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 gamma-aminobutyric acid type A (GABA(A)) 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 beta subunit, in the beta-alpha 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 beta subunit, in the alpha-beta interface (or beta-beta interface, in the case of homomeric beta receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the beta(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 beta subunit on activation of the alpha 1 beta 3 GABAA 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 alpha-beta interface leads to strong reduction in gating efficacy for propofol. Conclusion: We conclude that alpha 1 beta 3 GABAA receptors can be activated by propofol interactions with the beta-beta, alpha-beta, and beta-alpha 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.