Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-22
2004-01-20
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100, C435S320100, C435S325000, C536S023100, C536S023500
Reexamination Certificate
active
06680180
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
The invention provides isolated nucleic acid and amino acid sequences of Kv6.2, antibodies to Kv6.2, methods of detecting Kv6.2, methods of screening for voltage-gated potassium channel activators and inhibitors using biologically active Kv6.2, and kits for screening for activators and inhibitors of voltage gated potassium channels comprising Kv6.2.
BACKGROUND OF THE INVENTION
Potassium channels are involved in a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Potassium channels are thus found in a wide variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are made by alpha subunits that fall into 8 families, based on predicted structural and functional similarities (Wei et al.,
Neuropharmacology
35(7):805-829 (1997)). Three of these families (Kv, Eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families, CNG and SK/IK, also contain this motif but are gated by cyclic nucleotides and calcium, respectively. The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al.,
Proc. Natl. Acad. Sci. U.S.A.
94(25):14066-71 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al.,
J. Biol. Chem.
273:3509-16 (1998)). Another family, the inward rectifier potassium channels (Kir), belong to a structural family containing 2 transmembrane domains (see, e.g., Lagrutta et al.,
Jpn. Heart. J.
37:651-660 1996)), and an eighth functionally diverse family (TP, or “two-pore”) contains 2 tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels made from Kv, KQT and Slo or BK subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These beta subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels. formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al.,
J. Physiol.
493:625-633 (1996); Shi et al.,
Neuron
16(4):843-852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al.,
Nature
384:80-83 (1996)).
The Kv superfamily of voltage-gated potassium channels includes both heteromeric and homomeric channels that are typically composed of four subunits (see, e.g., Salinas et al.,
J. Biol. Chem.
272:8774-8780 (1997); Salinas et al.,
J. Biol. Chem.
272:24371-24379 (1997); Post et al.,
FEBS Letts.
399:177-182 (1996)). Voltage-gated potassium channels have been found in a wide variety of tissues and cell types and are involved in processes such as neuronal integration, cardiac pacemaking, muscle contraction, hormone section, cell volume regulation, lymphocyte differentiation, and cell proliferation (see, e.g., Salinas et al.,
J. Biol. Chem.
39:24371-24379 (1997)). Some alpha subunits of the Kv superfamily, of which the channels are composed, have been cloned and expressed, e.g., Kv5.1, Kv6.1 (Drewe et al.,
J. Neurosci.
12:538-548 (1992); Post et al.,
FEBS Letts.
399:177-182 (1996)); Kv8.1 (Hugnot et al.,
EMBO J.
15:3322-3331 (1996)); and Kv9.1 and 9.2 (Salinas et al.,
J. Biol. Chem.
39:24371-24379 (1997)). Expression patterns of some of these genes has also been examined (see, e.g., Verma-Kurvari et al.,
Mol. Brain. Res.
46:54-62 (1997); Maletic-Savatic et al.,
J. Neurosci.
15:3840-3851 (1995); Du et al.,
Neurosci.
84:37-48 (1998)).
SUMMARY OF THE INVENTION
The present invention thus provides for the first time Kv6.2, a polypeptide monomer that is an alpha subunit of an heteromeric voltage-gated potassium channel. Kv6.2 has not been previously cloned or identified, and the present invention provides the nucleotide and amino acid sequences for mouse and human Kv6.2.
In one aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having a monomer subunit association region that has greater than 70% amino acid sequence identity to a Kv6.2 subunit association region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In one aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having an S4-S6 region that has greater than 85% amino acid sequence identity to a Kv6.2 S4-S6 region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In one embodiment, the nucleic acid encodes mouse or human Kv6.2. In another embodiment, the nucleic acid encodes SEQ ID NO:1 or SEQ ID NO: 17. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:18.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of:
ATGCCCATGTCTTCCAGAGACAGG (SEQ ID NO:3), GATGTCTAGAGGGAGTTACATGTAGCG (SEQ ID NO:4) and GGCACTACGCATCCTCTACGTAATGCGC (SEQ ID NO:5), GATGATGGCCCACCAATAGGATGCGG (SEQ ID NO:6) and ATGCCCATGCCTTCCAGAGACGG (SEQ ID NO:7), TTACATGTGCATGATAGGCAAGGCTG (SEQ ID NO:8) and GTCCAGGCCCAAGACAAGTGTCAG (SEQ ID NO:9), GGGAGAAGGTGTGGAAGATAGACG (SEQ ID NO:10).
In one embodiment, the nucleic acid encodes a polypeptide monomer having a molecular weight of between about 53 kDa to about 65 kDa. In one embodiment, the nucleic acid selectively hybridizes under moderately stringent hybridization conditions to a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:18.
In another aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer, wherein the nucleic acid specifically hybridizes under highly stringent conditions to SEQ ID NO:2 or SEQ ID NO:18.
In another aspect, the present invention provides an isolated polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the potassium channel: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having a monomer subunit association region that has greater than 70% amino acid sequence identity to amino acids a Kv6.2 subunit association region; and (iv) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In another aspect, the present invention provides an isolated polypeptide monomer comprising an alpha s
Icagen, Incorporated
Kunz Gary
Seharaseyon Jegatheesan
Townsend and Townsend / and Crew LLP
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