Human theta subunit of the GABAa receptor
AssigneeMerck Sharp & Dohme Corp.
InventorsTimothy Peter Bonnert, Paul John Whiting
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Abstract
This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABAA receptor. In addition, the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.
Claims24
A stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor, which receptor comprises a θ receptor subunit of SEQ ID NO:4 and at least one receptor subunit.
A process for the preparation of an eukaryotic cell line capable of expressing a human GABAA receptor, which comprises stably co-transfecting a eukaryotic host cell with at least two expression vectors, one such vector comprising the cDNA sequence encoding the human θ GABAA receptor subunit of SEQ ID NO:4 and one such vector comprising the cDNA sequence encoding an α GABAA receptor subunit.
A recombinant nucleic acid molecule encoding the θ subunit of the human GABAA receptor comprising a sequence selected from the group consisting of the sequence depicted in SEQ ID NO:3;
- the complement of said sequence, and sequences having at least 95% homology to SEQ ID NO:3 or its complement, wherein a human GABAA receptor having the subunit combination α2β1θ1γ1 has a lower affinity for GABA as compared to a human GABAA receptor having the subunit combination α2β1θ1γ1.
A recombinant expression vector comprising a human GABAA receptor θ subunit nucleotide sequence selected from the group of SEQ ID NO:3, the complement of said sequence, and sequences having 95% homology to SEQ ID NO:
- 3 or its complement, together with additional sequences capable of directing the synthesis of said human GABAA receptor θ subunit in cultures of transfected eukaryotic cells, wherein a human GABAA receptor having the subunit combination α2β1θ1γ1 has a lower affinity for GABA as compared to a human GABAA receptor having the subunit combination α2β1θ1γ1.
A recombinantly produced protein preparation comprising a GABAA receptor having subunit combinations comprising a θ receptor subunit of SEQ ID NO:4 and at least one a receptor subunit.
A recombinantly produced membrane preparation comprising a GABAA receptor having subunit combinations comprising a θ receptor subunit of SEQ ID NO:4 and at least one α receptor subunit.
A method for determining whether a ligand, not known to be capable of binding to a human GABAA receptor comprising a θ subunit, can bind to a human GABAA receptor comprising a θ subunit, which comprises
- (a) contacting a mammalian cell comprising DNA molecules encoding at least one a receptor subunit and a θ receptor subunit of SEQ ID NO:4 with the ligand under conditions permitting binding of ligands known to bind to the GABAA receptor,
- (b) detecting the presence or absence of any of the ligand bound to the GABAA receptor comprising a θ subunit and
- (c) thereby determining whether the ligand binds to the GABAA receptor comprising a θ subunit.
A method of screening drugs to identify drugs which specifically interact with, and bind to, a human GABAA receptor comprising a θ subunit on the surface of a cell which comprises
- (a) contacting a mammalian cell comprising a human GABAA receptor comprising at least one α receptor subunit and a θ receptor subunit of SEQ ID NO:4, on the surface of said cell with a plurality of drugs,
- (b) determining which of those drugs bind to the mammalian cell, and
- (c) thereby identifying drugs specifically interact with, and bind to, human GABAA receptors comprising the θ subunit.
A recombinant GABAA receptor θ subunit polypeptide which has an amino acid sequence depicted in SEQ ID NO:4.
A stably co-transfected eukaryotic cell line which expresses a human GABAA receptor, which receptor comprises the subunit combination α2β1θγ1, wherein the θ receptor subunit has the sequence of SEQ ID NO:4.
A process for the preparation of an eukaryotic cell line capable of expressing a human GABAA receptor, said receptor comprising the subunit combination α2β1θγ1, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4, said process comprising stably co-transfecting a eukaryotic host cell with at least two expression vectors, one such vector comprising the cDNA sequence encoding the human θ GABAA receptor subunit and one such vector comprising the cDNA sequence encoding an alpha GABAA receptor subunit.
A recombinant nucleic acid molecule encoding the θ subunit of the human GABAA receptor, said θ subunit having the sequence depicted in SEQ ID NO:4.
A recombinant expression vector comprising the nucleotide sequence of the human GABAA receptor theta subunit sequence depicted in SEQ ID NO:4, together with additional sequences capable of directing the synthesis of the said human GABAA receptor θ subunit in cultures of transfected eukaryotic cells.
A recombinantly produced protein preparation comprising a GABAA receptor having the subunit combination α2β1θγ1, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4.
A recombinantly produced membrane preparation comprising a GABAA receptor having the subunit combination α2β1θγ1, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4.
A method for determining whether a ligand not known to be capable of binding to a human GABAA receptor can bind to said receptor, said receptor having the subunit combination α2β1θγ12, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4, which method comprises
- (a) contacting a mammalian cell expressing a human GABAA receptor having the subunit combination α2β1θγ1, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4, with the ligand under conditions permitting binding of ligands known to bind to the GABAA receptor,
- (b) detecting the presence or absence of any of the ligand bound to the GABAA receptor comprising the θ subunit and
- (c) thereby determining whether the ligand binds to the GABAA receptor.
A method of screening drugs to identify drugs which specifically interact with, and bind to, a human GABAA receptor on the surface of a cell, the receptor having the subunit combination α2β1θγ1, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4, which method comprises
- (a) contacting a mammalian cell expressing a human GABAA receptor having the subunit combination α2β1θγ1, wherein said θ receptor subunit has the sequence depicted in SEQ ID NO:4, on the surface of said cell with a plurality of drugs,
- (b) determining which of those drugs bind to the mammalian cell, and
- (c) thereby identifying drugs specifically interact with, and bind to, the human GABAA receptor.
A recombinant GABAA receptor theta subunit polypeptide which has an amino acid sequence depicted in SEQ ID NO:4.
Dependencies are inferred from the claim text.