Structural Mechanisms Underlying Benzodiazepine Modulation of the GABA_AReceptor

Abstract

Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABA A receptors and modulating their function. The structural mechanisms by which BZD binding is transduced to potentiation or inhibition of GABAinduced current (I GABA ) are essentially unknown. Here, we explored the role of the ␥ 2 Q182-R197 region (Loop F/9) in the modulation of I GABA by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy-␤-carboline (DMCM)] BZD ligands. Each residue was individually mutated to cysteine, coexpressed with wild-type ␣ 1 and ␤ 2 subunits in Xenopus oocytes, and analyzed using two-electrode voltage clamp. Individual mutations differentially affected BZD modulation of I GABA . Mutations affecting positive modulation span the length of this region, whereas ␥ 2 W183C at the beginning of Loop F was the only mutation that adversely affected DMCM inhibition. Radioligand binding experiments demonstrate that mutations in this region do not alter BZD binding, indicating that the observed changes in modulation result from changes in BZD efficacy. Flurazepam and zolpidem significantly slowed covalent modification of ␥ 2 R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that ␥ 2 Loop F is a specific transducer of positive BZD modulator binding. Therefore, ␥ 2 Loop F plays a key role in defining BZD efficacy and is part of the allosteric pathway allowing positive BZD modulator-induced structural changes at the BZD binding site to propagate through the protein to the channel domain.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…It is not known whether Glu and IVM induce the same conformational change in the coupling loops. In the heteromeric α1β2γ2 GABA A receptor, for example, it was demonstrated that positive benzodiazepine modulators induce movements in loop F (β8β9 loop) of the γ2 subunit near the transmembrane channel domain (63). Such movements were not triggered by the binding of GABA, the allosteric modulator pentobarbital, or the inverse agonist methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (63).…”

An Epilepsy-Related Region in the GABAA Receptor Mediates Long-Distance Effects on GABA and Benzodiazepine Binding Sites

“…Likewise, the most direct path from ␤ 2 Arg117 to the neighboring BZD binding site is in the opposite direction across the ␥ 2 /␤ 2 interface and through the ␥ 2 subunit. It is noteworthy that if ␥ 2 Arg43, ␥ 2 Glu178, and ␤ 2 Arg117 interact, then ␥ 2 Glu178 is in a position to transmit perturbations along its backbone ␤-strand into loop F, which has been implicated in BZD efficacy (Hanson and Czajkowski, 2008). Thus, a simple explanation for the effects of the mutations ␥ 2 R43Q and ␤ 2 R117K on both GABA unbinding and DZ dissociation is that ␥ 2 Arg43 and ␤ 2 Arg117 participate in interactions between the ␥ 2 and ␤ 2 subunits that mediate ligand affinities at the two neighboring ␤/␣ and ␣/␥ intersubunit interfaces.…”

Structural and electronic effects of the C2’ substituent in 1,4–benzodiazepines

“…The compounds with -NO2 groups on C7 and -Cl at C2' present anticonvulsant activities and may have more affinity for another subtype receptor, such as the α3β2γ2 receptor [8,10,30]. This study contributes, with relevant data, to the systematic three-dimensional quantitative relations between molecular properties and their biological activity (3D-QSAR relationships) and aids the understanding of structural mechanisms of benzodiazepine modulation at the GABA receptors [31].…”