Alphaxalone Binds in Inner Transmembrane ß+-α- Interfaces of α1ß3γ2 γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of γ-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αßγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane ß-α interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in ß-α interfaces. METHODS: The authors created 12 single-residue ß3 cysteine mutations: ß3T262C and ß3T266C in ß3-M2; and ß3M283C, ß3Y284C, ß3M286C, ß3G287C, ß3F289C, ß3V290C, ß3F293C, ß3L297C, ß3E298C, and ß3F301C in ß3-M3 helices. The authors coexpressed α1 and γ2L with each mutant ß3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. RESULTS: Eleven mutant ß3 subunits, when coexpressed with α1 and γ2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with ß3V290C, ß3F293C, ß3L297C, or ß3F301C mutations. Both etomidate and propofol protected receptors with ß3M286C or ß3V290C mutations. Etomidate also protected ß3F289C. In α1ß3γ2L structural homology models, all these protected residues are located in transmembrane ß-α interfaces. CONCLUSIONS: Alphaxalone binds in transmembrane ß-α pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.

Tryptophan and Cysteine Mutations in M1 Helices of α1ß3γ2L γ-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site.

BACKGROUND: γ-Aminobutyric acid type A (GABAA) receptors mediate important effects of intravenous general anesthetics. Photolabel derivatives of etomidate, propofol, barbiturates, and a neurosteroid get incorporated in GABAA receptor transmembrane helices M1 and M3 adjacent to intersubunit pockets. However, photolabels have not been consistently targeted at heteromeric αßγ receptors and do not form adducts with all contact residues. Complementary approaches may further define anesthetic sites in typical GABAA receptors. METHODS: Two mutation-based strategies, substituted tryptophan sensitivity and substituted cysteine modification-protection, combined with voltage-clamp electrophysiology in Xenopus oocytes, were used to evaluate interactions between four intravenous anesthetics and six amino acids in M1 helices of α1, ß3, and γ2L GABAA receptor subunits: two photolabeled residues, α1M236 and ß3M227, and their homologs. RESULTS: Tryptophan substitutions at α1M236 and positional homologs ß3L231 and γ2L246 all caused spontaneous channel gating and reduced γ-aminobutyric acid EC50. Substituted cysteine modification experiments indicated etomidate protection at α1L232C and α1M236C, R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid protection at ß3M227C and ß3L231C, and propofol protection at α1M236C and ß3M227C. No alphaxalone protection was evident at the residues the authors explored, and none of the tested anesthetics protected γ2I242C or γ2L246C. CONCLUSIONS: All five intersubunit transmembrane pockets of GABAA receptors display similar allosteric linkage to ion channel gating. Substituted cysteine modification and protection results were fully concordant with anesthetic photolabeling at α1M236 and ß3M227 and revealed overlapping noncongruent sites for etomidate and propofol in ß-α interfaces and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid and propofol in α-ß and γ-ß interfaces. The authors' results identify the α-γ transmembrane interface as a potentially unique orphan modulator site.

A Cysteine Substitution Probes ß3H267 Interactions with Propofol and Other Potent Anesthetics in α1ß3γ2L γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Anesthetic contact residues in γ-aminobutyric acid type A (GABAA) receptors have been identified using photolabels, including two propofol derivatives. O-propofol diazirine labels H267 in ß3 and α1ß3 receptors, whereas m-azi-propofol labels other residues in intersubunit clefts of α1ß3. Neither label has been studied in αßγ receptors, the most common isoform in mammalian brain. In αßγ receptors, other anesthetic derivatives photolabel m-azi-propofol-labeled residues, but not ßH267. The authors' structural homology model of α1ß3γ2L receptors suggests that ß3H267 may abut some of these sites. METHODS: Substituted cysteine modification-protection was used to test ß3H267C interactions with four potent anesthetics: propofol, etomidate, alphaxalone, and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (mTFD-MPAB). The authors expressed α1ß3γ2L or α1ß3H267Cγ2L GABAA receptors in Xenopus oocytes. The authors used voltage clamp electrophysiology to assess receptor sensitivity to γ-aminobutyric acid (GABA) and anesthetics and to compare p-chloromercuribenzenesulfonate modification rates with GABA versus GABA plus anesthetics. RESULTS: Enhancement of low GABA (eliciting 5% of maximum) responses by equihypnotic concentrations of all four anesthetics was similar in α1ß3γ2L and α1ß3H267Cγ2L receptors (n > 3). Direct activation of α1ß3H267Cγ2L receptors, but not α1ß3γ2L, by mTFD-MPAB and propofol was significantly greater than the other anesthetics. Modification of ß3H267C by p-chloromercuribenzenesulfonate (n > 4) was rapid and accelerated by GABA. Only mTFD-MPAB slowed ß3H267C modification (approximately twofold; P = 0.011). CONCLUSIONS: ß3H267 in α1ß3γ2L GABAA receptors contacts mTFD-MPAB, but not propofol. The study results suggest that ß3H267 is near the periphery of one or both transmembrane intersubunit (α+/ß- and γ+/ß-) pockets where both mTFD-MPAB and propofol bind.

Mutations at beta N265 in γ-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.

Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane ß+/α- inter-subunit interfaces. Early experiments in heteromeric receptors identified ßN265 (M2-15') on ß2 and ß3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the ß1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the ßN265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between ßN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous α1ß2N265Cγ2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the ß-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on α1ß2N265Cγ2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive ßN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in α1ß2/3γ2L GABAA receptors. Using α1M236C as a modifiable and anesthetic-protectable site occupancy reporter in ß+/α- interfaces, we found that ßN265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. ßN265M also impaired the transduction of gating effects associated with α1M236W, a mutation that mimics ß+/α- anesthetic site occupancy. Our results show that ßN265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in ß+/α- sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for ßN265 in anesthetic binding within the ß+/α- transmembrane sites.