Tryptophan scanning mutagenesis in TM2 of the GABAA receptor α subunit: effects on channel gating and regulation by ethanol

Authors

  • Susumu Ueno,

    1. Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, 2500 Speedway, MBB 1.124, Austin, Texas, TX 78712, U.S.A.
    2. Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan
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  • Audrey Lin,

    1. Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois, IL 60637, U.S.A
    2. Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut, CT 06510, U.S.A.
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  • Natalia Nikolaeva,

    1. Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois, IL 60637, U.S.A
    2. Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, IL 60637, U.S.A.
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  • James R Trudell,

    1. Department of Anesthesia and Program for Molecular and Genetic Medicine, Stanford University, Stanford, California, CA 94305, U.S.A.
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  • S John Mihic,

    1. Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, 2500 Speedway, MBB 1.124, Austin, Texas, TX 78712, U.S.A.
    2. Department of Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, NC 27157, U.S.A.
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  • R Adron Harris,

    Corresponding author
    1. Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, 2500 Speedway, MBB 1.124, Austin, Texas, TX 78712, U.S.A.
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  • Neil L Harrison

    1. Department of Anesthesiology, A-1050, Weill Medical College of Cornell University, New York, NY 10021, U.S.A.
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Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, 2500 Speedway, MBB 1.124, Austin, Texas, TX 78712, U.S.A. E-mail: harris@mail.utexas.edu

Abstract

  • Each residue in the second transmembrane segment (TM2) of the human GABAA receptor α2 subunit was individually mutated to tryptophan. The wild-type or mutant α2 subunits were expressed with the wild-type human GABAA receptor β2 subunit in Xenopus oocytes, and the effects of these mutations were investigated using two-electrode voltage-clamp recording.

  • Four mutations (V257W, T262W, T265W and S270W) produced receptors which were active in the absence of agonist, and this spontaneous open channel activity was blocked by both picrotoxin and bicuculline, except in the α2(V257W)β2 mutant receptor, which was not sensitive to picrotoxin.

  • Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10–100 times compared with the wild-type α2β2 receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the receptor to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA-induced currents.

  • The S270W mutation eliminated potentiation of GABA by ethanol, whereas T261W markedly increased the action of ethanol. The T262W mutation produced direct activation (10% of maximal GABA response) by ethanol in the absence of GABA, while other mutations did not alter the action of ethanol significantly.

  • These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABAA receptor channel function, in which specific residues within TM2 are critical for the regulation of channel gating (S270, L264), while other residues (L269, I271 and S272) have little effect on these functions and may be non-critical structural residues.

British Journal of Pharmacology (2000) 131, 296–302; doi:10.1038/sj.bjp.0703504

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