Abstract
Pharmacological analyses of γ-aminobutyric acidA(GABAA) receptor subtypes have suggested that both the α and γ subunits, but not the β subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different γ subunits in the receptor macromolecule to identify amino acids γ2Phe77 and γ2Met130 as key determinants of the benzodiazepine binding site. γ2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-β-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, γ2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218,872, or methyl-β-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of γ1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that γ2Phe77 and γ2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the α subunit, we demonstrated a critical role for the γ subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, γ2Phe77 is homologous to α1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.
Footnotes
- Received June 11, 1997.
- Accepted August 8, 1997.
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Send reprint requests to: Dr. Paul Whiting, Neuroscience Research Centre, Terlings Park, Merck Sharp & Dohme, Eastwick Road, Harlow, Essex CM20–2QR, England. E-mail:paul_whiting{at}merck.com
- The American Society for Pharmacology and Experimental Therapeutics
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