TWIK-5 potassium channel polypeptides and uses therefor

Details TWIK-5 potassium channel polypeptides and uses therefor TWIK-5 potassium channel polypeptides and uses therefor

2000-04-28

2003-12-16

Eyler, Yvonne (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S069100, C435S007200

Reexamination Certificate

active

06664373

ABSTRACT:

BACKGROUND OF THE INVENTION
Potassium (K
+
) channels are ubiquitous proteins which are involved in the setting of the resting membrane potential as well as in the modulation of the electrical activity of cells. In excitable cells, K
+
channels influence action potential waveforms, firing frequency, and neurotransmitter secretion (Rudy, B. (1988)
Neuroscience,
25, 729-749; Hille, B. (1992)
Ionic Channels of Excitable Membranes,
2nd Ed.). In non-excitable cells, they are involved in hormone secretion, cell volume regulation and potentially in cell proliferation and differentiation (Lewis et al. (1995)
Annu. Rev. Immunol.,
13, 623-653). Developments in electrophysiology have allowed the identification and the characterization of an astonishing variety of K
+
channels that differ in their biophysical properties, pharmacology, regulation and tissue distribution (Rudy, B. (1988)
Neuroscience,
25, 729-749; Hille, B. (1992)
Ionic Channels of Excitable Membranes,
2nd Ed.). More recently, cloning efforts have shed considerable light on the mechanisms that determine this functional diversity. Furthermore, analyses of structure-function relationships have provided an important set of data concerning the molecular basis of the biophysical properties (selectivity, gating, assembly) and the pharmacological properties of cloned K
+
channels.
Functional diversity of K
+
channels arises mainly from the existence of a great number of genes coding for pore-forming subunits, as well as for other associated regulatory subunits. Two main structural families of pore-forming subunits have been identified. The first one consists of subunits with a conserved hydrophobic core containing six transmembrane domains (TMDs). These K
+
channel a subunits participate in the formation of outward rectifier voltage-gated (Kv) and Ca
2+
-dependent K
+
channels. The fourth TMD contains repeated positive charges involved in the voltage gating of these channels and hence in their outward rectification (Logothetis et al. (1992)
Neuron,
8, 531-540; Bezanilla et al. (1994)
Biophys. J.
66, 1011-1021).
The second family of pore-forming subunits have only two TMDs. They are essential subunits of inward-rectifying (IRK), G-protein-coupled (GIRK) and ATP-sensitive (K
ATP
) K
+
channels. The inward rectification results from a voltage-dependent block by cytoplasmic Mg
2+
and polyamines (Matsuda, H. (1991)
Annu. Rev. Physiol.,
53, 289-298). A conserved domain, called the P domain, is present in all members of both families (Pongs, O. (1993)
J. Membr. Biol.,
136, 1-8; Heginbotham et al. (1994)
Biophys. J.
66,1061-1067; Mackinnon, R. (1995)
Neuron,
14, 889-892; Pascual et al., (1995)
Neuron.,
and 14, 1055-1063). This domain is an essential element of the aqueous K
+
-selective pore. In both groups, the assembly of four subunits is necessary to form a functional K
+
channel (Mackinnon, R. (1991)
Nature,
350, 232-235; Yang et al., (1995)
Neuron,
15, 1441-1447.
In both six TMD and two TMD pore-forming subunit families, different subunits coded by different genes can associate to form heterotetramers with new channel properties (Isacoffet al., (1990)
Nature,
345, 530-534). A selective formation of heteropolymeric channels may allow each cell to develop the best K
+
current repertoire suited to its function. Pore-forming &agr; subunits of Kv channels are classified into different subfamilies according to their sequence similarity (Chandy et al. (1993)
Trends Pharmacol. Sci.,
14, 434). Tetramerization is believed to occur preferentially between members of each subgroup (Covarrubias et al. (1991)
Neuron,
7, 763-773). The domain responsible for this selective association is localized in the N-terminal region and is conserved between members of the same subgroup. This domain is necessary for hetero- but not homomultimeric assembly within a subfamily and prevents co-assembly between subfamilies. Recently, pore-forming subunits with two TMDs were also shown to co-assemble to form heteropolymers (Duprat et al. (1995)
Biochem. Biophys. Res. Commun.,
212, 657-663. This heteropolymerization seems necessary to give functional GIRKs. IRKs are active as homopolymers but also form heteropolymers.
New structural types of K
+
channels were identified recently in both humans and yeast. These channels have two P domains in their functional subunit instead of only one (Ketchum et al. (1995)
Nature,
376, 690-695; Lesage et al. (1996)
J. Biol. Chem,
271, 4183-4187; Lesage et al. (1996)
EMBO J.,
15, 1004-1011; Reid et al. (1996)
Receptors Channels
4, 51-62). The human channel called TWIK-1, has four TMDs. TWIK-1 is expressed widely in human tissues and is particularly abundant in the heart and the brain. TWIK-1 currents are time independent and inwardly rectifying. These properties suggest that TWIK-1 channels are involved in the control of the background K
+
membrane conductance (Lesage et al. (1996)
EMBO J.,
15, 1004-1011).
Table of Contents
Summary of the Invention
Brief Description of the Drawings
Detailed Description of the Invention
I. Isolated Nucleic Acid Molecules
II. Isolated TWIK Proteins and Anti-TWIK Antibodies
III. Recombinant Expression Vectors and Host Cells
IV. Pharmaceutical Compositions
V. Uses and Methods of the Invention
A. Screening Assays
B. Detection Assays
1. Chromosene Mapping
2. Tissue Typing
3. Use of Partial TWIK Sequences in Forensic Biology
C. Predictive Medicine
1. Diagnostic Assays
2. Prognostic Assays
3. Monitoring of Effects During Clinical Trials
D. Methods of Treatment
1. Prophylactic Methods
2. Therapeutic Methods
3. Pharmacogenomics
Examples
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel members of the TWIK (for Tandem of P domains in a Weak Inward rectifying K
+
channel) family of potassium channels, referred to herein as TWIK-2, TWIK-3, TWIK-4, and TWIK-5 nucleic acid and protein molecules. The TWIK-2, TWIK-3, TWIK-4, and TWIK-5 molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding TWIK proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of TWIK-encoding nucleic acids.
In one embodiment, a TWIK nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:10, SEQ ID NO:12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1640, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-9 of SEQ ID NO:1. In yet another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1507-3452 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 1644 nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1-121 of SEQ ID NO:4. In yet another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1118-1575 of SEQ ID NO:4. In another preferred embodiment, the nucleic acid molecule has the

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