Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes

Abstract

Intravenous general anesthetics including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing gamma-aminobutyric acid type A (GABA-A) receptor activation. Here we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification-protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABA-A receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites. These results suggest strategies for developing new drugs that selectively modulate distinct GABA-A receptor subtypes.

Tryptophan and Cysteine Mutations in M1 Helices of α1β3γ2L γ-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site

“…These results suggest that α1β3γ2L receptors form overlapping etomidate and propofol sites in β3 + – α1 − interfaces adjacent to α1-M1s, and overlapping mTFD-MPAB and propofol sites in homologous α1 + – β3 − and γ2 + – β3 − pockets abutting β3-M1s (Fig 1). However, we don’t know how propofol sites overlap with vs. differ from etomidate and mTFD-MPAB sites, because photolabels don’t adduct all residues that contact parent drugs 13,14 . Additionally, no anesthetics have photolabeled γ2-M1, and because receptors photolabeled by aziPm and 6-aziP lacked γ2 subunits, propofol or neurosteroid interactions with γ2 remain untested.…”

“…Tryptophan substitutions in putative anesthetic sites have been reported to both mimic the channel-gating effects of anesthetic binding and impair anesthetic modulation 15–19 . Cysteines substituted at putative contact residues have been covalently modified by probes such as p -chloromercuricbenzenesulfonate (pCMBS) and protected from modification by anesthetics 14,20–24 . However, neither of these approaches has been rigorously compared to photolabeling.…”

Stabilizing membrane domains antagonizes n-alcohol anesthesia

“…XTC-2 cells (24) were maintained in L-15 Media diluted 1:1.5 with water for amphibian cells with 10% FBS, sodium bicarbonate (2.47 g/liter), pen strep (100 units/ml) at room temperature in 5% CO 2 . Freshly seeded cells were incubated in 000 00(00) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] complete media for at least 18h at the growth temperature indicated prior to GPMV isolation. All culture reagents were purchased from Fisher Scientific (Hampton, New Hampshire).…”

“…XTC-2 cells As with previous studies varying only temperature (19), we find that the fraction of phase separated vesicles present in a population of vesicles at a given temperature/pressure does not depend on the order in which temperature or pressures are sampled, suggesting the transition is fully reversible. 000 00(00) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Tadpole loss of righting reflex (LRR) measurements: Studies with Xenopus laevis tadpoles were conducted in compliance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were approved by the University of Michigan IACUC. Xenopus laevis embryos were collected, fertilized, and dejellied as described previously (30).…”

“…000 00(00) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Our understanding of the structure and function of the animal plasma membrane has grown dramatically since most membrane theories of anesthesia were put forward. It is now appreciated that animal plasma membranes have a thermodynamic tendency to separate into coexisting liquid domains, sometimes referred to as 'lipid rafts' or 'lipid shells' (14,15) and that this heterogeneity localizes and regulates ion channels, sometimes in a subtype specific manner (16).…”