Mutations at Beta N265 in γ-Aminobutyric Acid Type A Receptors Alter Both Binding Affinity and Efficacy of Potent Anesthetics

Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane beta+/alpha- inter-subunit interfaces. Early experiments in heteromeric receptors identified beta N265 (M2-15') on beta 2 and beta 3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the beta 1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the beta N265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between beta N265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous alpha 1 beta 2N265C gamma 2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the beta-265 sidechain. Using up to 300 mu M etomidate, we found both an absence of etomidate effects on alpha 1 beta 2N265C gamma 2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive beta N265M mutants, we applied indirect structurefunction strategies, exploiting second mutations in alpha 1 beta 2/3 gamma 2L GABAA receptors. Using alpha 1M236C as a modifiable and anesthetic-protectable site occupancy reporter in beta+/alpha- interfaces, we found that beta N265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. beta N265M also impaired the transduction of gating effects associated with alpha 1M236W, a mutation that mimics beta+/alpha- anesthetic site occupancy. Our results show that beta N265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in beta+/alpha- sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for beta N265 in anesthetic binding within the beta+/alpha- transmembrane sites.