Stably transfected rodent fibroblast cell lines expressing human

Details Stably transfected rodent fibroblast cell lines expressing human Stably transfected rodent fibroblast cell lines expressing human

1994-11-17

1997-07-29

Ulm, John

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 22, 435 641, 435352, 435357, C12Q 168, G01N 3353

Patent

active

056521006

DESCRIPTION:

BRIEF SUMMARY
This invention concerns a cell line, and in particular relates to a stable cell line capable of expressing human or animal GABA.sub.A receptors. The invention further concerns the cloning of novel cDNA sequences encoding particular subunits of the human GABA.sub.A receptor. In addition, the invention relates to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.
Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABA.sub.A receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates and .beta.-carbolines, in addition to sites for the agonist ligand GABA (for reviews see Stephenson, Biochem. J., 1988, 249, 21; Olsen and Tobin, Faseb J., 1990, 4, 1469; and Sieghart, Trends in Pharmacol. Sci., 1989, 10, 407).
Molecular biological studies demonstrate that the receptor is composed of several distinct types of subunit, which are divided into four classes (.alpha., .beta., .gamma., and .delta.) based on their sequence similarities. To date, six types of .alpha. (Schofield et al., Nature (London), 1987, 328, 221; Levitan et al., Nature (London), 1988, 335, 76; Ymer et al., EMBO J., 1989, 8, 1665; Pritchett & Seeberg, J. Neurochem., 1990, 54, 802; Luddens et al., Nature (London), 1990, 346, 648; and Khrestchatisky et al., Neuron, 1989, 3, 745), three types of .beta. (Ymer et al., EMBO J., 1989, 8, 1665), two types of .gamma. (Ymer et al., EMBO J., 1990, 9, 3261; and Shivers et al., Neuron, 1989, 3, 327) and one .delta. subunit (Shivers et al., Neuron, 1989, 3, 327) have been identified.
The differential distribution of many of the subunits has been characterised by in situ hybridisation (Sequier et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 7815; Malherbe et al., J. Neurosci., 1990, 10, 2330; and Shivers et al., Neuron, 1989, 3, 327) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex.
Various combinations of subunits have been co-transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide GABA.sub.A receptors in vivo (Pritchett et al., Science, 1989, 245, 1389; Malherbe et al., J. Neurosci., 1990, 10, 2330; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802; and Luddens et al., Nature (London), 1990, 346, 648). This approach has revealed that, in addition to an .alpha. and .beta. subunit, either .gamma..sub.1 or .gamma..sub.2 (Pritchett et al., Nature (London), 1989, 338, 582; Ymer et al., EMBO J., 1990, 9, 3261; and Malherbe et al., J. Neurosci., 1990, 10, 2330) or .gamma..sub.3 (Herb et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1433; Knoflach et al., FEBS Lett., 1991, 293, 191; and Wilson-Shaw et al., FEBS Lett., 1991, 284, 211) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the .alpha. and .gamma. subunits present. Receptors containing a .delta. subunit (i.e. .alpha..beta..delta.) do not appear to bind benzodiazepines (Shivers et al., Neuron, 1989, 3, 327). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ.sub.1 type receptor (.alpha..sub.1 .beta..sub.1 .gamma..sub.2) and a BZ.sub.2 type receptor (.alpha..sub.2 .beta..sub.1 .gamma..sub.2 or .alpha..sub.3 .beta..sub.1 .gamma..sub.2, Pritchett et al., Nature (London), 1989, 338, 582), as well as two GABA.sub.A receptors with a novel pharmacology, .alpha..sub.5 .beta..sub.2 .gamma..sub.2 (Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802) and .alpha..sub.6 .beta..sub.2 .gamma..sub.2 (Luddens et al., Nature (London), 1990, 346, 648). Although the pharmacology of these expressed receptors appears similar to that of those identified in brain t

REFERENCES:
patent: 5166066 (1992-11-01), Carter
S.J. Moss et al., Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties, European Journal of Pharm., Molec. Pharm., 189: pp. 77-88 (1990).
Pritchett et al, Nature 338:582-585, 13 Apr. 1989.
Wagstaff et al. Am. J. Hum. Genet. 49:330-337, 1991.

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