Structural determinants of benzodiazepine allosteric regulation of GABAA receptor currents

Benzodiazepine enhancement of GABA(A) receptor current requires a gamma subunit, and replacement of the gamma subunit by the delta subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABAA receptors at the junction between alpha and gamma subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABAA receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using gamma 2L-delta GABA(A) receptor subunit chimeras coexpressed with beta 1 and beta 3 subunits in human embryonic kidney 293T cells. Different domains of the gamma 2L subunit were replaced by delta subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.