Benzodiazepine actions: Teasing apart drug binding from drug efficacy

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Benzodiazepines (BZDs) are clinically important drugs that exert their CNS actions by binding to the GABAA receptor (GABAAR) and allosterically modulating GABA‐activated chloride currents (IGABA). Their actions depend on residues in the BZD binding site that mediate their high‐affinity binding as well as residues that mediate local movements in the site important for coupling BZD binding to modulation of IGABA (efficacy). To identify and distinguish residues involved in each of these functions, we made 22 mutations in and surrounding the BZD binding pocket. To assess BZD binding, mutant α1β2γ2 GABAARs were expressed in HEK293T cells and radioligand binding assays used to measure BZD affinity. To identify residues that contribute to drug efficacy, mutant receptors were expressed in Xenopus laevis oocytes, characterized using two‐electrode voltage clamp and BZD maximal potentiation of IGABA was measured. We identified six residues whose mutation altered BZD efficacy without altering BZD binding affinity, three residues whose mutation altered binding but had no effect on efficacy, and four residues whose mutation affected both binding and efficacy. These data advance our understanding of allosteric modulation and will aid in future rational drug design.