Substituted Cysteine Modification and Protection with n-Alkyl- Methanethiosulfonate Reagents Yields a Precise Estimate of the Distance between Etomidate and a Residue in Activated GABA Type A Receptors
| Autor: | Ryan J. Fantasia, Stuart A. Forman, Anahita Nourmahnad, Elizabeth A Halpin |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Xenopus 03 medical and health sciences chemistry.chemical_compound Xenopus laevis 0302 clinical medicine Etomidate Sulfhydryl reagent medicine Animals Humans Cysteine Binding site Receptor gamma-Aminobutyric Acid Pharmacology Mesylates Methionine biology GABAA receptor Articles biology.organism_classification Receptors GABA-A 030104 developmental biology chemistry Biophysics Molecular Medicine Female Indicators and Reagents 030217 neurology & neurosurgery Anesthetics Intravenous medicine.drug |
| Zdroj: | Mol Pharmacol |
| Popis: | The anesthetic etomidate modulates synaptic α1β2/3γ2 GABA(A) receptors via binding sites located in transmembrane β+/α− interfaces. Various approaches indicate that etomidate binds near β2/3M286 side chains, including recent cryogenic electron microscopy images in α1β2γ2L receptors under nonphysiologic 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 side chains in activated functional receptors. Using voltage-clamp electrophysiology in Xenopus oocytes expressing α1β3M286Cγ2L GABA(A) 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 GABA(A) 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, and 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 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 n-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 β+/α− interface. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|