CDNA encoding the human alpha2 delta4 calcium channel subunit
AssigneeOrtho-McNeil Pharmaceutical, Inc.
InventorsNing Qin, Ellen Codd
Life of this patent
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Abstract
The present invention provides nucleic acid and polypeptide sequences describing an isoform of the α2δ-4 subunit of a voltage gated calcium channel. The nucleic acids described herein can be used to produce functional α2δ-4 protein. The calcium channel α2δ-4 protein may be isolated for the purposes of binding experiments or may be used in cells to form a functional calcium channel complex.
Claims22
An isolated and purified nucleic acid molecule encoding an α2δ-4 calcium channel subunit protein, said nucleic acid molecule comprising a member selected from the group consisting of:
- (a) a nucleic acid molecule encoding a protein having at least a 95% identity to a polypeptide comprising amino acids 1 to 1090 of SEQ ID NO:10;
- (b) a nucleic acid molecule that is complementary to the polynucleotide of (a);
- (c) a nucleic acid molecule comprising at least 15 sequential bases of the polynucleotide of (a) or (b);
- (d) a nucleic acid molecule that hybridizes under stringent conditions to the polynucleotide molecule of (a) and has at least a 95% identity to the nucleic acid encoding a polypeptide comprising amino acids 1 to 1090 of SEQ ID NO:10;
- (e) a nucleic acid molecule that encodes a splice variant of a human alpha 2 calcium channel comprising exon 1B;
- (f) a nucleic acid molecule that encodes a splice variant of a human alpha 2 calcium channel comprising exon 37B;
- and
- (g) a nucleic acid molecule that encodes a splice variant of a human alpha 2 calcium channel comprising exon 1B and exon 37B.
An expression vector to express an α2δ-4 calcium channel subunit protein in a recombinant host, wherein said vector contains a nucleic acid sequence encoding a α2δ-4 calcium channel subunit protein.
A protein, in substantially pure form having at least a 95% identity with a polypeptide comprising amino acids 1-1090 of SEQ ID NO.:10.
A monospecific antibody immunologically reactive with an α2δ-4 calcium channel subunit protein.
A method for expressing an α2δ-4 calcium channel subunit protein in a recombinant host cell, comprising the steps of:
- (a) transferring an expression vector capable of encoding an α2δ-4 calcium channel subunit protein into a cell;
- and
- (b) culturing the cells under conditions that allow expression of the α2δ-4 calcium channel subunit protein from the expression vector.
A method for identifying compounds that alter α2δ-4 calcium channel subunit protein activity in a cell, comprising the steps of:
- a) contacting a compound with a cell containing an α2δ-4 calcium channel subunit, and
- b) measuring a change in the cell in response to the contacting step.
A method comprising the steps of:
- (a) incubating a cell membrane from a cell expressing recombinant α2δ-4 with radioactive gabapentin (GBP) and a candidate compound, wherein the membrane comprises an α2δ-4 subunit of calcium channel and wherein the incubating step is for sufficient time to allow GBP binding to the α2δ-4 subunit of calcium channels in the cell membranes,
- (b) separating the cell membranes from unbound radioactive GBP,
- (c) measuring binding of the radioactive GBP to the cell membranes, and
- (d) identifying a compound that inhibits GBP binding by a reduction of the amount of radioactive GBP in step (c) to an established control.
A method for identifying compounds that alters α2δ-4 calcium channel subunit protein activity, comprising the steps of:
- (a) combining a compound, a measurably labeled ligand for the α2δ-4 calcium channel subunit protein, and a α2δ-4 calcium channel subunit protein, and
- (b) measuring binding of the compound to the subunit protein by a reduction in the amount labeled ligand binding to the α2δ-4 calcium channel subunit protein.
Dependencies are inferred from the claim text.