Two etomidate sites in α1β2γ2 γ-aminobutyric acid type a receptors contribute equally and noncooperatively to modulation of channel gating
Background: Etomidate is a potent hypnotic agent that acts via γ-aminobutyric acid receptor type A (GABAA) receptors. Evidence supports the presence of two etomidate sites per GABAA receptor, and current models assume that each site contributes equally and noncooperatively to drug effects. These assumptions remain untested. Methods: We used concatenated dimer (β2-α1) and trimer (γ2-β2-α1) GABAA subunit assemblies that form functional α1β2γ2 channels, and inserted α1M236W etomidate site mutations into both dimers (β2-α1M236W) and trimers (γ2-β2-α1M236W). Wild-type or mutant dimers (D wt or Dαm236w) and trimers (T wt or T αm236w) were coexpressed in Xenopus oocytes to produce four types of channels: DT wt, DT wt, D αm236wT wt, and D αm236wT. For each channel type, two-electrode voltage clamp was performed to quantitatively assess GABA EC 50, etomidate modulation (left shift), etomidate direct activation, and other functional parameters affected by αM236W mutations. Results: Concatenated wild-type DT channels displayed etomidate modulation and direct activation similar to α1β2γ2 receptors formed with free subunits. DT receptors also displayed altered GABA sensitivity and etomidate modulation similar to mutated channels formed with free subunits. Both single-site mutant receptors (DT and DT) displayed indistinguishable functional properties and equal gating energy changes for GABA activation (-4.9 ± 0.48 vs.-4.7 ± 0.48 kJ/mol, respectively) and etomidate modulation (-3.4 ± 0.49 vs.-3.7 ± 0.38 kJ/mol, respectively), which together accounted for the differences between DT and DT channels. Conclusions: These results support the hypothesis that the two etomidate sites on α1β2γ2 GABAA receptors contribute equally and noncooperatively to drug interactions and gating effects. © 2012 the American Society of Anesthesiologists, Inc. Lippincott Williams &amp; Wilkins. Anesthesiology.