Newly Published
Perioperative Medicine  |   November 2017
Alphaxalone Binds in Inner Transmembrane β+–α Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors
Author Notes
  • From the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts. Current affiliations: Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York (A.M.Z.); School of Medicine, Case Western Reserve University, Cleveland, Ohio (A.S.); Department of Biology, Brandeis University, Waltham, Massachusetts (M.H.); Keck School of Medicine, University of Southern California, Los Angeles, California (A.T.S.); and Harvard Medical School, Boston, Massachusetts (A.N.).
  • Submitted for publication April 03, 2017. Accepted for publication October 12, 2017.
    Submitted for publication April 03, 2017. Accepted for publication October 12, 2017.×
  • Acknowledgments: The authors thank Youssef Jounaidi, Ph.D. (Instructor, Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston Massachusetts) for his help with molecular biology. Karol Bruzik, Ph.D. (Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois) provided mTFD-MPAB. Keith W. Miller, D.Phil. (Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital) and Jonathan B. Cohen, Ph.D. (Department of Neurobiology, Harvard Medical School, Boston, Massachusetts) provided helpful comments on the manuscript.
    Acknowledgments: The authors thank Youssef Jounaidi, Ph.D. (Instructor, Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston Massachusetts) for his help with molecular biology. Karol Bruzik, Ph.D. (Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois) provided mTFD-MPAB. Keith W. Miller, D.Phil. (Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital) and Jonathan B. Cohen, Ph.D. (Department of Neurobiology, Harvard Medical School, Boston, Massachusetts) provided helpful comments on the manuscript.×
  • Research Support: This work was supported by grant Nos. GM089745 and GM058448 from the National Institutes of Health, Bethesda, Maryland.
    Research Support: This work was supported by grant Nos. GM089745 and GM058448 from the National Institutes of Health, Bethesda, Maryland.×
  • Competing Interests: The authors declare no competing interests.
    Competing Interests: The authors declare no competing interests.×
  • Correspondence: Address correspondence to Dr. Forman: Department of Anesthesia, Critical Care, and Pain Medicine, Jackson 444, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts, 02114. saforman@mgh.harvard.edu. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Pharmacology
Perioperative Medicine   |   November 2017
Alphaxalone Binds in Inner Transmembrane β+–α Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors
Anesthesiology Newly Published on November 30, 2017. doi:10.1097/ALN.0000000000001978
Anesthesiology Newly Published on November 30, 2017. doi:10.1097/ALN.0000000000001978
Abstract

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 αβγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane β+–α interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in β+–α interfaces.

Methods: The authors created 12 single-residue β3 cysteine mutations: β3T262C and β3T266C in β3-M2; and β3M283C, β3Y284C, β3M286C, β3G287C, β3F289C, β3V290C, β3F293C, β3L297C, β3E298C, and β3F301C in β3-M3 helices. The authors coexpressed α1 and γ2L with each mutant β3 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 β3 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 β3V290C, β3F293C, β3L297C, or β3F301C mutations. Both etomidate and propofol protected receptors with β3M286C or β3V290C mutations. Etomidate also protected β3F289C. In α1β3γ2L structural homology models, all these protected residues are located in transmembrane β+–α interfaces.

Conclusions: Alphaxalone binds in transmembrane β+–α pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.