Modification and protection at α1M236C reflects anesthetic site occupancy.

Panel A) Data represent mean ± SD peak current responses to GABA from oocytes (n = 4) expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, normalized to maximal GABA responses. Lines represent logistic fits to responses using GABA alone (solid circles; EC50 = 58 µM) and GABA with 3.2 µM etomidate (open circles; EC50 = 3.2 µM). Panel B) Data represent mean ± SD peak current responses to etomidate from oocytes (n = 3) expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, normalized to maximal GABA responses. The line represents a logistic fit with etomidate EC50 = 47 µM. Panel C) A single voltage-clamp current trace illustrating maximal GABA (3 mM; white bar above trace) efficacy in oocyte-expressed β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, enhanced with addition of 10 µM etomidate (black bar). Panel D) Data from panels A and B were renormalized to maximal GABA efficacy (methods; Eq. 2) and fitted with a global MWC equilibrium co-agonist equation (methods; Eq. 3). Lines through data points represents the fitted MWC model: L0 = 10,000; KG = 42±8.7 µM; c = 0.0075±0.00048; KE = 50±12 µM; d = 0.0089±0.00096. Panel E) Traces are from a single oocyte expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, demonstrating the effects of repeated pCMBS applications on the relative responses to low versus high GABA stimulation. Panel F) Points represent response ratios to low (EC10) vs. high GABA, normalized to pre-modification control values. Lines through data represent linear fits used to determine relative bimolecular modification rates: GABA+pCMBS (circles; 1200±57 M−1s−1); GABA+pCMBS +5 µM alphaxalone (triangles; 3700±270 M−1s−1); and GABA+pCMBS +32 µM etomidate (squares; 106±8.4 M−1s−1). Panel G) Summary of modification rate results (mean ± se) for all oocytes expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors under different conditions. The rate with pCMBS alone is significantly accelerated with addition of GABA and GABA/alphaxalone. Relative to GABA+alphaxalone, modification in the presence of GABA is slowed 65% by 3 µM etomidate, 95% by 32 µM etomidate, and 91% by 30 µM propofol. * p<0.05; ** p<0.01.