Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol

Background: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the ?-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the "+" of the ? subunit, in the ?-? interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the "-" side of the ? subunit, in the ?-? interface (or ?-? interface, in the case of homomeric ? receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the ?(H267) residue lines a cavity that docks propofol with favorable interaction energy. Method: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the ? subunit on activation of the ?1?3 GABA A receptor by propofol. Results: We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the ?-? interface leads to strong reduction in gating efficacy for propofol. Conclusion: We conclude that ?1?3 GABA A receptors can be activated by propofol interactions with the ?-?, ?-?, and ?-? interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.