TY - JOUR T1 - Monod-Wyman-Changeux Allosteric Shift Analysis in Mutant α1β3γ2L GABAA Receptors Indicates Selectivity and Cross-Talk Among Intersubunit Transmembrane Anesthetic Sites JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.118.115048 SP - mol.118.115048 AU - Andrea Szabo AU - Anahita Nourmahnad AU - Elizabeth Halpin AU - Stuart A Forman Y1 - 2019/01/01 UR - http://molpharm.aspetjournals.org/content/early/2019/01/28/mol.118.115048.abstract N2 - Background: Propofol, etomidate, and barbiturate anesthetics are allosteric co-agonists at pentameric α1β3γ2 GABAA receptors, modulating channel activation via four biochemically established inter-subunit transmembrane pockets. Etomidate selectively occupies the two β+/α— pockets, the barbiturate photolabel R-mTFD-MPAB occupies homologous α+/β— and γ+/β— pockets, and propofol occupies all four. Functional studies of mutations at M2-15′ or M3-36′ loci abutting these pockets provide conflicting results regarding their relative contributions to propofol modulation. Methods: We electrophysiologically measured GABA-dependent channel activation in α1β3γ2L or receptors with single M2-15′ (α1S270I, β3N265M, γ2S280W) or M3-36′ (α1A291W, β3M286W, γ2S301W) mutations, in the absence and presence of equi-potent clinical range concentrations of etomidate, R-mTFD-MPAB, and propofol. Estimated open probabilities were calculated and analyzed using global two-state Monod-Wyman-Changeux models to derive log(d) parameters proportional to anesthetic-induced channel modulating energies. Results: All mutations reduced log(d)s for anesthetics occupying both abutting and non-abutting pockets. The Δlog(d)s [log(d, mut) – log(d, wt)] for M2-15′ mutations abutting an anesthetic’s biochemically established binding sites were consistently larger than Δlog(d)s for non-abutting mutations, although this was not true for M3-36′ mutant Δlog(d)s. The sums of anesthetic-associated Δlog(d)s for sets of M2-15′ or M3-36′ mutations were all much larger than wild-type log(d)s. Conclusions: Mutant Δlog(d)s qualitatively reflect anesthetic site occupancy patterns. However, the lack of Δlog(d) additivity undermines quantitative comparisons of distinct site contributions to anesthetic modulation, because the mutations impaired both abutting anesthetic binding effects and positive cooperativity between anesthetic binding sites. ER -