Small and intermediate conductance, calcium-activated potassium channels and uses thereof
AssigneesIcagen, Inc., Oregon Health Sciences University
InventorsJOHN P. ADELMAN, JAMES MAYLIE, CHRIS T. BOND, CHRISTOPHER P. SILVIA
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Abstract
This invention relates to small and intermediate conductance, calciumactivated potassium channel proteins. More specifically, the invention relates to compositions and methods for making and detecting calciumactivated potassium channel proteins and the nucleic acids encoding calciumactivated potassium channel proteins. The invention also provides methods and compositions for assaying compounds which increase or decrease potassium ion flux through a calciumactivated potassium channel.
Claims55
An isolated nucleic acid encoding a monomer of a calcium-activated potassium channel, said monomer:
- i. having a calculated molecular weight of between 40 and 80 kDa;
- ii. having a unit conductance of between 2 and 60 pS when the monomer is in the functional polymeric form of a potassium channel and is expressed in a Xenopus oocyte;
- and;
- iii. specifically binding to antibodies generated against SEQ ID NOS:30 or 42.
An isolated nucleic acid encoding at least 15 contiguous amino acids from a calcium-activated potassium channel protein, said protein having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:47 and conservatively modified variants thereof.
An isolated nucleic acid sequence of at least 20 nucleotides in length which specifically hybridizes, under stringent conditions, to a nucleic acid encoding an intermediate calcium-activated potassium channel protein, said protein selected from the group consisting of SEQ ID NO:32.
An isolated calcium-activated potassium channel protein having at least 15 contiguous amino acids from a sequence selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:47 and conservatively modified variants thereof, wherein said variants specifically react, under immunologically reactive conditions, with an antibody reactive to a protein selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO.3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.
An isolated intermediate conductance calcium-activated potassium channel protein encoded by a nucleic acid a portion of which when amplified by primer pairs produces an amplified fragment which selectively hybridizes, under stringent hybridization conditions to SEQ ID NO:31 wherein said primer pairs are selected from the group consisting of:
- 2 5′ GCCGTGCGTGCAGGATTTAGG 3′ (SEQ ID NO:34) 5′ CCAGAGGCCAAGCGTGAGGCC 3′ (SEQ ID NO:35) 5′ TCCAAGATGCACATGATCCTG 3′ (SEQ ID NO:36);
- and, 5′ GGACTGCTGGCTGGGTTCTGG 3′ (SEQ ID NO:37).
An antibody specifically reactive, under immunologically reactive conditions, to a calcium-activated potassium channel protein, said protein having a sequence selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.
An expression vector comprising a nucleic acid encoding a calcium-activated potassium channel protein, said channel protein having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:
- 43, and SEQ ID NO:
- 47, and conservatively modified variants thereof, wherein said conservatively modified variant is a protein which when expressed in an oocyte leads to formation of a calcium-activated potassium channel having a conductance of between 2 and 80 pS, a molecular weight of between 40 and 80 kilodaltons, and specifically reacts, under immunologically reactive conditions, with an antibody reactive to the channel protein selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.
An isolated nucleic acid sequence of at least 20 nucleotides in length which specifically hybridizes, under stringent conditions, to a nucleic acid encoding a calcium-activated potassium channel protein, said protein selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3. SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47.
A method for detecting the presence of a calcium-activated potassium channel protein in a biological sample, said method comprising:
- (a) contacting said biological sample with an antibody, wherein said antibody specifically reacts, under immunologically reactive conditions, to the channel protein having a sequence selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47;
- (b) allowing said antibody to bind to said protein under immunologically reactive conditions, wherein detection of said bound antibody indicates the presence of said channel protein.
A method for detecting the presence, in a biological sample, of a nucleic acid sequence encoding a calcium-activated potassium channel protein of at least 25 amino acids in length, said method comprising:
- (a) contacting said sample, under stringent hybridization conditions, with a nucleic acid probe comprising a nucleic acid segment that selectively hybridizes to a nucleic acid encoding said channel protein having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47;
- (b) allowing said nucleic acid encoding the channel protein to selectively hybridize to said probe to form a hybridization complex, wherein detection of said hybridization complex is an indication of the presence of said nucleic acid sequence in said sample.
An isolated calcium-activated potassium channel protein encoded by a nucleic acid amplified by primers which selectively hybridize, under stringent hybridization conditions, to the same sequence as primers selected from the group consisting of:
- 3 for hSK1 ATGCCGGGTCCCCGGGCGGCCTGC (SEQ ID NO:5) TCACCCGCAGTCCGAGGGGGCCAC (SEQ ID NO:6) for rSK2 ATGAGCAGCTGCAGGTACAACGGG (SEQ ID NO:7) CTAGCTACTCTCAGATGAAGTTGG (SEQ ID NO:8) for rSK3 ATGAGCTCCTGCAAATACAGCGGT (SEQ ID NO:9) TTAGCAACTCTGTGAACTTG (SEQ ID NO:10) for rSK1 TCAGGGAAGCCCCCGACCGTCAGT (SEQ ID NO:11) TCACCCACAGTCTGATGCCGTGGT (SEQ ID NO:12) for hSK2 ATGAGCAGCTGCAGGTACAACG (SEQ ID NO:23) CTAGCTACTCTCTGATGAAGTTG (SEQ ID NO:24) for hSK3 ATGAGCTCCTGCAAGTATAGC (SEQ ID NO:25) TTAGCAACTGCTTGAACTTGTG (SEQ ID NO:26) for hlK1 GCCGTGCGTGCAGGATTTAGG (SEQ ID NO:34) CCAGAGGCCAAGCGTGAGGCC (SEQ ID NO:35);
- and, for hlK1 TCCAAGATGCACATGATCCTG (SEQ ID NO:36) GGACTGCTGGCTGGGTTCTGG (SEQ ID NO:37).
A method of identifying a compound which increases or decreases the potassium ion flux through a calcium-activated potassium channel, the steps of:
- a) contacting said compound with a eukaryotic host cell or cell membrane in which has been expressed a calcium-activated potassium channel protein having a sequence selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47 conservatively modified variants thereof, wherein said conservatively modified variants specifically bind to antibodies specifically reactive with an antigen having an amino acid sequence selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47, have a conductance of between 2 and 80 pS, and a molecular weight between 40 and 80 kilodaltons;
- and
- b) determining the functional effect of the compound upon the cell or cell membrane expressing said channel.
An isolated eukaryotic nucleic acid encoding a calcium-activated channel protein of at least 400 amino acid residues in length, wherein said channel protein comprises an amino acid sequence having at least 60% similarity over at least the length of a core region of a protein selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47, and wherein said channel protein has a conductance of between 2 and 80 pS.
A method of identifying a compound that increases or decreases the potassium ion flux through a calcium-activated potassium channel, the steps of:
- a) contacting said compound with a host cell or cell membrane in which has been expressed a calcium-activated potassium channel protein of at least 400 amino acid residues in length, wherein said SK channel protein has an amino acid sequence having at least 60% similarity over the length of the core region of a protein selected from the group consisting of:
- SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:43, and SEQ ID NO:47, and wherein said channel protein has a conductance of between 2 and 80 pS;
- and
- b) determining the functional effect of the compound upon the cell or cell membrane expressing the channel.
In a computer system, a method of screening for mutations of SK and IK genes, the method comprising the steps of:
- (i) receiving input of a first nucleic acid sequence encoding a calcium-activated channel protein having a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 19, 20, 32, 43, 47 and conservatively modified versions thereof;
- (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity to the first nucleic acid sequence;
- and
- (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In a computer system, a method for identifying a three-dimensional structure of SK and IK proteins, the method comprising the steps of:
- (i) receiving input of an amino acid sequence of a calcium-activated channel protein or a nucleotide sequence of a gene encoding the protein, the protein having an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 19, 20, 32, 43, 47, and conservatively modified versions thereof;
- and
- (ii) generating a three-dimensional structure of the protein encoded by the amino acid sequence.
Dependencies are inferred from the claim text.