Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-10-28
2003-08-26
Pak, Michael (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06610827
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to potassium channel subunit polypeptide and polynucleotide compositions, to the production of these compositions, and to the use of the compositions in the diagnosis, prevention, and treatment of disease.
BACKGROUND OF THE INVENTION
Potassium channels are a heterogeneous group of ion channels that allow selective permeation of potassium ions across the plasma membrane, but differ in details of activation mechanism, voltage range of activity, and kinetic properties. (Hille, B. (1992)
Ionic Channels of Excitable Membranes
, 2nd Ed., Sinauer, Sunderland, Mass.; Latorre, R. and Miller, C. (1983) J. Memb. Biol. 7:11-30). They contribute to numerous physiological functions, for example, action potential repolarization, cardiac pacemaking, neuron bursting, muscle contraction, hormone secretion, vascular tone regulation, renal ion reabsorption, learning and memory, and cell growth and differentiation.
Voltage-gated potassium (Kv) channels are critical determinants of excitability in nerve and muscle cells, where they regulate impulse conduction, rhythmicity, and synaptic transmission. These channels form the largest and most diversified family of ion channels. At least six subfamilies of these channels have been identified: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), Kv4 (Shal), KvLQT and EAG. Such channels are formed by the association of channel subunits, either of the same subunit (forming homomeric channels) or of different subunits (forming heteromeric channels). Kv channel subunits are characterized structurally by the presence of six transmembrane domains (S1-S6), one of which is highly positively charged, and a pore region between S5 and S6.
Regulation of Kv channel function can occur by association of different Kv subunits, resulting in heteromeric Kv channels having different conductance properties. Electrically silent Kv subunits, which by themselves do not form active homomeric channels, can modulate the conductance properties of Kv channels by associating with electrically active Kv subunits. For example, electrically-silent Kv subunits Kv6.1 and Kv8.1 modulate the conductance properties of Kv2 (Shab) channels by associating with Kv2.1 and Kv2.2 subunits, forming heteromeric Kv channels with conductance properties differing from the homomeric Kv2.1 and Kv2.2 channels (Post, M. A., et al. (1996) FEBS Lett. 399:177-182; Salinas. M. et al. (1997) J. Biol. Chem. 272:8774-8780)
Potassium channels are associated with a variety of disease states. In some diseases and disorders, abnormal ion channels are believed to be causative factors, while other diseases appear to arise from inappropriate regulation of otherwise normal ion channels. Diseases believed to have a particular association with potassium channels include neurological, cardiovascular, musculoskeletal, and proliferative disorders.
Potassium channel proteins are useful targets for drug therapy in a variety of neurological and vascular disease conditions. One such neurological condition is epilepsy, fundamentally a disease of neuronal overexcitability, resulting from excessive and synchronous firing of a large population of neurons in the cerebral cortex. In addition, many other neuropsychiatric diseases are consequences of abnormal neuronal activity in the cerebral cortex. Ideally, it would be desirable to treat epilepsy and related neuropsychiatric conditions with compounds that act selectively on ion channels in the cerebral cortex, to reduce side effects related to more generalized effects on the patient's nervous system. To this end, it would be desirable to isolate, characterize, and recombinantly express human ion channel proteins (i) whose activities are related to neuronal excitability, and (ii) which are localized in the cerebral cortex.
A vascular condition associated with potassium ion current is pulmonary artery (PA) hypertension. Reduced voltage-dependent potassium current in PA smooth muscle cells leads to PA vasoconstriction, pulmonary hypertension and heart failure. It would therefore be desirable to treat pulmonary hypertension with compounds that restore potassium current in PA smooth muscle. Furthermore, it has been demonstrated that the appetite suppressant fenfluramine, which potentiates serotonin release in the brain, inhibits vascular smooth muscle potassium currents and is a causative agent in pulmonary hypertension (Weir E. K. et al. (1996) Circulation 94:2216-2220). It would therefore be desirable to isolate, characterize, and recombinantly express human ion channel proteins which are expressed in muscle tissues such as pulmonary artery smooth muscle, for use as a screening target to identify channel modulators useful in treating pulmonary hypertension and other cardiovascular and musculoskeletal disorders. Such channel proteins would also be useful in identifying compounds, such as serotonin release potentiators, which do not modulate such channels and thus lack hypertensive side-effects.
SUMMARY OF THE INVENTION
The invention includes proteins having sequence similarity to the Shab subfamily of voltage-gated potassium channel subunits and identified herein as Shab-like voltage-gated potassium channel subunits-1 and -2 (Kv-SL1 and Kv-SL2, and collectively as Kv-SL). The invention includes a substantially purified KV-SL protein having an amino acid sequence at least 85% identical to the sequence identified as SEQ ID NO:3 or SEQ ID NO:7 and which is capable of associating with one or more Kv subunits to form a Kv channel. In other embodiments, Kv-SL protein has a sequence at least 90% identical, preferably at least 95% identical to SEQ ID NO:3 or SEQ ID NO:7. In another embodiment, Kv-SL1 protein includes a portion between about amino acids 223 and 481 having a sequence selected from the group consisting of (a) the sequence between amino acids 223 and 481 of SEQ ID NO:3, (b) the sequence SEQ ID NO:5, and (c) internally consistent variations between sequences (a) and (b). In another embodiment, Kv-SL2 protein includes a portion between about amino acids 170 and 491 having a sequence selected from the group consisting of (a) the sequence between amino acids 170 and 491 of SEQ ID NO:7, (b) the sequence SEQ ID NO:9, and (c) internally consistent variations between sequences (a) and (b). In a more specific embodiment, Kv-SL1 protein has the sequence SEQ ID NO:3. In another specific embodiment, Kv-SL2 protein has the sequence SEQ ID NO:5 or SEQ ID NO:12. In another embodiment, Kv-SL protein is a human protein. The invention also includes fragments of Kv-SL protein, which are antigenic or which are capable of interacting with other proteins, peptides, or chemicals, such interaction which alters the functional properties or cellular/subcellular localization of Kv-SL protein or a Kv channel comprising Kv-SL. In one embodiment, the fragment corresponds to an intracellular domain of Kv-SL protein.
In another aspect the invention includes an isolated nucleic acid having a sequence which encodes Kv-SL as described above, or a sequence complementary to the Kv-SL coding sequence, and a composition comprising the nucleic acid. The nucleic acid may be mRNA, cRNA, DNA, cDNA, genomic DNA, or an antisense analog thereof. In various embodiments the nucleic acid may encode a Kv-SL protein having an amino acid sequence at least 85%, 90%, 95%, or 97% identical to SEQ ID NO:3 or SEQ ID NO:7. In another embodiment, the nucleic acid may encode a Kv-SL1 protein which includes a portion between about amino acids 223 and 481 having a sequence selected from the group consisting of (a) the sequence between amino acids 223 and 481 of SEQ ID NO:3, (b) the sequence SEQ ID NO:5, and (c) internally consistent variations between sequences (a) and (b). In another embodiment, the nucleic acid may encode a Kv-SL2 protein which includes a portion between about amino acids 170 and 491 having a sequence selected from the group consisting of (a) the sequence between amino acids 170 and 491 of SEQ ID NO:7, (b) the sequence SEQ ID NO:9, and (c) internally consistent variations between sequence
Chavez Raymond A.
Forsayeth John R.
Zhao Byron
Elan Pharmaceuticals Inc.
Gorthey LeeAnn
Pak Michael
Perkins Coie LLP
Petithory Joanne R.
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