Mechanism of inhibition of GluA2 AMPA receptor channel opening by 2,3-benzodiazepine derivatives: Functional consequences of replacing a 7,8-methylenedioxy with a 7,8-ethylenedioxy moiety

2,3-Benzodiazepine (2,3-BDZ) compounds are a group of AMPA receptor inhibitors and are drug candidates for treating neurological diseases involving excessive AMPA receptor activity. We investigated the mechanism by which GluA2Q _flip receptor channel opening is inhibited by two 2,3-BDZ derivatives, i.e., 1-(4-aminophenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3- benzodiazepin-4-one (2,3-BDZ-11-2) and its 1-(4-amino-3-chlorophenyl) analogue (2,3-BDZ-11-4). Both compounds have a 7,8-ethylenedioxy moiety instead of the 7,8-methylenedioxy feature present in the structure of GYKI 52466, the prototypic 2,3-BDZ compound. Using a laser-pulse photolysis approach with a time resolution of ∼60 μs and a rapid solution flow technique, we characterized the effect of the two compounds on the channel opening process of the homomeric GluA2Q _flip receptor. We found that both 2,3-BDZ-11-2 and 2,3-BDZ-11-4 are noncompetitive inhibitors with specificity for the closed-channel conformation of the GluA2Q _flip receptor. However, 2,3-BDZ-11-4 is ∼10-fold stronger, defined by its inhibition constant for the closed-channel conformation (i.e., K _I = 2 μM), than 2,3-BDZ-11-2. From double-inhibitor experiments, we determined that both compounds bind to the same site, but this site is different from two other known, noncompetitive binding sites on the GluA2Q _flip receptor previously reported. Our results provide both mechanistic clues to improve our understanding of AMPA receptor regulation and a structure-activity relationship for designing more potent 2,3-BDZ compounds with predictable properties for this new noncompetitive site.