Substituted Cysteine Modification and Protection with -Alkyl- Methanethiosulfonate Reagents Yields a Precise Estimate of the Distance between Etomidate and a Residue in Activated GABA Type A Receptors.

The anesthetic etomidate modulates synaptic 12/32 GABA receptors via binding sites located in transmembrane +/- interfaces. Various approaches indicate that etomidate binds near 2/3M286 side chains, including recent cryogenic electron microscopy images in 122L receptors under nonphysiologic conditions with ∼3.5-Å resolution. We hypothesized that substituted cysteine modification and protection experiments using variably sized -alkyl-methanethiosulfonate (MTS) reagents could precisely estimate the distance between bound etomidate and 3M286 side chains in activated functional receptors. Using voltage-clamp electrophysiology in oocytes expressing 13M286C2L GABA receptors, we measured functional changes after exposing GABA-activated receptors to -alkyl-MTS reagents, from methyl-MTS to -decyl-MTS. Based on previous studies using a large sulfhydryl reagent, we anticipated that cysteine modifications large enough to overlap etomidate sites would cause persistently increased GABA sensitivity and decreased etomidate modulation and that etomidate would hinder these modifications, reducing effects. Based on altered GABA or etomidate sensitivity, ethyl-MTS and larger -alkyl-MTS reagents modified GABA-activated 13M286C2L GABA receptors. Receptor modification by -propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased etomidate modulation. Receptor-bound etomidate blocked 3M286C modification by -propyl-MTS, -butyl-MTS, and -hexyl-MTS. In contrast, GABA sensitivity was unaltered by receptor exposure to methyl-MTS or ethyl-MTS, and ethyl-MTS modification uniquely increased etomidate modulation. These results reveal a "cut-on" between ethyl-MTS and -propyl-MTS, from which we infer that --(-propyl) is the smallest 3M286C appendage that overlaps with etomidate sites. Molecular models of the native methionine and --ethyl and --(-propyl) modified cysteines suggest that etomidate is located between 1.7 and 3.0 Å from the 3M286 side chain. SIGNIFICANCE STATEMENT: Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. This study combined electrophysiology, a cysteine substitution, and -alkyl-methanethiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1β3γ2L GABA type A receptor residue and etomidate bound in the transmembrane +/- interface.