Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a Mutation in Anesthetic Binding Sites on GABA Type A Receptors

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate a1b2/3c2 GABAA receptors by binding in overlapping transmembrane inter-subunit sites near bM286 and aL232 side chains. 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 a1b3M286Cc2L receptors to estimate the distance from etomidate to b3M286 with precision near 1.3 angstrom. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in a1L232Cb3c2L receptors; (ii) studies in a1L232Wb3M286Cc2L receptors assessed whether a1L232W displaces etomidate relative to b3M286C; 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 a1L232Cb3c2L 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 a1L232Wb3M286Cc2L 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 a1b3M286Cc2L reveals that a1L232W, which increases maximal sidechain length by 2.1 angstrom, displaces etomidate closer to b3M286C by about 1.3 angstrom. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 angstrom of a1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT We combined electrophysiology, cysteine substitutions, and n-alkylmethanethiosulfonate modifiers in functional GABAA receptors to enable precise estimates of the distance between 33M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane b+/a-inter-subunit pockets. Comparing results in a133M286Cg2L and a1L232W33M286Cg2L receptors reveals that a1L232W mutations displace both anesthetics toward 33M286C, indicating that these anesthetics bind within 1 angstrom of the a1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.