Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a...

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate α1β2/3γ2 GABA_A receptors by binding in overlapping transmembrane inter-subunit sites near βM286 and αL232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in α1β3M286Cγ2L receptors to estimate the distance from etomidate to β3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in α1L232Cβ3γ2L receptors; (ii) studies in α1L232Wβ3M286Cγ2L receptors assessed whether α1L232W displaces etomidate relative to β3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in Xenopus oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In α1L232Cβ3γ2L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In α1L232Wβ3M286Cγ2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in α1β3M286Cγ2L reveals that α1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to β3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of α1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABA_A receptors to enable precise estimates of the distance between β3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane β+/α- inter-subunit pockets. Comparing results in α1β3M286Cγ2L and α1L232Wβ3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward β3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.