amino acid sequence identity of 20%, and have several regions of closer sequence identity, particularly in the conserved PI and P2 domains, which share about 45% identity (10/22 identical residues) and 56% identity (14/25 identical residues), respectively.
- the substantially purified polypeptides of the invention includes a polypeptide comprising 0 an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90% or 95% identity to the sequence identified as SEQ ID NO: 2 or to the sequence identified as SEQ ID NO: 6.
- TWIK family 2PD polypeptides of the invention are composites of the KT4 and KT5 sequences SEQ ID NO: 2 and SEQ ID NO: 6. 5
- composite is meant that the consensus sequences formed when the two sequences are aligned using one or more of the various sequence alignment programs known in the art.
- An exemplary alignment of KT4 and KT5 is shown in FIGS. 5 A and 5B herein. It was produced using the CLUSTAL-W alignment program of MacVectorTM software (ver. 6.01; Oxford Molecular Ltd, Oxford, UK) using the default pairwise parameters.
- positions 4 and 5 are identical between KT4 and KT5, and are indicated as "Match” in the upper line of each comparison row.
- positions 4 and KT5 are conserved or “fixed” in the consensus composite sequence and do not vary.
- Other positions, such as position 6 (alanine, A, in KT4; proline, P, in KT5) are different, and the composite composition allows variation between the two selections A or P in this position.
- positions 4-6 5 shown in FIG. 5 A the composite sequence would read: ...RG(A or P)...
- Similar comparisons and composites are readily determined for the remainder of the sequence and composite sequences are readily formed from such comparison.
- test sequence falls within such KT4/KT5 composite sequence by comparing such test sequence falls within the conserved and variable residues along the length of the o composite sequence, as described above. Such comparison can be conveniently made, for example, with the aid of a sequence analysis program, such as the CLUSTAL-W program refe ⁇ ed to above.
- the polypeptide may be a recombinant protein, a natural protein or a synthetic protein, preferably a recombinant protein.
- the polypeptide may be in mature and/or modified form, also as defined above.
- fragments derived from KT4 or KT5 or composites thereof, 5 which are capable of interacting with other polypeptides, proteins, or other molecules, such interaction which alters the functional properties or the cellular/subcellular localization of the TWIK family channel protein.
- the invention also includes substantially purified potassium channel proteins which contain at least one KT4 or one KT5 subunit.
- polypeptide sequence variations may include those that are considered conserved and non-conserved substitutions with reference to KT4 or KT5, as defined above.
- a protein with a sequence having at least 80% sequence identity with the protein identified as SEQ ID 5 NO:2 (313 amino acids) contains up to 62 amino acid substitutions when optimally aligned as defined above.
- the protein contains a sequence substantially identical to SEQ ID NO:2.
- KT4 may be (i) a polypeptide in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) a polypeptide in which one or more of 0 the amino acid residues includes a substituent group, or (iii) a polypeptide in which the KT4 is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol (PEG)), or (iv) a polypeptide in which additional amino acids are fused to KT4, or (v) an isolated fragment of the polypeptide.
- PEG polyethylene glycol
- polypeptide with a sequence having at least 80% sequence identity with the polypeptide identified as SEQ ID NO: 6 (499 amino acids) contains up to 99 amino acid substitutions when optimally aligned as defined above.
- the polypeptide has a sequence substantially identical (97-100% identical) to SEQ ID NO: 6.
- KT5 o may therefore be considered (i) a polypeptide in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) a polypeptide in which one or more of the amino acid residues includes a substituent group, or (iii) a polypeptide in which the KT5 is fused with another compound, such as a compound to increase the half-life of the polypeptide (for 5 example, polyethylene glycol (PEG)), or (iv) a polypeptide in which additional amino acids are fused to KT5, or (v) an isolated fragment of the polypeptide.
- PEG polyethylene glycol
- the polypeptide may also be obtained by isolation from natural sources, e.g., by affinity purification using the antibodies described in the section below.
- fragment or fragments corresponding to extracellular regions and/or the mtracellular regions of KT4 or KT5 may be cleaved from the membrane-bound regions using limited proteolysis techniques known to those of skill in the art.
- the amino acid sequence of a fragment so obtained may be used to design nucleotide coding sequence for recombinant production of the fragment.
- polypeptides of the invention has uses in (1) therapeutic treatment methods and (2) drug screening.
- the sections below provide guidance for such methods.
- Polypeptide of the invention are generally useful in treating diseases and disorders associated with ion channel dysfunction, including, for example, renal, musculoskeletal, and proliferative diseases. These include, but are not limited to, renal failure, nephrosis, cirrhosis, dysphagia, and gastritis; myotonias and muscular distrophy; atherosclerosis and cancers.
- KT5 may be present as a monomer, may self-associate to form a homomeric KT5 channel protein, or may associate with other potassium channel polypeptides, including KT4, to form heteromeric KT5 channel proteins.
- a polypeptide fragment preferably a soluble fragment which binds an agonist of the particular channel, may be employed to inhibit activity of the channel by binding an agonist which is necessary for channel activity, in effect competing with the channel for agonist.
- an mtracellular fragment of a channel polypeptide such as an intracellular fragment of KT5, preferably a soluble fragment, may be used to block the interaction of intracellular effector molecules with an intracellular domain of the KT5 channel, thus preventing the cellular response induced by the interaction of the intracellular effector molecule with the channel.
- agonists and antagonists of TWIK family such as an intracellular fragment of KT5
- 2PD channels may also be selected using one or more screening assays, as described in the section that follows.
- screening assays can be carried out using high throughput screening techniques, in order to screen large numbers of compounds.
- Exemplary "libraries" of compounds may include, but are not limited to, a phage display library. See, e.g., Devlin, W0 91/18980; Key, B.K., et al, eds., Phage Display of Peptides and Proteins, A Laboratory Manual, Academic Press, San Diego, C A, 1996. Phage display is a powerful technology that allows one to use phage genetics to select and amplify peptides or
- the peptide substrate library containing 10 8 different sequences is fused to a protein (such as a gene III protein) expressed on the surface of the phage and a sequence that can be used for binding, such as biotin.
- the phage are digested with protease, and undigested phage are removed by binding to appropriate immobilized binding protein, such as streptavidin. This selection is repeated until a population of phage encoding substrate peptide sequences is recovered.
- the DNA in the phage is sequenced to yield the substrate sequences. These substrates are then used for further development of peptidomimetics, particularly peptidomimetics having inhibitory properties.
- Combinatorial libraries and other compounds are initially screened for suitability by determining their capacity to bind to, or preferably, to enhance or inhibit potassium channel activity in any of the assays described herein or otherwise known in the art. Compounds identified by such screens are then further analyzed for potency in such assays. Antagonist or agonist compounds can then be tested for prophylactic and therapeutic efficacy in appropriate in vitro and in vivo animal models of disease, such as the epilepsy model described herein, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art.
- test compound has an acceptable toxicity profile, e.g., in a variety of in vitro cells and animal model(s). It may also be useful to search the tested and identified compound(s) against existing compound databases to determine whether the compound or analogs thereof have been previously employed for pharmaceutical purposes, and if so, optimal routes of administration and dose ranges.
- routes of administration and dosage ranges can be determined empirically, using methods well known in the art (see, e.g., Benet, L.Z., et al. Pharmacokinetics in Goodman & Gilman 's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman, J.G., et al, Eds., McGraw-Hill, New York, 1966) applied to standard animal models, such as the transgenic PDAPP animal model (e.g., Games, D., et al. Nature 373: 523-527, 1995; Johnson- Wood, K, et al., Proc. Natl. Acad. Sci. USA 94: 1550-1555, 1997).
- a library of near-neighbor analogs to search for analogs with greater specificity and/or activity.
- Methods for synthesizing near-neighbor and/or targeted compound libraries are well-known in the combinatorial library field.
- the practitioner is also provided ample guidance for further refinement of the ligand binding site of the channel, for example, by crystallizing the purified channel in accord with the methods provide herein. Noting the success in this area that has been enjoyed in the area of HIV protease inhibitor development, it is contemplated that such efforts will lead to further optimization of test compounds.
- the present invention includes potassium channel modulatory agents discovered by any of the methods described herein, particularly the inhibitor assays and the 0 crystallization/optimization protocols. Such agents are therapeutic candidates for treatment of diseases associated with abnormal potassium conductance, such as epilepsy.
- potassium channel antagonist compounds e.g., 4-aminopyridine
- agents which enhance conductivity through potassium channels of the present invention particularly of KT4 channels found in the brain, are considered therapeutic candidates.
- Potassium channel agonist or antagonist compositions may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue.
- Peptide-based therapeutic compositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones.
- Therapeutic o compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means.
- Compositions may also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
- a polypeptide may be given topically to the skin or epithelial linings of body 5 cavities, for infections in such regions.
- Examples of treatable body cavities include the vagina, the rectum and the urethra.
- the composition would be formulated into suppository form for administration to these areas.
- the composition can also be given via intravenous or intraperitoneal injection.
- the composition may be injected to other localized regions of the body.
- polypeptide compositions may also be administered via nasal insufflation. o Enteral administration is also possible.
- the polypeptide should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
- polypeptides be formulated into an appropriate carrier, including ointments, gels, suppositories.
- Appropriate 5 formulations are well known to persons skilled in the art.
- Dosage of therapeutic compositions will vary, depending upon the potency and therapeutic index of the particular polypeptide selected. These parameters are easily determinable by the skilled practitioner. As an example, if the composition inhibits potassium channel activity in vitro at a given concentration, the practitioner will know that the final desired therapeutic concentration will be this range, calculated on the basis of expected biodist ⁇ bution. An approp ⁇ ate target concentration is m the ng/kg to low mg/kg range, e.g., 50 ng/kg to 1 mg/kg body weight, for IV administration.
- a therapeutic composition for use m the treatment method can include the polypeptide in a sterile mjectable solution, the polypeptide m an oral delivery vehicle, or the polypeptide in a nebulized form, all prepared according to well known methods.
- Such compositions comp ⁇ se a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
- a carrier includes but is not limited to salme, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof.
- the present invention also includes an assay for identifying molecules, such as synthetic drugs, antibodies, peptides, or other molecules, which have a modulating effect on the activity of TWIK family potassium channels, e.g. agonists or antagonists of the KT5 channel which comprises the KT5 polypeptide of the present invention.
- Such an assay comprises the steps of providing a functional KT4 or KT5 potassium channel comp ⁇ smg polypeptides encoded by the polynucleotides of the present invention, contacting the channel with one or more molecules to determine its modulating effect on the activity of the channel, and selecting from the molecules a candidate molecule capable of modulating KT5 channel activity.
- Such compounds are useful in the treatment of disease conditions associated with activation or depression of KT5 channel activity.
- such assays can be carried out with KT4 or KT4/KT5 composite channels.
- KT4, KT5, or the hgand-bmdmg, catalytic, lmmunogemc fragments, or ohgopeptides thereof can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
- the protein employed m such a test may be membrane-bound, free in solution, affixed to a solid support, borne on a cell surface, or located lntracellularly.
- the formation of binding complexes between a TWIK family 2PD polypeptide channel and the agent being tested may be measured.
- Compounds that inhibit binding between a channel subunit and its agonists may also be measured.
- the screening system includes recombmantly expressed KT4 or KT5, and the compounds screened are tested for their ability to block (inhibit) or enhance (activate) the potassium current activity of the KT4 or KT5 channel.
- a functional screening assay mammalian cell lines or Xenopus oocytes which lack both KT4 and KT5 are used to express KT4 or KT5.
- the polypeptide subunit may be expressed individually or together with other potassium channel subunit polypeptides.
- Compounds are screened for their relative effectiveness as channel activators or inhibitors by comparing the relative channel occupancy to the extent of hgand-mduced activation or inhibition of potassium ion conductance.
- Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the KT5 channel is desc ⁇ bed in detail by Geysen in PCT Application WO 84/03564, published on Sep. 13, 1984.
- large numbers of different small peptide test compounds are synthesized on a solid substrate, such as 5 plastic pms or some other surface.
- the peptide test compounds are reacted with the channel KT4 or KT5 polypeptide subunit (for example, either a soluble extracellular fragment of KT4 or KT5, or intact subunit solubihzed in detergents or in lipid vesicles), and washed. Bound subunit is then detected by methods well known in the art.
- Substantially purified KT4 of KT5 can also be coated directly onto plates for use in the aforementioned drug screening techniques.
- non- 0 neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
- Antibodies to KT4 or KT5, as described in Section IV. below may also be used in screening assays according to methods well known m the art. For example, a "sandwich" assay may be performed, in which an anti- KT4 or KT5 antibody is affixed to a solid surface such as a microtiter plate and solubihzed KT4 or KT5 channel polypeptide is added, as approp ⁇ ate. Such an 5 assay can be used to capture compounds which bind to the channel. Alternatively, such an assay may be used to measure the ability of compounds to interfere with the binding of a ligand, such as an agonist, to the potassium channel.
- polypeptide subunit is o crystallized in order to carry out structural determinations useful m defining the conformation and size of the substrate binding site. This information can be used in the design and modeling of hgands of the channel, such as agonist or antagonist hgands.
- Exemplary buffers and precipitants forming an empirical g ⁇ d for determining crystallization conditions are available.
- the "Crystal Screen" kit (Hampton Research) provides a sparse matrix method of tnal conditions that is biased and selected from known o crystallization conditions for macromolecules. This provides a “gnd” for quickly testing wide ranges of pH, salts, and precipitants using a very small sample (50 to 100 microhters) of macromolecule.
- 1 ⁇ l of buffer/prec ⁇ p ⁇ tant(s) solution is added to an equal volume of dialyzed protein solution, and the mixtures are allowed to sit for at least two days to two weeks, with careful monito ⁇ ng of crystallization.
- Chemicals can be obtained from common commercial 5 suppliers; however, it is preferable to use punty grades suitable for crystallization studies, such as are supplied by Hampton Research (Laguna Niguel, CA).
- Common buffers include Citrate, TEA, CHES, Acetate, ADA and the like (to provide a range of pH optima), typically at a concentration of about 100 mM.
- Typical precipitants include (NH 4 ) 2 S0 4 , MgS0 4 , NaCl, MPD, Ethanol, polyethylene glycol of various sizes, isopropanol, KC1; and the like (Ducruix).
- Antibodies 5 In still another aspect of the invention, purified polypeptide is used to produce antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of KT4 and KT5.
- Antibodies to KT4 or KT5 may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single 0 chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which block ligand binding, are especially preferred for therapeutic use.
- KT4 or KT5 for antibody induction does not require biological activity; however, the polypeptide fragment or oligopeptide must be antigenic.
- Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least ten amino acids, preferably at least 20 5 amino acids. Preferably they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of a KT4 or a KT5 polypeptide may be fused with another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to KT4 or KT5.
- various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with KT5 or any portion, fragment or oligopeptide which retains immunogenic properties.
- various adjuvants may be used to increase immunological response.
- adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, 5 polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
- BCG Bacilli
- Monoclonal antibodies to KT4 or KT5 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975; o Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
- Antibody fragments which contain specific binding sites for KT5 may also be generated.
- such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
- Fab expression 0 libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al. (1989) Science 256:1275-1281).
- Antibodies which specifically bind KT4 or KT5 are useful for the diagnosis of conditions or diseases characterized by expression of KT4 or KT5.
- such antibodies may be used in assays to monitor patients being treated with KT4, KT5, or agonists or antagonists thereof.
- Diagnostic assays for the channel protein include methods utilizing the antibody and a label to detect KT 4 or KT5 or fragments thereof in extracts of cells, tissues, or biological fluids such as 5 sera.
- the proteins and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known in the art.
- a variety of protocols for measuring KT5, using either polyclonal or monoclonal o antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on KT4 or KT5 is prefe ⁇ ed, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al. 5 (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of KT5 expression.
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescent activated cell sorting
- Normal or standard values for channel polypeptide expression are established by combining cell extracts taken from normal subjects, preferably human, with antibody to KT5 under conditions suitable for complex formation which are well known in the art.
- the amount of standard complex formation may be quantified by various methods, preferably by photometric methods.
- standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
- the antibody assays are useful to determine the level of KT4 or KT5 present in a particular tissue, e.g., biopsied tumor tissue or neuronal tissue, as an indication of whether the channel polypeptide is being overexpressed or underexpressed in the tissue, or as an indication of how such channel levels levels are responding to drug treatment.
- tissue e.g., biopsied tumor tissue or neuronal tissue
- therapeutic value may be achieved by admmiste ⁇ ng an antibody specific against KT5, to inhibit, for example, binding of an agonist to the KT5 channel, or to block the ion pore.
- the antibody employed is preferably a humanized monoclonal antibody, or a human Mab produced by known globulm-gene library methods
- the antibody is administered typically as a sterile solution by IV injection, although other parenteral routes may be suitable.
- the antibody is administered m an amount between about 1-15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1-7 days, until a therapeutic improvement is seen. o From the foregoing, it can be seen how va ⁇ ous objects and features of the invention are met.
- Example 1 Identification of TWIK Family Potassium Channel Nucleic Acid Sequences 5 This example provides guidance for isolation of additional TWIK family 2PD polynucleotides, based on the exemplary KT4 and KT5 channel family members descnbed herein.
- cDNA from a human bram cDNA library was mixed with 50-80 ng biotmylated oligonucleotide, 50 ng of each clamp ohgo, and 1 ul of IN NaOH in a total volume of 10 ul.
- the oligonucleotide sequences were derived from human EST sequence AA604914 (SEQ ID NO:4). After the mixture was incubated at RT for 15-20 mm, 40 ul of neutralization solution (0.12 M T ⁇ s, pH 7, 2x SSPE, 0.1% Tween 20) was added, and further incubated at 37 - 42°C.
- Filters were screened using a labeled hyb ⁇ dization probe based on the above EST sequence.
- the filters were prehyb ⁇ dized without probe in the prehyb ⁇ dization solution (5x SSPE, 5xDenhardt's solution, 0.1% SDS) at 45-50 °C for 1 hour, and then hybridized with probe overnight.
- the filters were then washed twice for 20 minutes each at room temperature in 2x SSPE, 0.1% SDS, and twice for 20 minutes each at 45-50 °C in 2x SSPE, 0.1% SDS. Signals were detected by a few hours of exposure of the filters to X-ray film.
- Plasmid DNA was isolated from the positive cultures using a Qiagen mmiprep kit (Qiagen, Santa Cla ⁇ ta, CA) and sequenced, resulting m the identification of nucleic acid sequences of human KT4 potassium channel.
- the DNA sequence is provided herein as SEQ ID No: 1 (human KT4), and translated ammo acid sequence is given as SEQ ID NO: 2.
- KT5 Coding Sequences Similar methods were employed for isolation of KT5 coding sequences. Briefly, 10 to 20 ug cDNA from a human bram cDNA library was mixed with 50-80 ng biotmylated oligonucleotide, 50 ng of each clamp ohgo, and 1 ul of IN NaOH in a total volume of 10 ul. The oligonucleotide sequences were derived from human EST sequence AA533124 (SEQ ID NO: 7).
- the eluted plasmid cDNA was then transform into E coli cells and transformants were plated on one or more 15-cm dishes and bacte ⁇ al colonies were lifted onto Hybond N filters, as above. Filters were screened using a labeled hyb ⁇ dization probe based on the above EST sequence, as desc ⁇ bed in Part A, above. Positive colonies were subjected to secondary and tertiary screenings. Positive colonies from tertiary screening were cultured. Plasmid DNA was isolated from the positive cultures and sequenced as above, resulting in the identification of nucleic acid sequences of human KT5 potassium channel. The DNA sequence is provided herein as SEQ ID NO: 5 (human KT5), and translated ammo acid sequence is given as SEQ ID NO: 6.
- Northern Analysis Multiple tissue Northern blots were purchased from Clontech. High Efficiency Hyb ⁇ dization System (HS-114) was purchased from Molecular Research Center (Cincinnati, Ohio). B ⁇ efly, the blot was first soaked in prehyb ⁇ dization solution (1% SDS and 0 1M NaCl) for 30 mm at room temperature, and then was incubated in HS-114 solution with 100 ⁇ g/ml salmon sperm DNA in the absence of probe for a few hours 68 °C. The cDNA probe was then added and the blot was let to hybridize at 68 °C overnight.
- prehyb ⁇ dization solution 1% SDS and 0 1M NaCl
- the blot was then washed under the following conditions: twice in 2x SSC, 0.05% SDS, at room temperature; and twice in O.lx SSPE, 0.1% SDS, at 50 °C. After washing, the blot was exposed to X-ray film.
- Experiments performed as descnbed above showed that, among the tissues tested, expression of KT4 transc ⁇ pts was observed in pancreas, somewhat lower abundance in heart and placenta, and still lower abundance in liver, lung and bram. Expression of KT5 transc ⁇ pts was observed m liver, kidney, pancreas, placenta, and lung Example 3
- Potassium currents are measured by the patch clamp method in the whole cell configuration (Hamill, et al., 1981). Electrode resistances ranging from 2-6 M ⁇ are approp ⁇ ate. Recordings can be made with either an Axopatch 1C or Axopatch 200A amplifier (Axon Instruments, Foster City,
- Potassium currents are recorded utilizing an external bath solution consisting of (in mM): 140 sodium chloride, 5 potassium chloride, 10 HEPES, 2 calcium chlo ⁇ de, 1 magnesium chloride, and 12 glucose, adjusted to pH 7.4 with sodium hydroxide and 305 mOsM.
- the internal pipette solution consists of (in mM): 15 sodium chlo ⁇ de, 125 potassium methanesulphonate, 10 HEPES, 11 EGTA, 1 calcium chloride, 2 magnesium chlo ⁇ de and 59 glucose, adjusted to pH 7.4 with potassium hydroxide and 295 mOsM.
- cells are placed in a flow through chamber (0.5-1 ml/min).
- mice (-21 days old; approx. 7-10 g) are obtained from Jackson Laboratories, Bar Harbor, Maine, and are housed for a minimum of three days to acclimate them to laboratory conditions.
- mice are injected i.c.v. into the lateral ventricle with vehicle or test compound (total volume: 5 ⁇ l) according to standard methods (Jackson and Scheideler, 1996) 30 minutes prior to exposure to sound stimulus.
- vehicle or test compound total volume: 5 ⁇ l
- the mice are individually housed in observation chambers and are observed over the following 30 min. for evidence of shaking behavior (persistent whole body shakes) or any other abnormal behaviors.
- mice are exposed to a high intensity sound stimulus (100-110 dB sinusoidal tone at 14 Hz for 30 s). Mice are observed for the presence of clonic and tonic seizures with full hindlimb extension during the 30 s exposure to the sound. Test compounds are evaluated for ability to inhibit duration and/or intensity of such seizures or to prolong latency to onset of seizures.
Abstract
Description
Claims
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WO2005052576A1 (en) * | 2003-11-27 | 2005-06-09 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with potassium channel, subfamily k, member 6 (kcnk6) |
US9427441B2 (en) | 2014-02-19 | 2016-08-30 | Indiana University Research And Technology Corporation | Targeting primary cilia to treat glaucoma |
WO2023283646A1 (en) * | 2021-07-08 | 2023-01-12 | Baylor College Of Medicine | Recombinant microorganism-based methods and compositions for treatment of disease |
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US5599673A (en) * | 1995-03-09 | 1997-02-04 | University Of Utah Research Foundation | Long QT syndrome genes |
EP0930364A1 (en) * | 1998-01-16 | 1999-07-21 | Synthelabo | TWIK-1 related two domains potassium channel |
WO2000052164A2 (en) * | 1999-03-01 | 2000-09-08 | Millennium Pharmaceuticals, Inc. | Potassium channel molecules and uses therefor |
-
1999
- 1999-07-20 WO PCT/US1999/016471 patent/WO2000003687A2/en not_active Application Discontinuation
- 1999-07-20 AU AU57702/99A patent/AU5770299A/en not_active Abandoned
- 1999-07-20 EP EP99944996A patent/EP1097166A4/en not_active Withdrawn
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US5599673A (en) * | 1995-03-09 | 1997-02-04 | University Of Utah Research Foundation | Long QT syndrome genes |
EP0930364A1 (en) * | 1998-01-16 | 1999-07-21 | Synthelabo | TWIK-1 related two domains potassium channel |
WO2000052164A2 (en) * | 1999-03-01 | 2000-09-08 | Millennium Pharmaceuticals, Inc. | Potassium channel molecules and uses therefor |
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LESAGE FLORIAN ET AL: "TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure." EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, vol. 15, no. 5, 1996, pages 1004-1011, XP002017569 ISSN: 0261-4189 * |
REYES R ET AL: "CLONING AND EXPRESSION OF A NOVEL PH-SENSITIVE TWO PORE DOMAIN K+ CHANNEL FROM HUMAN KIDNEY" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 273, no. 47, 20 November 1998 (1998-11-20), pages 30863-30869, XP000919229 ISSN: 0021-9258 * |
See also references of WO0003687A2 * |
Also Published As
Publication number | Publication date |
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EP1097166A4 (en) | 2003-01-02 |
WO2000003687A3 (en) | 2000-04-20 |
WO2000003687A2 (en) | 2000-01-27 |
CA2337835A1 (en) | 2000-01-27 |
AU5770299A (en) | 2000-02-07 |
JP2002520039A (en) | 2002-07-09 |
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