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Molecular Pharmacology, Volume 52, Issue 5, 874-881
Subunit of the -Aminobutyric
AcidA Receptor that Determine Ligand Binding and Modulation
at the Benzodiazepine Site
Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Harlow, Essex CM20 2QR, United Kingdom
Pharmacological analyses of 
-aminobutyric acidA
(GABAA) receptor subtypes have suggested that both the 
and subunits, but not the 
subunit, contribute to the
benzodiazepine binding site. We took advantage of the different
pharmacological properties conferred by the inclusion of different subunits in the receptor macromolecule to identify amino acids
2Phe77 and 2Met130 as key determinants of the benzodiazepine
binding site. 2Phe77 was required for high affinity binding of the
benzodiazepine site ligands flumazenil, CL218,872, and
methyl--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, 2Met130
was required for high affinity binding of flunitrazepam, clonazepam,
and triazolam but not flumazenil, CL218,872, or
methyl--carboline-3-carboxylate and did not affect benzodiazepine
efficacy. Introduction of the phenylalanine and methionine into the
appropriate positions of 1 was not sufficient to confer high
affinity for the benzodiazepine site ligand zolpidem. These data show
that 2Phe77 and 2Met130 are necessary for high affinity binding
of a number of benzodiazepine site ligands. Although most previous
studies have focused on the contribution of the subunit, we
demonstrated a critical role for the subunit at the benzodiazepine
binding site, indicating that this modulatory site is located at the
interface of these two subunits. Furthermore, 2Phe77 is homologous
to 1Phe64, 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.
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