Benzodiazepines allosterically modulate GABAA 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 α1 subunit M3 membrane-spanning segment. With the cysteine-substitution mutant α1F296Cβ1γ2 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 α subunits; whereas, in the presence of GABA, pCMBS- reacts with the cysteine in the other α subunit, or with both cysteines. This implies that the two α 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 α subunits in a GABAA receptor and provides new insights into the mechanism of allosteric potentiation by benzodiazepines.
- Ion channel
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience