PT  - JOURNAL ARTICLE
AU  - Ryan J. Fantasia
AU  - Anahita Nourmahnad
AU  - Elizabeth A Halpin
AU  - Stuart A. Forman
TI  - Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Yields a Precise Estimate of the Distance Between Etomidate and a Residue in Activated GABA Type A Receptors
AID  - 10.1124/molpharm.120.000224
DP  - 2021 Jan 01
TA  - Molecular Pharmacology
PG  - MOLPHARM-AR-2020-000224
4099  - http://molpharm.aspetjournals.org/content/early/2021/03/24/molpharm.120.000224.short
4100  - http://molpharm.aspetjournals.org/content/early/2021/03/24/molpharm.120.000224.full
AB  - The anesthetic etomidate modulates synaptic α1β2/3γ2 GABAA receptors via binding sites located in transmembrane β+/α- interfaces. Various approaches indicate that etomidate binds near β2/3M286 sidechains, including recent cryogenic electron microscopy images in α1β2γ2L receptors under non-physiologic conditions with ~3.5 Å resolution. We hypothesized that substituted cysteine modification and protection experiments using variably sized n-alkyl-methanethiosulfonate (MTS) reagents could precisely estimate the distance between bound etomidate and β3M286 sidechains in activated functional receptors. Using voltage-clamp electrophysiology in Xenopus oocytes expressing α1β3M286Cγ2L GABAA receptors, we measured functional changes after exposing GABA-activated receptors to n-alkyl-MTS reagents from methyl-MTS to n-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 n-alkyl-MTS reagents modified GABA-activated α1β3M286Cγ2L GABAA receptors. Receptor modification by n-propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased etomidate modulation. Receptor-bound etomidate blocked β3M286C modification by n-propyl-MTS, n-butyl-MTS and n-hexyl-MTS. In contrast, GABA sensitivity was unaltered by receptor exposure to methyl-MTS or ethyl-MTS, while ethyl-MTS modification uniquely increased etomidate modulation. These results reveal a "cut-on" between ethyl-MTS and n-propyl-MTS, from which we infer that -S-(n-propyl) is the smallest β3M286C appendage that overlaps with etomidate sites. Molecular models of the native methionine and -S-ethyl and -S-(n-propyl) modified cysteines suggest that etomidate is located between 1.7 and 3.0 Å from the β3M286 sidechain. Significance Statement Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. We combined electrophysiology, a cysteine substitution, and n-alkyl-methanthiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1β3γ2L GABA type A receptor residue and etomidate bound in the transmembrane β+/α- interface.