| Benzodiazepines (BZDs) are clinically important drugs that exert their CNS actions by binding to the GABAA receptor 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 alpha1beta2gamma2 GABA A receptors 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.;Seven of the mutations assessed for their effects on BZD efficacy exhibit altered GABA kinetic effects apparent in two electrode voltage clamp experiments. This method is not as well suited to assess macroscopic kinetics and excised patch clamp recordings with rapid solution exchange were employed to evaluate these mutant receptors kinetically. &agr;S205C and &agr;G157C containing receptors both exhibited a decreased extent of desensitization after 1 sec in 10mM GABA and &agr;G157C containing receptors have a significantly reduce 10-90% rise time.;Taken together this data improves our knowledge of atypical BZDs interactions at the BZD binding site, but also provides some insight into how structural perturbations at subunit interfaces affect GABAA receptor functioning. These perturbations come from ligand occupancy, such as BZD presence in the BZD binding site, or by mutation, such as the cysteine mutations engineered into the BZD binding site. Also, this data paves the way for further experimentation to explore the effects of ligand orientation on channel function or for in silico modeling or drug screens. |
