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 12/32 GABA receptors by binding in overlapping transmembrane inter-subunit sites near M286 and L232 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 13M286C2L receptors to estimate the distance from etomidate to 3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in 1L232C32L receptors; (ii) studies in 1L232W3M286C2L receptors assessed whether 1L232W displaces etomidate relative to 3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in 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 1L232C32L 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 1L232W3M286C2L 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 13M286C2L reveals that 1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to 3M286C 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 1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABA receptors to enable precise estimates of the distance between β3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane +/ 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.