Monitoring Motions in GABA-A Receptor Intersubunit Transmembrane Cavities

Abstract

GABA-A receptors (GABAARs) are the target for many therapeutic drugs including benzodiazepines (BZDs), barbiturates, anesthetics and neurosteroids. These drugs bind to distinct sites and potentiate GABA-induced currents. They are often used in combination but little is known about mechanism(s) underlying their interactions. Recent crystal structures of GLIC and the glutamate-activated chloride channel suggest agonist-mediated channel opening is associated with expansion of transmembrane interfaces between M3 and M1 helices of adjacent subunits. Here, we examined if binding of different allosteric drugs and combinations of these drugs to α1β2γ2L GABAARs induce similar motions. We also tested whether potentiation of GABA-mediated current by pairs of drugs binding to distinct sites are additive or super-additive.We individually inserted a cysteine in the M1 helices of each GABAAR subunit at α1I227, β2L223 and γ2LI238 to monitor motions at different intersubunit interfaces. We expressed wild-type and mutant GABAARs in Xenopus oocytes. We measured rates of modification of the substituted cysteines in the absence and presence of different combinations of allosteric modulators that bind to different sites: the intravenous anesthetics pentobarbital (PB) and etomidate, the neurosteroid THDOC and the benzodiazepine flurazepam. When applied alone, flurazepam and THDOC induced distinct motions in the intersubunit interfaces, flurazepam increased rate of modification of only γI238C whereas THDOC only increased the rate of βL223C. When flurazepam and THDOC were co-applied, no change in rate of modification of γI238C was observed indicating that the presence of THDOC inhibited the ability of flurazepam to elicit structural rearrangements near γI238C. The data suggest structural mechanisms underlying the effects of combining drugs are different than mechanisms underlying their actions alone.