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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• This invention relates generally to a new family of potassium channels. More particularly, the present invention relates to the cloning and characterization of a family of distinct trans-membrane potassium ion channels, characterization of such channels, newly identified polynucleotide sequences, polypeptides encoded by such sequences, expression vectors capable of heterologous expression of such polynucleotide sequences, transformed host cells containing the expression vectors and assay methods for determining the expression of heterologous nucleotide sequences encoding all or a portion of said potassium channels in host cells, chromosome mapping, diagnostic methodologies and kits therefor.
• Genes encoding potassium channels representative of this family were cloned from Drosophila melanogaster, Caenorhabditis elegans, human and mouse ESTs, and human brain, heart, and kidney cDNA libraries. More particularly, the invention arises in part from the determination that the DNA sequences of these genes encode a structurally distinct potassium channel whose molecular architecture is characterized by four membrane spanning domains and two putative pore forming domains.
• Ion channels which include sodium (Na + ), potassium (K + ), and calcium (Ca ++ ), are present in both eukaryotic and prokaryotic cells and control a variety of physiological and pharmacological processes.
• Potassium channels comprise a large and diverse group of integral membrane proteins that are involved in the movement of potassium into and out of the cell. Such channels regulate the level of excitability and repolarization properties of neurons and muscle fibers (B. Hille, Ionic Channels of Excitable Membranes, 2d Ed., Sinauer, Sunderland, MA (1992)), and are implicated in a broad spectrum of processes in both excitable and non-excitable cells.
• K + channels play a role in determining the resting electrical membrane potential by setting the membrane permeability to K + ions.
• Potassium currents have been shown to be more diverse than sodium or calcium currents and play a role in determining the way a cell responds to external stimuli.
• K + channels Several classes of K + channels have been identified based on their pharmacological and electrophysiological properties. These include voltage-gated, ATP-sensitive, muscarinic-activated, S type, SK Ca ++ -activated, Na + -activated, and inward and/or outward rectifier types of K + channels. Prior to this work, and on the basis of membrane-spanning segments, potassium channels may be subdivided into topologically distinct classes.
• one well-known class of voltage-gated, calcium activated, and/or cyclic nucleotide-gated-channels is composed of six membrane scanning domains (S I -S6) one of which contains repeated positive charges presumed to be involved in the voltage sensing of these channels and hence in their functional outward rectification and a single pore forming domain (H5 or P region).
• a second class may be described as an inward rectifying potassium channel that passes tlirough the cellular membrane twice and also contains a single pore forming region (Y. Kubo, E. Reuveny, P.A. Slesinger, Y.N. Jan, LN. Jan, Nature 364:802-806 (1993); Y. Kubo, T.J. Baldwin, Y. ⁇ . Jan, LN. Jan, Nature 362:127-133 (1993); see also American Cyanamid copending U.S. patent application # 08/431,928 filed on 6/28/1995 for a description of "HIRK").
• K + channels are the voltage-gated outward rectifying channels (the K y family), the prototype being the protein which is coded for by the Shaker gene seen in Drosophila melanogaster, which is a voltage-gated channel.
• the proteins in this gene family contain a structural motif characterized by six membrane spanning segments (S1-S6), a putative voltage sensor (S4), and an S5- S6 linker (H5 or P region) involved in ion conductance.
• S1-S6 membrane spanning segments
• S4 putative voltage sensor
• H5 or P region S5- S6 linker
• K ir family Another well characterized class of potassium channel proteins, the inward rectifier potassium channels (K ir family) play a significant role in maintaining the resting potential of, and in controlling the excitability of a cell.
• These channels are characterized by two transmembrane domains and a pore-forming region and the lack of an S4 or voltage sensing region.
• Inward rectifying K + channels are generally characterized by two transmembrane domains and one pore-forming domain. The pore-forming domain is common to both groups of K + channels, the voltage-gated outward rectifier groups and the inward rectifying K + channels and is an essential element of the aqueous K + -selective pore.
• a functional channel is assembled in the membrane via the association of four K ir subunits, necessitating the presence of four P domains.
• a potassium channel from Saccharomyces cerevisiae, designated Tokl, (Ketchum et al, Nature 376:690-695 (1995)) or YORK (Lesage et al, J. Biol. Chem 271:4183-4187 (1996)) has recently been identified and is characterized by the presence of two pore (2P) domains and an outward rectifying K + -selective current which is coupled to potassium equilibrium (Ketchum et al, Nature 376:690-695 (1995)).
• the yeast channel comprises eight transmembrane domains, such domains resembling an assembly of an inward rectifying K + channel of the K ir family (two transmembrane domains) with an outward rectifying channel of the K ⁇ , family (six transmembrane domains).
• a first aspect of the present invention is the discovery of a new family of potassium channel genes and proteins encoded thereby.
• Potassium channels belonging to this new family comprise four hydrophobic domains capable of forming transmembrane helices, wherein a first pore-forming domain is interposed between the first and second transmembrane helices and a second pore-forming domain is interposed between the third and fourth transmembrane helices, and the channels fiirther contain various potassium selective peptide motifs.
• the channels contain a GXG motif in the first pore-forming region and preferably in both pore-forming regions, wherein X is an amino acid selected from the group consisting of Y, F, V, I, M, and L, and particularly L or I.
• the channels preferably contain a further peptide motif in the P ! and/or P 2 pore-forming regions, spanning several amino acids upstream of GXG, and particularly for about six (6) amino acids upstream of the first G.
• the preferred pore-forming region motif is XXXXXXGXG (SEQ ID NO:65), where X at positions 1, 4, and 5 are preferably the amino acids residues T or S, and X at position 6 is preferably I or V, and X at position 8 is an amino acid selected from the group consisting of Y, F, V, I, M, and L, again, with the amino acid residues L or I particularly preferred.
• the channels display yet a second peptide motif, XXXXGXPX (SEQ ID NO:66), wherein X at position 1 is the amino acid residue Y or F, and preferably Y, and X at positions 2, 3, 4, and 6 are amino acid residues, wherein residues at position 2 are A, S, or G, with A or S preferred, and X at positions 3, 4, 6, and 8 are the amino acid residues M, I, V, L, F, or Y, with L or I particularly preferred.
• this motif is "YALLGIP" (SEQ ID NO:67).
• This second peptide motif is located downstream of P t generally about 12-25 amino acids downstream, and preferably about 16 amino acids downstream of P ⁇
• the isolation and characterization of invertebrate (i.e. insect and nematode) potassium channel genes belonging to this new family is presented.
• the present invention further provides the isolation and characterization of polynucleotides from invertebrates and vertebrates, which encode amino acid sequence elements unique to this potassium gene family and specifically sourced from Drosophila melanogaster, Caenorhabditis elegans, avian libraries, murine and various other mammalian libraries, and libraries from all human tissues including human heart and brain.
• the aforementioned channels are mutated so as to confer improved inward potassium flux under acidic conditions.
• these mutations cluster around the second pore-forming domain.
• the mutations may arise at one or more of amino acid positions 256, 270, 272, and 274.
• Such mutations should preferably confer upon selected yeast host cells containing heterologous potassium channel expression plasmids the ability to grow on low pH, low potassium concentration medium.
• Such yeast host cells are unable to grow in medium containing low potassium concentration in the absence of expression of a heterologous potassium channel (CY 162 for example, see J.A. Anderson et al., Proc. Natl. Acad Aci. USA 89:3736-3740 (1992).
• Potassium channels of any type may be used, with TPCK1 being particularly preferred.
• FIGURE 1 Growth ofCY162 cells bearing pDmORFl.
• CY162 cells transformed with plasmids isolated from survivors of a primary library screen for plasmids that support the growth of CY162 on medium contain low potassium concentration.
• Six individual transformants of each plasmid-bearing strain are cultured in patches on the indicated medium.
• CY 162 cells bearing pDmORFl are found in the upper left-hand corner of each plate while pKATl containing cells are found in the lower right hand corner.
• FIGURE 2 A and 2B DNA sequence and deduced amino acid sequence of Dm ORFl (SEQ ID NOS:l and 2).
• the nucleotide sequence of the 2.4 kb cDNA revealed a single long open reading frame proximal to the GAL1 promoter. Segments corresponding to putative transmembrane (M1-M4) and pore-forming H5 domains in the predicted polypeptide are underlined. The single amino-terminal asparagine linked glycosylation site is indicated by a G.
• FIGURE 3 A and 3B DNA sequence and deduced amino acid sequence of the F22b7.7 segment of the Caenorhabditis elegans genome (SEQ ID NO:3 and SEQ ID NO:4, respectively). Segments corresponding to putative transmembrane (M1-M4) and pore-forming H5 domains in the predicted polypeptide are underlined.
• FIGURE 4 Alignment of DmORFl and F22b7.7 sequences.
• Protein-coding regions of DmORFl SEQ ID NO:37
• F22b7.7 SEQ ID NO:38
• CeORF- 1 in this FIGURE
• FIGURE 5 A Comparison of the pore-forming domains of DmORFl and F22b7.7. Amino acid sequences from the six cloned Drosophila melanogaster potassium channels and three inward rectifier channels (SEQ ID NOS:7 through 21) are compared to DmORFl and F22b7.7 within the pore-forming H5 regions. Amino acid identities are indicated by a vertical line and conserved substitutions indicated by a dot. Amino acid substitutions deemed acceptable are indicated.
• FIGURE 5B Hydropathy plot analysis of the DmORFl and F22b7.7 polypeptide sequence.
• FIGURE 6 Predicted membrane spanning topology of DmORFl.
• FIGURE 7 Heterologous potassium channel-dependent growth of plasmid bearing CY162 (trk ⁇ ) strains.
• CY162 bearing pYES2, pKATl, pDmORFl, and pRATRAK are cultured at 30° C for four days on arginine phosphate agar medium containing 0 mM, 0.2 mM, or 100 mM added KC1.
• FIGURE 8 Inhibition of growth of yeast cells containing heterologous potassium channels.
• CY162 cells (10 5 ) bearing the indicated plasmids are plated in arginine phosphate agar medium containing 0.2 mM potassium chloride.
• Sterile filter disks were placed on the surface of the agar and saturated with 20 ⁇ l of a 1 M solution of potassium channel blocking compound.
• Clockwise from upper left-hand comer is BaCl 2 , CsCl, TEA, and RbCl.
• KC1 is applied to the center disk.
• FIGURE 9 A and 9B DNA sequence and deduced amino acid sequence of CORK (SEQ ID NO:36 and SEQ ID NO:74).
• the nucleotide sequence of the 1.4 kb cDNA revealed a single long open reading frame proximal to the GALl promoter. Segments corresponding to pore-forming H5 domains in the predicted polypeptide are underlined. Asparagine-linked glycosylation sites are indicated by a G.
• Figure 10 Depicts a schematic representation of a preferred motif of the potassium channels of the invention.
• FIGS 11 A- 11 D Depicts a biophysical analysis of TPKCl expressed inXenopus laevis oocytes.
• TPKCl currents mXenopus oocytes injected with TPKCl cRNA were measured by two-electrode clamp. Displayed are current traces measured at voltages adjusted stepwise from the -90 mN resting potential and the corresponding translation to an I/V Plot of current- voltage relationship. Additionally, current- voltage relationships for currents measured in ⁇ D96 containing 2, 5, 10, 50, or 96 mM KC1 are depicted. Also, the figures indicate that TPKCl confers potassium selective currents. Finally, current-voltage relationship for currents measured in the presence of 0.5 mM and 1 mM BaCl 2 are depicted.
• Ade Ade; A-Adenine G-Guanine Ura; U-Uracil
• Amino acid residues are abbreviated herein to either three letters or a single letter as follows:
• mammalian refers to any mammalian species (e.g., human, mouse, rat, and monkey).
• heterologous refers to nucleotide sequences, proteins, and other materials originating from organisms other than the host organism used in the expression of the potassium channels or portions thereof, or described herein (e.g., mammalian, avian, amphibian, insect, plant), or combinations thereof not naturally found in the host organism.
• upstream and downstream are used herein to refer to the direction of transcription and translation, with a sequence being transcribed or translated prior to another sequence being referred to as “upstream” of the latter.
• channel and the nucleotide sequences encoding same, is intended to encompass all potassium channels, and mutants, derivatives, homologs, and other variations thereof.
• EST refers to an expressed sequence tag.
• Potassium channels belonging to this family may be derived from a wide variety of animal species, both vertebrate and invertebrate. This family is structurally and functionally novel, as manifested by the presence of two-pore forming domains (2P) in conjunction with a four membrane spanning domain configuration.
• 2P two-pore forming domains
• Nucleotide sequences encoding various representative members of this new family of two-pore K + channels were cloned by expression in yeast cells from Drosophila melanogaster (dORK or DmORF), and also by degenerate PCR from human brain, heart, and kidney cDNA (TPKCl), and from human and mouse ESTs. Preliminary analyses of expression by a Northern blotting procedure indicates that TPKCl is present primarily in human brain. Genes encoding structural homologues are present in the genome of Drosophila melanogaster (dORK), Caenorhabditis elegans (cORK), avian tissue, and various mammalian tissue such as human (TPKCl) and murine.
• dORK Drosophila melanogaster
• cORK Caenorhabditis elegans
• avian tissue avian tissue
• various mammalian tissue such as human (TPKCl) and murine.
• the potassium channel family of the present invention may be structurally characterized in that the potassium channels have four hydrophobic domains capable of forming transmembrane helices. These channels are fiirther characterized in that they comprise two pore-forming domains, one of which is interposed between the first helix and the second helix, and the other of which is interposed between the third helix and the fourth helix. " While the present inventor does not wish to be bound by theory, it is hypothesized that the 2P channels organize as dimers in the plasma membrane, consistent with a requirement for four (4P) domains to form a functional channel.
• the pore-forming domains further contain a potassium selective motif, which serves to confer upon the channel the ability to pass potassium ions to the exclusion of other ions, such as sodium calcium, and the like.
• this motif contains the peptide Y/G, and particularly in either a dipeptide or tripeptide motif, and frequently with Y/F-G bonding.
• the motif comprises GXG, wherein X is an amino acid selected from the group consisting of V, L, Y, F, M, and I, and preferably L or I, such motif generally being found between the first two transmembrane domains.
• a second GXG motif wherein X is an amino acid selected from the aforementioned group, is found between the third and fourth transmembrane domain as well.
• the channels preferably contain a further peptide motif in the P j and/or P 2 pore-forming regions, spanning several amino acids upstream of GXG and particularly for about six (6) amino acids upstream of the first G.
• the preferred pore-forming region motif is XXXXXXGXG (SEQ ID NO:65), where X at positions 1, 4, and 5 are preferably the amino acids residues T or S, and X at position 6 is preferably I or V, and X at positions 2, 3, and 8 is an amino acid selected from the group consisting of V, L, Y, F, M, and I, again, with the amino acid residues L or I particularly preferred.
• the potassium channels of the invention comprise a second peptide motif, which in terms of the DNA encoding it, is located downstream of the first GXG motif, and within the second transmembrane domain (see Figure 13 for a schematic depiction).
• This is the XXXXGXPX (SEQ ID NO:66) motif wherein X at position lis the amino acid residue Y or F, and preferably Y, and X is an amino acid residue wherein X at position 2 is A, S, or G, with A or S preferred, and X at positions 3, 4, 6, and 8 are the amino acid residues M V, L, F, or Y, with L or I particularly preferred.
• the preferred XXXXGXPX (SEQ ID NO: 66) motif is flanked by the first GXG motif (that is located between the first and second transmembrane domain) and is located in the second transmembrane, and a second pore-forming peptide motif is located downstream of the first pore-forming motif, between the third and fourth transmembrane domains.
• the preferred XXXXGXPX (SEQ ID NO:66) motif is located downstream of the first pore-forming peptide motif by about 12-25 amino acids. In other preferred embodiments the first pore-forming peptide motif is within about 16 amino acids.
• the topological configuration of the potassium channels of the invention is such that one may presume that a regulatory domain of indeterminate length often may be interposed between the second transmembrane domain (TM2) and the third transmembrane domain (TM3).
• TM2 second transmembrane domain
• TM3 third transmembrane domain
• XXXXGXPX comprise the amino acids YALLGXP (SEQ ID NO:68), where X at position 6 is M, I, V, L, F, or Y, and particularly "YALLGIP" (SEQ ID NO:67).
• the aforementioned channels are mutated so as to confer improved inward potassium flux under acidic conditions.
• these mutations cluster around the second pore-forming domain.
• the mutations may arise at one or more of amino acid positions 256, 270, 272, and 274.
• the mutation at amino acid position 256 can be a substitution of T for the wild type A (SEQ ID NO: 57).
• the mutation can be at position 272 alone, wherein H is substituted for the wild type Y (SEQ ID NO: 58), or that substitution can be coupled with a substitution at position 274 of V for the wild type A (SEQ ID NO:59).
• a further embodiment is a substitution at position 270 of R for the wild type G (SEQ ID NO:60).
• Such mutations should preferably confer upon selected yeast host cells containing heterologous potassium channel expression plasmids the ability to grow on low pH, low potassium concentration medium.
• the two pore potassium channels described above are mutated so as to confer improved inward potassium flux under acidic conditions.
• these mutations cluster around the second pore-forming domain at amino acids 256, 270, 272, and 274.
• the potassium channels of the present invention further comprise a glycosylation site. This site may be an amino-terminal glycosylation site and may also be asparagine-linked.
• the potassium channels of the present invention possess certain properties in common with known potassium channels including voltage-gated channels, calcium activated channels, cyclic nucleotide gated channels, inward rectifier channels, and the like, and especially with regard to electrophysiological properties.
• a hallmark of the potassium channels of the invention are that they exhibit either outward current rectification or both inward and outward current rectification, in each case affected by potassium concentration.
• Potassium channels play an essential role in determining the resting electrical membrane potential by setting the membrane permeability to K + ions.
• the cloned 2P channels confer potassium selective currents when expressed mXenopus oocytes.
• the dORK channels encode instantaneous open-pore channel activity.
• the potassium ions flow either into or out of the cell, depending on the magnitude and direction of the electrochemical driving force.
• the human 2P channel designated herein as TPKCl is functionally distinguishable from dORK in that the TPKCl channel permits potassium flow primarily in an outward direction. Even when external potassium concentration is raised to the point where the electrochemical potential will drive potassium flux into oocytes containing dORK, little inward potassium current observed in TPKCl -containing oocytes.
• the dORK and TPKCl potassium channels When expressed in yeast host cells that require heterologous potassium channel expression for survival on low potassium medium, the dORK and TPKCl potassium channels exhibit distinguishable growth promoting properties. Yeast host cells of this type containing dORK are able to grow on low potassium medium, likely as a manifestation of the ability of the dORK potassium channel to promote potassium ion flow into the yeast cell. Lacking the capacity to promote efficient inward potassium ion flux, the TPKCl channel fails to support the growth of the yeast host cells. This failure certain potassium channels to promote growth of the yeast host cells limits the usefulness of the potassium channels and the expression system for use in high-throughput screening applications.
• modified potassium channel proteins that can support the growth of the yeast host cells can be obtained by mutating their genes and phenotypically selecting for growth on low potassium and/or low pH medium, then the modified potassium channels and expression system would be more useful as a drug discovery tool.
• the invention is not limited to the specific nucleotide and amino acid sequences depicted in the Sequence Listing, but also includes sequences that hybridize to such depicted sequences. Further, the invention also encompasses modifications to the depicted sequences, such as deletions, insertions, or substitutions in the sequence which produce changes in the resulting protein molecule that are not detrimental to the protein's activity. For example alterations in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a biologically equivalent amino acid at a given site, are contemplated.
• a codon for the amino acid alanine, a hydrophobic amino acid may substituted by a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
• a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
• changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.
• assembly of 2P channel into functional dimers may require disulfide formation, and should take that into consideration when making modifications as taught herein (see e.g. , Lesage et al.
• the present invention further provides functional derivatives of the nucleotide sequences encoding the potassium channels of the invention.
• the term "functional derivative” is used to define any DNA sequence which is derived from the original DNA sequence and which still possesses at least one of the biological activities present in the parent molecule.
• a functional derivative can be an insertion, deletion, or a substitution of one or more bases in the original DNA sequence.
• Functional derivatives of the nucleotide sequences as presented herein, having an altered nucleic acid sequence can be prepared by mutagenesis of the DNA.
• preparation of functional derivatives may be achieved by random mutagenesis. Random mutagenesis allows the production of functional derivatives through the use of mutator E. coli strains (e.g., XLlRed (Stratagene)) which introduce mutations during cloning and amplification of expression plasmids. This can be accomplished using one the mutagenesis procedures known in the art.
• preparation of functional derivatives may be achieved by site-directed mutagenesis.
• Site-directed mutagenesis allows the production of functional derivatives through the use of a specific oligonucleotide which contains the desired mutated DNA sequence.
• Site-directed mutagenesis typically employs a phage vector that exists in both a single- stranded and double-stranded form.
• Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage, as disclosed by Messing et al, Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), the disclosure of which is incorporated herein by reference. These phage are commercially available and their use is generally well known to those skilled in the art.
• plasmid vectors containing a single- stranded phage origin of replication may be employed to obtain single-stranded DNA.
• the mutation per se need not be predetermined.
• random mutagenesis may be conducted at a target region and the newly generated sequences can be screened for the optimal combination of desired activity.
• Biologically equivalent refers to those modified nucleic acid and amino acid sequences in which the modified sequence at least substantially maintains the biological activity of the unmodified sequence; i.e., in the case of a nucleic acid sequence, the protein expressed therefrom at least substantially maintains the biological activity.
• the present invention also relates to the biologically equivalents of the potassium channel proteins whether specifically modified as described above or other isolated proteins.
• Biologically equivalent as used herein means protein having some homology with the TPCKl protein, wherein such protein maintains all or substantially all of the biological activity of the TPCKl protein, and contain the pore-forming peptide motif and, preferably, also the XXXXGXPX (SEQ ID NO:66) motif.
• the percentage of homology can vary from at least about 20% up to about 99.95%. Certainly percentage homologies of at least about 40%, at least about 70%, at least about 90%, or at least about 95% can be employed based on the retention of biological activity. One skilled in this art will note that forty percent (40%) homology at amino acid level is usually consistent with retention of comparable 2° and 3 ° structure amongst homologs.
• mRNA encoded by a functional derivative made by site-directed mutagenesis can be injected into an oocyte as described in the EXAMPLES and the oocyte tested for channel activity. Other target constructs may also be tested in this manner.
• any eukaryotic organism can be used as a source for a protein which is a member of the potassium channel family as described herein, or the genes encoding same, so long as the source organism naturally expresses such a protein or contains genes encoding same.
• source organism refers to the original organism from which the amino acid or DNA sequence of the protein is derived, regardless of the organism the protein is expressed in and ultimately isolated from.
• a member of the TPCKl family of channel proteins expressed in hamster cells, yeast cells, or the like is of human origin as long as the amino acid sequence is that of a human protein which is a member of this family.
• the protein is purified from tissues or cells which naturally produce the protein.
• One skilled in the art can readily follow known methods for isolating proteins in order to obtain a member of the protein family, free of natural contaminants. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immunoaffinity chromatography.
• the invention provides further methods of obtaining other members of this novel family of potassium channels, i.e., those sharing significant homology to one or more regions of the proteins described herein. Specifically, by using the sequences disclosed herein as probes or as primers, and techniques such as PCR cloning and colony/plaque hybridization, one skilled in the art can obtain other members of the family of potassium channel proteins as well as genomic sequences encoding such additional family members.
• Region specific primers or probes derived from any of the sequences in the Sequence Listing can be used to prime DNA synthesis and PCR amplification, as well as to identify colonies containing cloned DNA encoding a member of this family using known methods.
• primers derived from one of the nucleotide sequences for amplification When using primers derived from one of the nucleotide sequences for amplification, one skilled in the art will recognize that by employing high stringency conditions, annealing at 50° -60° C, sequences which are greater than 75% homologous to the primer will be amplified. By employing lower stringency conditions, annealing at 35°-37° C, sequences which are greater than 40-50% homologous to the primer will be amplified.
• tissue can be used as the source for the genomic DNA or RNA encoding members of the TPCKl family of potassium channels.
• the most preferred source is tissues which express elevated levels of the desired potassium channel family member.
• using the sequences as taught herein it is now possible to identify such cells using the dORK, cORK, or TPCKl sequence as a probe in Northern blot or in situ hybridization procedures, thus eliminating the necessity to obtain RNA/DNA from a tissue which expresses elevated levels of such protein.
• Genes encoding the potassium channels of the present invention may be expressed in a recombinant host.
• Heterologous DNA sequences are typically expressed in a host by means of an expression vector.
• An expression vector is a replicable DNA construct in which a DNA sequence encoding the heterologous DNA sequence is operably linked to suitable control sequences capable of affecting the expression of a protein or protein subunit coded for by the heterologous DNA sequence in the intended host.
• control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and (optionally) sequences which control the termination of transcription and translation.
• Vectors useful for practicing the present invention include plasmids, viruses (including bacteriophage), and integratable DNA fragments (i.e., fragments integratable into the host genome by genetic recombination).
• the vector may replicate and function independently of the host genome, as in the case of a plasmid, or may integrate into the genome itself, as in the case of an integratable DNA fragment.
• Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host.
• a promoter operable in a host cell is one which binds the RNA polymerase of that cell
• a ribosomal binding site operable in a host cell is one which binds the endogenous ribosomes of that cell.
• DNA regions are "operably associated" when they are functionally relate to each other.
• a promoter is operably linked to a coding sequence if it controls the transcription of the sequence;
• a ribosome binding site is operably linked to coding sequence if it is positioned so as to permit translation.
• operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase.
• Transformed host cells of the present invention are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques and express the protein or protein subunit coded for by the heterologous DNA sequences.
• the novel nucleic acid sequences of the invention and fragments thereof can be used to express protein in a variety of host cells, both prokaryotic and eukaryotic.
• suitable eukaryotic cells include mammalian cells, plant cells, yeast cells, and insect cells.
• Suitable prokaryotic hosts include Escherichia coli and Bacillus subtilis.
• ehinese hamster ovary CHO cell COS cells
• human embryonic kidney cells ehinese hamster ovary (CHO) cell COS cells
• human embryonic kidney cells ehinese hamster ovary (CHO) cell COS cells
• NIH3T3 fibroblasts ehinese hamster ovary (CHO) cell COS cells
• mouse Ltk cells ehinese hamster ovary (CHO) cell COS cells
• human embryonic kidney cells ehinese hamster ovary (CHO) cell COS cells
• NIH3T3 fibroblasts ehinese hamster ovary
• mouse Ltk cells ehinese hamster ovary
• insect cells ehinese hamster ovary (CHO) cell COS cells
• SP9 cells ehinese hamster ovary
• Suitable expression vectors are selected based upon the choice of host cell.
• Numerous vectors suitable for use in transforming host cells are well known.
• plasmids and bacteriophages such as ⁇ phage, are the most commonly used vectors for bacterial hosts, and for E. coli in particular.
• plasmid and virus vectors are frequently used to obtain expression of exogenous DNA.
• mammalian cells are commonly transformed with conventional viral vectors, or transfected with plasmids, such as the pcDNAI vector series from Invitrogen Corporation (San Diego, CA) and the pMAM vector series from Clontech, and insect cells in culture may be transformed with baculovirus expression vectors.
• Yeast vector system include yeast centromere plasmids, yeast episomal plasmids, and yeast integrating plasmids.
• the invention encompasses any and all host cells transformed or transfected the claimed nucleic acid sequences or fragments thereof, as well as expression vectors used to achieve this.
• the transformed host cells are yeast.
• yeast cultures and suitable expression vectors for transforming yeast cells, are known. See e.g., U.S. Patent No. 4,745,057; U.S. Patent No. 4,797,359; U.S. Patent No. 4,615,974; U.S. Patent No. 4,880,734; U.S. Patent No. 4,711,844; and U.S. Patent No. 4,865,989. Saccharoniyces cerevisiae is the most commonly used among the yeasts, although a number of other yeast species are commonly available. See, e.g., U.S. Patent No.
• a heterologous potassium channel may permit a yeast strain unable to grow in medium containing low potassium concentration to survive (CY 162, for example, see J.A. Anderson et al, Proc. Natl. Acad. Sci. USA 89:3736-3740 (1992)).
• Yeast vectors may contain an origin of replication from the endogenous 2 micron (2 ⁇ ) yeast plasmid or an autonomously replicating sequence (ARS) which confer on the plasmid the ability to replicate at high copy number in the yeast cell centromeric (CEN) sequences which limit the ability of the plasmid to replicate at only low copy number in the yeast cell, a promoter, DNA encoding the heterologous DNA sequences, sequences for polyadenylation and transcription termination, and a selectable marker gene.
• An exemplary plasmid is Yrp7, (Stinchcomb et al, Nature 282:39 (1979); Kingsman et al, Gene 7:141 (1979); Tschemper etal, Gene 10:157 (1980)).
• This plasmid contains the TRP1 gene, which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in the absence tryptophan, for example ATCC No. 44076.
• the presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Suitable promoting sequences in yeast vectors include the promoters for metallothionein (Yep52), 3 -phosphoglycerate kinase (pPGKH, Hitzeman et al., J. Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (pYSK153, Hess et al, J. Adv. Enzyme Reg.
• promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2 (pAD4M), isocytochrome C, acid phosphates, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and gly ceraldehyde-3 - phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose (pYES2) utilization.
• pAD4M alcohol dehydrogenase 2
• isocytochrome C acid phosphates
• degradative enzymes associated with nitrogen metabolism and the aforementioned metallothionein and gly ceraldehyde-3 - phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose (pYES2) utilization.
• pYES2 metallothionein and gly ceraldehyde-3 - phosphate dehydrogenase
• the nucleic acid sequences of the invention are used to express proteins in a bacterial host.
• Protein expressed in bacteria can be used in raising antisera (both polyclonal and monoclonal) by standard methodology.
• antisera both polyclonal and monoclonal
• Such antibodies are useful in immunohistochemical studies to determine the level of expression of the channel protein in various tissues and cell lines.
• the channel can be purified from bacterial cells if found in inclusion bodies, for example, by isolation of inclusion bodies by standard techniques, followed by electrophoresis in SDS-PAGE gels and isolation of the protein band from the gel.
• the potassium channel proteins, or portions thereof can be expressed as a fusion protein, e.g., with glutatliione-s-transferase, or maltose binding protein, and then purified by isolation of the protein to which it is fused.
• the predicted amino acid sequence can be used to design synthetic peptides unique to the potassium channels as herein described, which peptides can then be used to raise antibodies to the channels.
• the present invention further provides methods of identifying cells or tissues which express a member of the family of channel proteins presented herein.
• a probe comprising a DNA sequence of hORKl, a fragment thereof, or a DNA sequence encoding another member of the TPKCl family of channel proteins can be used as a probe or amplification primer to detect cells which express a message homologous to the probe or primer.
• One skilled in the art can readily adapt currently available nucleic acid amplification or detection techniques so that it employs probes or primers based on the sequences encoding a member of this family.
• kits which is compartmentalized to receive in close confinement, one or more containers which comprises: (a) a first container comprising one or more probes or amplification primers based on the TPCKl sequence or any of the other sequences, or simply a fragment containing nucleic acids that encode XXXXXXGXG (SEQ ID NO:65) and XXXXGXPX (SEQ ID NO:66); and (b) one or more other containers comprising one or more of the following: a sample reservoir, wash reagents, reagents capable of detecting presence of bound probe from the first container, or reagents capable of amplifying sequences hybridizing to the amplification primers.
• a compartmentalized kit includes any kit in which reagents are contained in separate containers.
• Such containers include small glass containers, plastic containers or strips of plastic or paper.
• Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
• Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris buffers, etc.), and containers which contain the reagents used to detect the bound probe or amplified product.
• Types of detection reagents include labeled secondary probes, or in the alternative, if the primary probe is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled probe.
• probes and amplification primers based on the sequence disclosed in the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
• sequences of the present invention are also valuable for chromosome identification.
• the sequence may be specifically targeted to and hybridize with a particular location on an individual chromosome, for example, the human chromosome.
• the mapping of DNA to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease, or tracking other possible disease pathways. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
• PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
• sublocalization 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 hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific- cDNA libraries.
• Fluorescence in situ hybridization (FISH) of a cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
• This technique can be used with cDNA as short as 500 or 600 bases; however clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
• FISH requires use of the large clones from which the cDNA was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
• the physical position of the sequence on the chromosome can be correlated with genetic map data.
• genetic map data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).
• the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
• linkage analysis coinheritance of physically adjacent genes.
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
• Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms
• a yeast expression system wherein yeast cells bear heterologous potassium channels.
• Cloning and expression of potassium channels from heterologous species such as those described herein are useful in the discovery of new pesticides, and animal and human therapeutics. Discovery of such compounds will necessarily require screening assays of high specificity and throughput. For example, new pesticides directed at potassium channels require high selectivity for insect channels and low activity against non- insect species. Screening assays utilizing yeast strains genetically modified to accommodate functional expression of heterologous potassium channels offer significant advantages in this area.
• these channels expressed in heterologous yeast cells are dORK, RAK (as described below), Shal, Shaw, Eag, cORK, or TPKCl.
• transformed host cells of the present invention express the proteins or protein subunits coded for by the heterologous DNA sequences.
• the potassium channel is located in the host cell membrane (i.e., physically positioned therein in proper orientation for both the stereoselective binding of ligands and passage of potassium ions).
• the potassium channel is positioned within a cell membrane in such a manner as to allow it to function as a modulator of the flow of potassium ions into and out of the cell.
• at least one pore-forming domain may be positioned proximal to a exterior portion of the cell membrane.
• a transformed yeast cell is presented, containing a heterologous DNA sequence which codes for a potassium channel, as herein presented, cloned into a suitable expression vector.
• Various other useful potassium channels may be utilized in the screening assay embodiments of the present invention, such as a delayed rectifier potassium channel referred to as "RAK or RATRAK” (Paulmichl et al , Proc. Natl. Acad. Sci, USA 88-7892-7895 (1991), reporting the cloning of this potassium channel from rat cardiac tissue).
• RAK is capable of complementing the potassium- dependent phenotype of Saccharomyces cerevisiae strain CY 162 on medium containing low potassium concentration.
• the present invention provides methods of obtaining and identifying agents capable of binding to or otherwise interacting with the potassium channels of the invention.
• the method comprises:
• the screened substances in the above assay can be, but are not limited to, proteins, peptides, peptidomimetics, carbohydrates, vitamin derivatives, compounds, or other pharmaceutical agents or any mixtures thereof.
• the substances can be selected and screened at random or rationally selected or designed using protein modeling techniques.
• a substance is said to be "rationally selected or designed" when the substance is chosen based on the configuration of the particular member of the claimed family of channel proteins. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides.
• the present invention further provides methods for modulating the expression of TPCKl, or a member of the TPCKl family of channel proteins. Specifically, anti- sense RNA expression is used to disrupt the translation of the mRNA encoding the TPCKl protein.
• a cell is modified using routine procedures such that if expresses an antisense mRNA, an mRNA which is complementary to mRNA encoding the TPCKl family member.
• an antisense mRNA an mRNA which is complementary to mRNA encoding the TPCKl family member.
• the translation of the TPCKl family member rnRNA can be regulated.
• the cloning of the members disclosed herein now makes possible the screening capability which enables the identification of agonists (potassium channel openers) and antagonists (potassium channel closers) of this family of channel proteins.
• the two-pore K + channels described herein in humans can be used as targets for novel human therapeutics.
• the primary target for such therapeutic agents will be conditions related to alterations in the plasma membrane resting potential and/or the duration of the action potential in excitable cells.
• Potassium channels influence action waveforms and firing frequency of cells and therefore play a role in neuronal integration, muscle contraction, and hormone secretion in excitable cells.
• Potassium channels play the vital role of determining resting electrical membrane potential by setting membrane permeability to potassium ions in the cell.
• this class of potassium channels may be of use in the discovery of new agents for the treatment of atrial and ventricular arrhythmias, heart failure including associated arrhythmias and cardiac ischemia.
• the action of such agents would be effected through the modulation of the kinetics duration of the cardiac action potential.
• the delayed rectifier potassium current in heart cells regulates the duration of the plateau of the cardiac action potential by countering the depolarizing, inward calcium current. Delayed rectifier potassium currents characteristically are activated upon depolarization from rest, display a sigmoidal or delayed onset, and have a nonlinear, or rectifying, current- voltage relationship.
• Delayed rectifier potassium currents characteristically are activated upon depolarization from rest, display a sigmoidal or delayed onset, and have a nonlinear, or rectifying, current- voltage relationship.
• Several types of delayed potassium conductances have been identified in cardiac cells based on measured single-channel conductances. Heart-rate and contractility are regulated by second messenger modification of delayed rectifier potassium conductances, and species differences in the shape of the plateau may be influenced by the type and level of channel expression.
• Potassium channel openers may also function as smooth muscle relaxants, functioning a vasodilators, vasospasmolytics, and other smooth muscle spasmolytic.
• vasodilators these compounds have use as dilators of peripheral vasculature, coronary arteries, renal vasculature, cerebral vasculature, and mesenteric vasculature.
• vasospasmolytics these compounds have use in the treatment of coronary artery spasm, peripheral vascular spasm, cerebral vascular spasm and impotence.
• Other smooth muscle spasmolytics have use as bronchodilators, in the control of urinary bladder and gall bladder spasm, and in the control of esophageal, gastric, and intestinal smooth muscle spasm.
• Potassium channel closers may function in the pancreas to enhance release of insulin, in the kidney as diuretics and renal epithelial anti-ischemic agents, as hypertensive agents for promoting vasoconstriction for use in hypotensive states as antiarchythmic agents, and as agents for modifying cardiac muscle contractility.
• Other uses for potassium channel agonists or antagonists include anticonfulsants, hair growth promoting agents, and agents effective in preventing or reducing skeletal muscle damage or fatigue.
• methods of modulating cellular activity to provide therapeutic value are provided, by applying to a patient in need of such modulation, a substance capable of interacting with a potassium channel contained in the relevant cells of such patient and modulating the activity of same (a good example of which are cardiac cells, useful for cardiac modulation purposes).
• a substance capable of interacting with a potassium channel contained in the relevant cells of such patient and modulating the activity of same (a good example of which are cardiac cells, useful for cardiac modulation purposes).
• Certain substances whether biological or chemical in nature may be applied cell membranes having as an integral part of their structure, one or more potassium channels presented herein, and particularly those comprising the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:36, SEQ ID NO:46, or RAK, in an amount and for a time sufficient to affect the ability of the potassium channel to so regulate the flow of ions.
• Substances that are potassium channel blockers will inhibit the ability of the channel to regulate the flow of such ions.
• Substances that enhance such ability may be considered potassium channel "activators.”
• compositions may comprise conventional delivery/carrier systems, e.g., liposome or phospholipid encapsulation, water or saline solutions, polymeric compositions, and the like.
• Such assays may be performed in vitro and extrapolated to in vivo conditions, or in some cases may be easily established directly in vivo.
• the field of insecticides is instructive for this purpose.
• in vivo tests can be run by applying the substance directly to a test sample comprising the target insect pest (whole organism) and noting the appropriate parameters at which an acceptable per cent of insect death is attained.
• methods of selectively inhibiting insect pests are presented by applying to such insect pests a substance capable of selectively inhibiting the activity of a potassium channel contained in the cells of such insect, and comprising the amino acid sequence of SEQ ID NO:2, or a potassium channel biologically equivalent thereto.
• the inhibitor will inhibit the activity of the aforementioned potassium channel without inhibition of other, non-homologous or otherwise non-equivalent potassium channels that may be present in species other than the targeted insect pest. It is envisioned that such other species may also be present at the site of application of the inhibitor, such as in a garden, crop, or other site wherein it is desired to control insect pests.
• methods of selectively inhibiting nematode pests are presented much in the same manner as discussed for control of insect pests, by applying to such pests a substance capable of selectively inhibiting the activity of a potassium channel contained in the cells of such pest, said potassium channel comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:36, or potassium channels biologically equivalent thereto.
• the present invention further provides methods for generating chimeric or transgenic animals 1) in which the animal contains one or more exogenously supplied genes which are expressed in the same temporal and spatial manner as a member of the family of channel proteins as presented herein, or 2) in which such member of this family of channel proteins has been deleted of overexpressed.
• Such chimeric and transgenic animals are useful in the further elucidation of the mechanisms of potassium channel function as well as their effect an animal physiology.
• These transgenic and chimeric animals are produced by utilization of techniques which are well known and well described in the technical literature, e.g., see U.S. Patent No. 5,434,340 and scientific references cited therein discussing, among other things, the introduction of transgenes into the gumline of a non-human animal, herein incorporated by reference.
• Dm ORFl an invertebrate source
• Dm ORFl contains a single long open reading frame encoding a protein of 618 amino acids (SEQ ID NO:2) that exhibits substantial amino acid identity to the pore-forming regions of other potassium channels.
• the DmORFl contains structural features that distinguish it from other classes of potassium channels, including four hydrophobic domains capable of forming transmembrane helices (M1-M4) and two putative pore forming H5 domains found between transmembrane helices Ml and M2, and M3 and M4. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity (Heginbotham et al, Science 258:1152-1155, (1992)). This-work was expanded to clone a construct derived from C. elegans having a single open reading frame sufficient to encode a protein of 434 amino acids, designated pCORK.
• the DNA sequence contained a single long open reading frame sufficient to encode a protein of 336 amino acids (predicted MW 38.5 kDa) with substantial homology to known potassium channel sequences.
• CeORFl contains a single long open reading frame encoding a protein that exhibits substantial amino acid identity to pore- forming regions of other potassium channels.
• DNA sequences encoding a human putative two-pore potassium channel were cloned by polymerase chain reaction (PCR) from human brain cDNA.
• oligonucleotides (5'and 3' oligo) used in the analysis were designed from a compilation of nucleotide sequences encoding the pore- forming domains of putative two pore potassium channels identified in a search of the GENBANK DNA sequence database.
• CeORFl and pCORK each contain structural features similar to DmORFl, including two putative pore forming H5 domains. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity (Heginbotham et al, Science 258:1152-1155. (1992)). These features form the basis of the designation of a new subfamily of potassium channels comprising DmORFl, CORK, CeORFl, TPCKl, and various other homologs. The particulars of this discovery is set forth in more detail below:
• Saccharomyces cerevisiae strain CY 162 is described in Anderson, J.A. el al, Proc. Natl. Acad. Sci. USA 89:3736-3740 (1992). Growth of bacterial strains and plasmid manipulations are performed by standard methods (Maniatis T., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1982). Media conditions for growth of yeast, isolation of plasmid DNA from yeast, and DNA-mediated transformation of yeast strains are as described in Rose M. D., Methods in yeast genetics, Cold Spring Harbor Laboratory Press, 1990. A multifunctional expression library constructed in pYES2 and containing cDNA made from 3rd instar male Drosophila melanogaster mRNA is used as described in S.J.
• CY162 cells are transformed with plasmid DNA from each library to give 3 x 10 6 transformants from each library on SCD-ura (synthetic complete dextrose (2 %) medium containing all necessary nutritional supplements except uracil) containing 0.1 M KC1 agar medium.
• Transformants are replica-plated to SCG-ura (synthetic complete galactose (2 %) medium containing all necessary nutritional supplements except uracil) agar medium. Colonies that grow on this selective agar medium are transferred to SCG-ura agar medium to obtain single colonies clones and while reassaying suppression of the potassium-dependent phenotype. Plasmid DNA is isolated from surviving colonies and used to transform CY162.
• Plasmids that confer suppression of the potassium-dependent phenotype are subjected to automated DNA sequence analysis performed by high temperature cycle sequencing (Applied Biosystems). Geneworks DNA sequence analysis software (Intelligenetics) is used to align raw DNA sequence information and to identify open reading frames.
• the DNA sequence of the 2.4 kb insert in pDmORFl is displayed in FIGURE 2A and 2B (SEQ ID NO:l).
• the 5' untranslated sequences of the cDNA contain long poly A and poly T tracts not likely to be found in protein coding regions.
• the first ATG proximal to the 5' end is present in a consensus Drosophila melanogaster translational initiation site (D.R.
• a single long open reading frame sufficient to encode a protein of 618 amino acids is encoded in pDmORFl.
• the DmORFl contains structural features that distinguish it from other classes of potassium channels, including four hydrophobic domains capable of forming transmembrane helices (M1-M4) and two pore forming H5 domains found between transmembrane helices Ml and M2, and M3 and M4.
• Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity (Heginbotham et al, Science 258:1152-1155. 0992)).
• a search of the GENBANK database protein sequences similar to DmORFl reveals significant matches with several known potassium channel sequences.
• the closest match is to a putative protein coding DNA sequence, F22b7.7, reported in the Coenorhabditis elegans genome sequencing project (Wilson et al, Nature 368:32-38 (1994)).
• the DNA sequence and predicted amino acid sequence assembled from putative expns recognized by a GENBANK exon identification algorithm is displayed in FIGURE 3 A and 3B (SEQ ID NOS:3 and 4).
• the DNA sequence contains a single long open reading frame sufficient to encode a protein of 336 amino acids (predicted MW 38.5 kDa) with substantial homology to known potassium channel sequences.
• the F22b7.7 sequence contains structural features that distinguish it from other classes of potassium channels, including three of four hydrophobic domains capable of forming transmembrane helices (M1-M4) identified in DmORFl and two pore forming H5 domains found between transmembrane helices a predicted Ml and M2, and M3 and M4. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity (Heginbotham et al, Science 258:1152-1155, (1992)).
• the lack of an amino terminal transmembrane domain homologous to DmORFl Ml in the F22b7.7 sequence may be due to failure of the search algorithm to identify exon(s) encoding the amino terminus.
• an amino terminal coding sequence may be added by trans-splicing, which occurs frequently in Caenorhabditis elegans.
• Oligonucleotides corresponding to DNA sequences encoding the two pore forming domains of F22b7.7 are synthesized using an Applied Biosystems DNA synthesizer.
• the oligos were labeled at their 5' ends with 32 P using a 5'-end labelling kit according to the manufacturer's instructions (New England Nuclear).
• the labeled oligos are pooled and used to screen 6 x 10 5 plaques from a ⁇ ZAP '-Caenorhabditis elegans cDNA library (obtained from Clontech) by published methods (T.N. Davis and J. Thorner, Meth Enzymol. 139:246-262 (1987)).
• Hybridization is at 42 °C for 16 hours. Positive clones are plaque-purified by twice repeating the hybridization screening process.
• Plasmid DNAs excised from phage DNA according to the manufacturer's instructions, are subjected to automated DNA sequence analysis performed by high temperature cycle sequencing (Applied Biosystems). Geneworks DNA sequence analysis software (Intelligenetics) is used to align raw DNA sequence data and to identify open reading frames.
• FIGURE 4 Predicted amino acid sequences of DmORFl and F22b7.7 are aligned and displayed in FIGURE 4 (SEQ ID NOS:37 and 38). Only limited overall amino acid homology is exhibited by these two proteins with regions of greatest homology existing in the pore forming H2-1 and H2-2 domains.
• FIGURE 5 A shows a comparison of the pore forming domains of DmORFl and F22b7.7 with those of the known Drosophila melanogaster potassium channel and inward rectifier sequences (SEQ ID NOS:7 through 21). Amino acid identities greater than 50 % are observed with all potassium channel sequences.
• FIGURE 5B shows hydropathy plot analysis of DmORFl and F22b7.7.
• the two proteins which show remarkable topological similarity through their length, are predicted to be composed of four membrane- spanning hydrophobic domains (M1-M4), and two pore forming H2 domains. These data suggest the predicted topology shown in FIGURE 6. Both proteins are predicted to span the membrane four times with amino and carboxyl termini residing within the cell. This topology places the single amino-terminal asparagine-linked glycosylation site and H2 domains on the cell exterior permitting permeation of the membrane by the pore forming domains from the outside, an absolute requirement for the formation of a functional potassium channel.
• EXAMPLE 6 Functional expression of a rat atrial delayed rectifier potassium channel in yeast.
• CY162 transformants containing plasmids pKATl, which encodes a plant inward rectifier potassium channel, pRATRAK, which encodes a rat atrial delayed rectifier potassium channel, pDmORFl, and control plasmid pYES are cultured on arginine-phosphate-dextrose agar medium lacking ura medium (A. Rodriguez-Navarro and J. Ramos, J. Bacteriol. 159:940-945, (1984)) containing various KC1 concentrations (FIGURE 7).
• pRATRAK is constructed by modifying the protein-coding sequences of RATRAK to add 5' Hindlll and 3' Xbal sites using PCR. In addition, four A residues are added to the sequences immediately 5' proximal to the initiator ATG to provide a good yeast translational initiation site. The modified fragment is cloned into the Hindlll and Xbal sites in the yeast expression vector pYES2 (Invitrogen), forming pRATRAK.
• Yeast strains dependent on heterologous potassium channels for growth should be sensitive to non-specific potassium channel blocking compounds.
• a convenient agar plate bioassay is employed. Strains containing pKATl, pRATRAK, pDmORFl, and pYES2 are plated in arginine-phosphate-dextrose agar medium lacking ura and containing various amounts of potassium chloride. Arginine-phosphate-dextrose medium is used to avoid interference from potassium and ammonium ions present in standard synthetic yeast culture medium. Sterile filter disks were placed on the surface of the agar and saturated with potassium channel blocking ions CsCl, BaCl 2 , and TEA.
• heterologous potassium channel containing strains is inhibited by potassium channel blocking ions in a channel dependent manner.
• DmORFl -dependent growth is blocked by BaCl 2 but not by CsCl or TEA.
• KAT- dependent growth is blocked by BaCl 2 , CsCl, and TEA.
• RATRAK-dependent growth is blocked by BaCl 2 , CsCl, and TEA to a much greater extent than pKATl , reflecting in part a slower growth rate of pRATRAK-containing cells.
• Yeast strains made capable of growing on medium containing low potassium concentration by expression of heterologous potassium channels are used to screen libraries of chemical compounds of diverse structure for those that interfere with channel function.
• CY162 cells containing pKATl, pRATRAK, pDmORFl, pCeORFl, and pYES2-TRKl (lOVml) are plated in 200 ml of arginine-phosphate- dextrose agar medium lacking ura and containing 0.2 mM potassium chloride in 500 cm 2 plates.
• the CY162 cells bearing pYES2-TRKl are included in the assay as a control to identify compounds that have non-specific effects on the yeast strain and are therefore not specifically active against the heterologous potassium channels.
• Samples of chemical compounds of diverse structure (2 ⁇ l of 10 mg/ml solution in DMSO) are applied to the surface of the hardened agar medium in a 24 x 24 array.
• the plates are incubated for 2 days at 30 °C during which time the applied compounds radially diffuse into the agar medium.
• the effects of applied compounds on strains bearing heterologous potassium channel genes are compared to the pYES2-TRKl bearing strain.
• Compounds that cause a zone of growth inhibition around the point of application that is larger on plates containing cells bearing the heterologous potassium channels than that observed around the pYES2-TRKl bearing strains are considered selective potassium channel blockers.
• Compounds that induce a zone of enhanced growth around the point of application that is larger on plates containing cells bearing the heterologous potassium channels than that observed around the pYES2-TRKl bearing strains are considered selective potassium channel openers.
• EXAMPLE 9 DmORFl-induced currents in X laevis oocytes assayed by two-electrode voltage Clamp.
• DNA sequences encoding the open reading frame of DmORFl were amplified by polymerase chain reaction (PCR) using the following oligonucleotides: MPO23: ATAAAGCTTAAAAATGTCGCCGAATCGATGGAT (SEQ ID NO:22) MPO24: AGCTCTAGACCTCCATCTGGAAGCCCATGT (SEQ ID NO:23)
• PCR polymerase chain reaction
• MPO23 ATAAAGCTTAAAAATGTCGCCGAATCGATGGAT
• MPO24 AGCTCTAGACCTCCATCTGGAAGCCCATGT (SEQ ID NO:23)
• the full length PCR product was cloned into corresponding sites in pSP64 poly A (Promega), forming pMP 147.
• Template DNA was linearized with EcoRI and RNA transcribed using the Message Machine (Arnbion) in vitro transcription kit according to the manufacturer's instructions.
• RNA content was estimated by ethidium bromide staining. The remainder was stored on dry ice.
• X laevis oocytes were isolated and injected with 50 nl of sterile T ⁇ containing 5-20 ng transcript according to published procedures. After three days, whole oocyte currents were recorded using a two-electrode voltage clamp. Electrodes contained 3M KC1 and had resistances of 0.3-1.0 MW. Recordings were performed with constant perfusion at room temperature in the presence of either low (10 mM) or high (90 mM) potassium chloride.
• Two electrode voltage clamp analysis of the DmORFl gene product expressed in X laevis oocytes demonstrates properties of a voltage- and potassium- dependent potassium channel. At low potassium concentrations, DmORFl exhibited outward current at depolarizing potentials. At high potassium concentration, DmORFl exhibits both inward and outward currents. The DmORFl channel displays a high preference for potassium and shows cation selectivity in the rank order K>Rb>NH 4 ,>Cs>Na>Li. Potassium currents were greatly attenuated by BaCl 2 .
• EXAMPLE 10 Developmental regulation of DmORFl expression in D. melanogaster determined by Northern blotting analysis.
• RNA from embryo, larvae, and adult forms was resolved in a MOPS-acetate-formaldehyde agarose gel according to standard procedures. The gel was stained with ethidium bromide and photographed to mark the positions of 18 S and 28 S ribosomal RNAs used as molecular weight markers. RNA was transferred by capillary action to nitrocellulose with 10 x SSPE. The blot was air-dried, baked for one hour at 80 °C, and prehybridized in 4x SSPE, 1% SDS, 2x Denhardf s, 0.1% single stranded DNA at 68°C for 2 hours.
• a 2.4 kb Xhol fragment of DmORFl was isolated from pDmORFl and labeled with ⁇ - 32 P dCTP using the Ready-to-Go kit (Pharmacia) according to the manufacturer's instructions.
• the probe was denatured by heating to 100°C for 5 minutes followed by quenching in an ice water bath. The probe was added to the prehybridization solution and hybridization continued for 24 hours at 68 °C.
• the blot was washed briefly with 2x SSPE, 0.1% SDS at room temperature followed by 0.5 x SSPE, 0.1% SDS at 65 °C for 2 hours.
• the blot was air-dried and exposed to Reflection X-ray film (NEN) using an intensifying screen at -70 °C for 48 hours.
• NNN Reflection X-ray film
• DNA sequence analysis of the pDmORFl insert reveals a single long ORF with conserved amino acid sequence domains in common with known potassium channels.
• the DNA sequence predicts an ORF sufficient to encode a protein of 618 amino acid in length.
• the DmORFl polypeptide contains four segments of at least 20 hydrophobic amino acids in length suggesting that the segments span the plasma membrane.
• the DmORFl protein sequence contains a putative N-linked glycosylation site (Asn-Thr-Thr) at amino acids 58-60.
• pDmORFl was used as template to drive coupled in vitro transcription/translation.
• Plasmid pMP147 was used as template to produce 35 S -labeled DmORFl gene product in vitro using a TnT coupled transcription-translation kit (Promega) according to the manufacturer's instructions. Glycosylation of the nascent DmORFl polypeptide was accomplished by addition of canine pancreatic microsomes (Promega) to the transcription-translation reaction. Samples of glycosylated DmORF protein were treated with endoglycosidase H to remove added carbohydrate moieties. Aliquots were precipitated with TCA and collected on GF/C filters, washed with ethanol, dried and counted. Equivalent counts per minute were resolved by SDS-PAGE. The gel was impregnated with soluble fluor Amplify (Amersham) and dried onto Whatman 3MM paper. The dried gel was exposed to Reflection X-ray film at room temperature.
• Translation of the DmORFl gene product in vitro produced a polypeptide of 68 kDa, consistent with the predicted molecular weight of the ORF.
• Translation of DmORFl in the presence of canine pancreatic microsomes results in synthesis of a protein with reduced electrophoretic mobility, consistent with glycosylation of the nascent polypeptide.
• Treatment of glycosylated DmORF with EndoH increased its relative mobility as expected upon removal of carbohydrate moieties.
• the pDmORFl insert is capable of directing the expression of a glycoprotein with the expected molecular weight. EndoH treatment removes carbohydrate residues consistent with the sugar added through N-linked glycosylation.
• DmORF permits CY 162 cells to grow on medium containing a low concentration of potassium, implying that DmORFl supplies high affinity potassium uptake capacity.
• DmOPFl supplies high affinity potassium uptake capacity.
• 86 Rb uptake studies were performed. Examination of the uptake of this potassium congener revealed important aspects of potassium uptake by DmORFl.
• Yeast strains containing heterologous potassium-expression plasmids CY162-DmORFl, CY162-pKAT and the control strain CY162-pYES2 (Clontech) were cultured overnight in SC Gal-ura containing, 0.1 M KC1.
• the cells were harvested, washed with sterile doubled distilled water and starved for K + for 6 hours in Ca-MES buffer. Cells were washed again and distributed to culture tubes (10 8 cells/tube) containing 86 RbCl in Ca-MES buffer. The tubes were incubated at room temperature, samples filtered at various time intervals and counted. 86 Rb uptake into cells was displayed.
• the high-affinity potassium uptake capacity encoded by DmORFl permits high-affinity uptake of the potassium Congener, 86 Rb, as well. Barium inhibited 86 Rb uptake. No high affinity 86 Rb uptake is observed in control CY162-pYES2 cells and 86 Rb uptake into CY162-pKAT cells is consistent with its published properties.
• EXAMPLE 13 Expression of Drosophila melanogaster potassium channels in Yeast.
• Voltage-gated potassium channel diversity in the fruitfly Drosophila melanogaster is encoded in large part by six genes, Shaker, Shab, Shal, Shaw, Eag, and Slo. Expression of these potassium channels in yeast will permit their introduction into screening assays for novel insecticidal compounds and facilitate characterization of their ion channel properties and sensitivity to compounds with activating and inhibitory properties.
• DNA sequences encoding Drosophila melanogaster potassium channels were amplified by PCR using synthetic oligonucleotides that add 5' Hindlll or Kpnl, sites and Xbal, Sphl, ox Xhol sites:
• Shaker 5' AAAAAGCTTAAAATGGCACACATCACG (SEQ IDNO:24)
• Shaker 3' AAACTCGAGTCATACCTGTGGACT (SEQ ID NO:25)
• Shab 5' AAAAAGCTTAAAATGGTCGGGCAATTG (SEQ ID NO:26)
• Shab 3' AAAAGCATGCTCATCTGGATGGGCA (SEQIDNO:27)
• Shal 5':AAAAAGCTTAAAATGGCCTCGGTCGCC Shal 3':TTTTCTAGACTACATCGTTGTCTT (SEQ ID NO:29)
• Eag 5' AAAAAGCTTAAAATGCCTGGCGGA (SEQ ID NO:32)
• Eag 3' AAATCTAGAGGCTACAGGAAGTCC (SEQ ID NO:33)
• Plasmids used as templates for the PCR reactions were: pBSc-DShakerH37, pBSc- dShabll, P BSc-dShal2+(A)36, pBScMXT-dShaw (A. Wei et al, Science 248:599-603 (1990), provided by L. Salkoff), pBScMXT-slo,v4 (Atkinson el al, Science 253:551 - 555, (1991), provided by L.
• CY162 cells were transformed with assembled Drosophila melanogaster potassium channel expression plasmids by the LiCl method and plated on SCD-ura containing 0.1 M KCl agar medium. Selected transformants were tested for growth on arginine-phosphate-galactose (2%)/sucrose (0.2%)-ura agar medium containing 1-5 mM KCl. CY162 cells containing pKATl or pDmORFl were cultured as positive controls and CY162 cells containing pYES2 were grown to provide a negative control.
• CY162 cells bearing Drosophila melanogaster potassium channel expression plasmids survive under conditions in which growth is dependent on functional potassium channel expression. At potassium ion concentrations between 1-3 mM, negative control CY162 cells containing pYES2 grow poorly. Expression of the Drosophila melanogaster potassium channels Shal, Shaw and Eag substantially improve growth of CY162. These results are consistent with the Drosophila melanogaster potassium channels providing gh-affinity potassium uptake capacity. This capacity is apparently sufficient to replace the native high-affinity potassium transport capacity encoded by TRK1 which is lacking in CY162 (trkl trlc2) cells.
• EXAMPLE 14 Cloning of a novel C. elegans sequence with homology to potassium channels.
• CY162 cells were transformed with a pYES2-based yeast expression library constructed using cDNA synthesized from C. elegans mRNA (Invitrogen). Plasmid DNA isolated from yeast cells that survived the selection scheme described in EXAMPLE 1 were subjected to automated DNA sequence analysis performed by high temperature cycle sequencing (Applied Biosystems). Geneworks DNA sequence analysis software (Inteligenetics) is used to align raw DNA sequence information and to identify open reading frames. The DNA sequence of the 1.4 kb insert in pCORK is displayed in FIGURE 9A and 9B (SEQ ID NO:36). The 5' untranslated sequences of the cDNA are present in this construct.
• a single long open reading frame sufficient to encode a protein of 434 amino acids is predicted in pCORK.
• a consensus polyadenylation site, AAT AAA occurs at position 1359-1364 in 3' untranslated sequences and is followed by a tract of 15 consecutive A residues.
• the CORK ORF contains structural features that resemble pore forming H5 domains found in potassium channels. Two putative pore forming H5 domains (residues 76-39 and 150-162) contain the G- Y/F-G tripeptide motif required for potassium selectivity (Heginbotham et al, Science 258: 1152-1155, (1992)).
• DNA sequences encoding a human putative two-pore potassium channel were cloned by polymerase chain reaction (PCR) from human brain cDNA.
• PCR polymerase chain reaction
• Degenerate oligonucleotides (5'and 3' oligo) used in the analysis were designed from a compilation of nucleotide sequences encoding the pore-forming domains of putative two pore potassium channels identified in a search of the GENBANK DNA sequence database.
• 5' oligo 5' TIG GAT (AT)(CT)G G(AT)G A(CT)(AT) T (SEQ ID NO:39)
• 3' oligo 5* (AG)TC (AT)CC (AG)(AT)A (ACT)CC (AGT)A(CT) (AGT)GT (SEQ ID NO:40)
• Clontech QUICK-Clone human brain cDNA was used as template (1 ng cDNA in 20 ⁇ l reaction) in a reaction mixture containing 1.25 U ArnpliTaq DNA Polymerase (Perkin-Elmer), 1 ⁇ M primers, 200 ⁇ M dNTPs. PCR was carried out by standard procedures using the cycles given below in a Perkin-Elmer 9600 thermocycler.
• the resulting PCR fragments were cloned into the Invitrogen TA cloning kit according to the manufacturer's instructions.
• the cloned DNA fragments were sequenced with ABI Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit on the ABI 373 Automated DNA sequencer according to the manufacturer's instructions.
• One fragment contained a 339 base pair (bp) open reading frame (ORF) with two consensus pore forming domains separated by two putative transmembrane domains.
• fragments corresponding to 5' and 3' sequences were isolated from fetal brain Marathon Ready cDNA (Clontech) using a rapid analysis of cDNA ends (RACE) procedure according to the manufacturer's instructions.
• RACE rapid analysis of cDNA ends
• a 1060 bp 5' fragment was isolated which, after DNA sequence analysis, was found to contain a 208 bp 5' untranslated region (UTR) and 852 bp ORF encoding 284 amino acids. Similarly, a 2000 bp 33' fragment was isolated which contained a 432 bp ORF capable of encoding an additional 144 amino acids along with an extensive 3'UTR.
• a DNA fragment containing the complete TPKCl ORF sequence was generated by PCR-mediated fusion of the 5' and 3' fragments.
• the isolated 5' and 3' fragments were added together to a PCR reaction mixture containing oligos corresponding to 14 nucleotides upstream of the ATG and the first 12 nucleotides of the ORF and the complement of the 20 nucleotides after the stop codon.
• TPKCl ORF fragment was isolated and cloned into the Invitrogen TA cloning kit according to the manufacturer's instructions. DNA sequence analysis confirmed the presence of a single ORF sufficient to encode a protein of 426 amino acids. The complete amino acid and DNA sequences are as follows: MLPSASRERPGYRAGVAAPDLLDPKSAAQNSKPRLSFSTKPTVLASRVESDTT
• VNHLTSERDVLPPLLKTESIYLNGLAPHCAGEEIAVIENIK (SEQ ID NO:45)
• the TPKCl ORF was amplified using oligos that added restriction endonuclease cleavage sites appropriate for insertion into the yeast expression vectors pLPlOO and pYES2 (Invitrogen).
• the corresponding TPKCl expression plasmids, pLP155 and pLP156 were constructed using standard molecular biological methodology and used to transform S. cerevisiae CY162 cells using the lithium acetate method.
• the resulting yeast strains were examined for their ability to grow on standard synthetic agar media containing a low concentration of KCl. Expression of TPKCl in CY162 cells supports their growth on low (2-3 mM KCl) potassium media.
• TPKCl -containing CY162 cells was inhibited by the known potassium channel blockers Ba2 + , Ca2 + , Cs + , and quinine, but not by TEA.
• the oligos used for the cloning of 5'and 3' RACE fragments were used in this analysis as well.
• Oligos used to clone the TPKCl ORF into pLP 100 5' AAA AGA TCT AAA ATG CTT CCC AGC GCC (SEQ ID NO:47) 3' AAA GTC GAC CTA TTT GAT GTT CTC AAT (SEQ ID NO:48)
• a fragment corresponding to the coding region of the TPCKl gene was generated by PCR with the 5' primer (5*-AAT GCT GCA TGC CTC ATG CTT CCC AGC-3') (SEQ ID NO:70) and the 3'-primer (5*-GGT TAT TTA AAG AGA GGG CT-3') (SEQ ID NO:44) and used to probe the Human Multiple Tissue Northern Blots I and II (Clontech).
• a fragment corresponding to nucleotide bases 900-1300 was generated by PCR with the 5' primer (5'-TAA GAG CAT CGG ACC ATC AG-3') (SEQ ID NO:71) and the 3' primer (5'-GGT TAT TTA AAG AGA GGG CT-3') (SEQ ID NO:44) and used to probe Human Brain Blot II and III (Clontech).
• 50 ng of DNA was labeled with Ready-To-Go DNA Labeling Beads (Pharmacia Biotech) with 32 P-dCTP (Amersham). Probes were purified over a NICKTM column (Pharmacia Biotech).
• Probes were hybridized with blots for 1 hour in the presence of ExpressHyb Hybridization Solution (Clontech) at 68 °C. Membranes were washed at room temperature in 2X SSC, 0.05% SDS for 20 minutes, and then at 50°C in O.1X SSC, 0.1% SDS for40 minutes. The blots were exposed to Kodak Biomax MS X-ray film at -70 °C for 24 hours with two Biomax MS intensifying screens.
• TPKCl expression in human tissues indicates that a 3.5 kb mRNA is expressed predominately in brain.
• the TPKCl transcript was not detected in heart, placenta, lung, liver, kidney or pancreas. Analysis of blots containing RNA from separate regions of the brain was examined and further localized high levels of TPKCl expression in the caudate nucleus, amygdala, putamen, frontal lobe, hippocampus, and spinal cord.
• the TPKCl transcript is present at significantly lower levels in other regions of the brain; cerebellum, cerebral cortex, medulla, occipital lobe, temporal lobe, corpus callosum, substantia nigra, subthalamic nucleus, and thalamus.
• EXAMPLE 16 TPCKl-induced currents in X laevis oocytes assayed by two-electrode voltage clamp.
• the TPKCl ORF was amplified by PCR with 5' primer (5'- AAA AAG CTT GCC ACC ATG CTT CCC AGC GCC-3') (SEQ ID ⁇ O:72) and 3' primer (5'-CTA TTT GAT GTT CTC-3') (SEQ ID NO:73) digested with Hindlll and inserted into the vector pLP160 digested with Hindlll and Smdl to give pLP163.
• This construct was linearized with Bgl ⁇ l for in vitro cRNA transcription with T7 RNA polymerase (Ambion). The cRNA was quantified by gel electrophoresis using RNA standards (Gibco BRL).
• TPKCl cRNA 23 nl of 40 ng/ l solution
• Oocytes were incubated at 17 °C with gentle shaking in ND96 medium.
• Whole cell electrophysiological recordings were taken 1-3 days post-injection at room temperature in a constantly-perfusing bath using a two-electrode voltage clamp protocol of 300 ms pulses from -150 to +60 mV from a holding potential of -90 mV. The interval between pulses was one second.
• TPCKl cRNA Injection of TPCKl cRNA results in a substantial outward current not present in the uninjected or water injected oocyte. Currents corresponding to the channel are rapidly responsive to changes in applied transmembrane membrane voltage, rising to their highest level with little apparent delay. Currents are non-inactivating and outwardly rectifying (FIGURE 11 A). TPKCl expression supplies a potassium selective pore, permitting movement of potassium ions in preference to sodium. Currents obtained after isotonic substitution of NaCl for KCl in the bath solution were in agreement with values predicted by the Nernst equation indicating a high degree of selectivity over both sodium and chloride ions (FIGURE 1 IB). Replacement of aspartate for chloride had no demonstrable effect (data not shown).
• EXAMPLE 17 Yeast expression in strains deficient in the transport of potassium.
• yeast molecular biological and genetic manipulations were performed by standard procedures, such as described by Rose, M. et al. in Methods in Yeast Genetics, Cold Spring Harbor Press,(1990).
• LY890 (MATtrkl:: LYS2 trk2::TRPl ura3-52 Iys2-S0l, ade2- ⁇ 0l, trpl-A63, his3-A200, Ieu2-Al) was constructed by deletion of the TRKI and TRK2 genes from the parent yeast strain YPH500 (Stratagene).
• TPKCl ORF fragment The full length TPKCl ORF fragment was generated by PCR; 5' primer (5'-AAA AGA TCT AAA ATG CTT CCC AGC GCC-3') (SEQ ID NO:47), 3* primer (AAA GTC GAC CTA TTT GAT GTT CTC AAT-3') (SEQ ID NO:48) and inserted into the yeast expression vector pLPl 00 (18) to yield pLP155.
• pLP155 was used to transform S. cerevisi ⁇ e LY890 cells using standard methods.
• CY162 was constructed as described in J.A. Anderson et al., Proc. N ⁇ tlAc ⁇ d. Sci USA 89:3736-3740 (1992).
• Yeast cells (10 5 ) containing the indicated plasmids were plated in RPD (arginine phosphate glucose) low potassium (2 mM) agar media and compounds applied to the surface of the agar.
• RPD arginine phosphate glucose
• TPKCl ORF fragment from plasmids capable of conferring growth under selective conditions were subcloned into unmutagenized pLP155, retransformed back into LY890 or Cyl62 and the resulting strains assayed on selective medium.
• the DNA sequences of TPKCl ORFs from these positive plasmids were determined.
• EXAMPLE 18 2P channels obtained by searching the EST database.
• the GENBANK expressed sequence tag database (dbEST) was searched for putative 2P channel coding sequences using the program TBLASTN to compare all open reading frames to the amino acid sequence of TPKCl. Several sequences corresponding, to TWIK were identified. In addition, one human and five murine cDNA sequences different than TWIK were identified. The five cDNAs were purchased (ATCC, Genome Systems Inc.) and subjected to automated DNA sequence analysis.
• a predicted open reading frame found in partial human cDNA sequence (GENBANK accession # n39619) apparently encodes a portion of a unique putative 2P channel.
• the predicted translation product contains amino acid motifs corresponding to pore forming domains, transmembrane domains, and XXXXGXPX (SEQ ID NO:66) consensus sequences:
• a predicted open reading frame found in partial murine cDNA sequence (GENBANK accession # wl8545) apparently encodes a portion of a unique putative 2P channel.
• DNA sequence analysis of the purchased cDNA clone (333546) revealed the presence of a single long open reading frame:
• the predicted translation product contains amino acid motifs corresponding to pore forming domains, transmembrane domains, and XXXXGXPX (SEQ ID NO: 66) consensus sequences: leu lys pro trp ala arg tyr leu leu leu leu met ala his leu leu ala met gly leu gly ala val val leu gin ala leu glu gly pro pro ala arg his leu gin ala gin val gin ala glu leu ala ser phe gin ala glu his arg ala cys leu pro pro glu ala leu glu glu leu leu gly ala val leu arg ala gin ala his gly val ser ser leu gly asn ser ser xxx thr ser asn trp asp leu pro ser ala leu leu phe thr ala ser ile

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