Structural Determinants of Benzodiazepine Allosteric Regulation of GABAAReceptor Currents

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Abstract
Benzodiazepine enhancement of GABAAreceptor current requires a γ subunit, and replacement of the γ subunit by the δ subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABAAreceptors at the junction between α and γ subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABAAreceptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using γ2L-δ GABAAreceptor subunit chimeras coexpressed withα1 andβ3 subunits in human embryonic kidney 293T cells. Different domains of the γ2L subunit were replaced by δ 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.