Evidence for distinct conformations of the two α1 subunits in diazepam-bound GABAA receptors

Benzodiazepines allosterically modulate GABA(A) receptors to increase currents induced by submaximal GABA concentrations. Benzodiazepine-induced conformational changes in the transmembrane domain increase the reactivity of cysteines substituted for a subset of residues in the alpha (1) subunit M3 membrane-spanning segment. With the cysteine-substitution mutant alpha (1)F296C beta (1)gamma (2) we previously noted that p-chloromercuribenzenesulfonate (pCMBS(-)) modification in the presence of diazepam potentiated subsequent GABA-induced currents. In contrast, pCMBS(-)) modification in the presence of GABA caused inhibition of subsequent responses. We now show that in the presence of diazepam, pCMBS(-) only reacts with the engineered cysteine in one of the two alpha subunits; whereas, in the presence of GABA, pCMBS(-) reacts with the cysteine in the other alpha subunit, or with both cysteines. This implies that the two alpha subunits have distinct conformations in the diazepam-bound state. Based on analysis of single channel kinetic data, others have hypothesized that diazepam only alters the GABA affinity of one of the two GABA binding sites. The results presented here provide structural evidence to support the hypothesis that diazepam binding only alters the conformation of one of the two alpha subunits in a GABA(A) receptor and provides new insights into the mechanism of allosteric potentiation by benzodiazepines. (C) 2001 Elsevier Science Ltd. All rights reserved.