Mutations at Beta N265 in c-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 b+/a2 inter-subunit interfaces. Early experiments in heteromeric receptors identified bN265 (M2-159) on b2 and b3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the b1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the bN265 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 bN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous a1b2N265Cc2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the b-265 sidechain. Using up to 300 mM etomidate, we found both an absence of etomidate effects on a1b2N265Cc2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive bN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in a1b2/3c2L GABAA receptors. Using a1M236C as a modifiable and anesthetic-protectable site occupancy reporter in b+/a2 interfaces, we found that bN265M reduced apparent anestheti