title: A transmembrane amino acid in the GABAA receptor β2 subunit critical for the actions of alcohols and anesthetics.
abstract: Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing α, β, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the β subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA(A) receptor β(2) subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α(1)β(2)(N265C)γ(2S) receptors [but not α(1)β(2)(I264C)γ(2S)], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the β(2) subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α(1)β(2)(N265C)γ(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, Texas 78712, USA.
authors.forename: Mandy L
authors.initials: ML
authors.lastname: McCracken
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Borghese
authors.forename: James R
authors.initials: JR
authors.lastname: Trudell
authors.forename: R Adron
authors.initials: RA
authors.lastname: Harris
mesh_terms: D020001:1-Butanol / Q000378:metabolism / Q000494:pharmacology; D020003:1-Octanol / Q000378:metabolism / Q000494:pharmacology; D000438:Alcohols / Q000378:metabolism / Q000494:pharmacology*; D019943:Amino Acid Substitution / Q000502:physiology; D000777:Anesthetics / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001216:Asparagine / Q000502:physiology*; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007530:Isoflurane / Q000378:metabolism / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000235:genetics; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013886:Thiosulfonic Acids / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000438:Alcohols; D000777:Anesthetics; C495710:GABRB2 protein, human; C546242:Gabra1 protein, rat; C546281:Gabrg2 protein, rat; D018075:RNA, Complementary; D011963:Receptors, GABA-A; D013886:Thiosulfonic Acids; D000431:Ethanol; D005680:gamma-Aminobutyric Acid; D001216:Asparagine; D020001:1-Butanol; D007530:Isoflurane; D003545:Cysteine; D020003:1-Octanol
references.citation: Neurosci Lett. 2007 Apr 18;416(3):275-8
references.pmid: 17350761
references.citation: J Biol Chem. 2005 Jan 7;280(1):308-16
references.pmid: 15522868
references.citation: Proc Natl Acad Sci U S A. 1998 May 26;95(11):6504-9
references.pmid: 9600996
references.citation: J Neurosci. 2001 Jul 15;21(14):4977-86
references.pmid: 11438573
references.citation: Alcohol Clin Exp Res. 1999 Oct;23(10):1563-70
references.pmid: 10549986
references.citation: J Neurosci. 1997 Apr 15;17(8):2728-37
references.pmid: 9092594
references.citation: J Biol Chem. 2010 Mar 19;285(12):8615-20
references.pmid: 20083606
references.citation: ACS Chem Neurosci. 2010 Jul;1(7):482
references.pmid: 21326622
references.citation: Nature. 2006 Nov 23;444(7118):486-9
references.pmid: 17108970
references.citation: Br J Pharmacol. 1999 May;127(2):377-82
references.pmid: 10385236
references.citation: Neuropharmacology. 2002 Mar;42(3):337-45
references.pmid: 11897112
references.citation: J Biol Chem. 1998 Feb 6;273(6):3314-9
references.pmid: 9452448
references.citation: Neuropharmacology. 2009 Jan;56(1):141-8
references.pmid: 18760291
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 1997 Sep;356(3):267-82
references.pmid: 9303562
references.citation: Curr Top Med Chem. 2002 Aug;2(8):795-816
references.pmid: 12171572
references.citation: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):11031-6
references.pmid: 9380754
references.citation: J Biol Chem. 2009 Oct 2;284(40):27304-14
references.pmid: 19656948
references.citation: Annu Rev Pharmacol Toxicol. 2001;41:23-51
references.pmid: 11264449
references.citation: Trends Neurosci. 1995 Mar;18(3):121-7
references.pmid: 7754520
references.citation: Mol Neurobiol. 1998 Aug;18(1):35-86
references.pmid: 9824848
references.citation: Nat Rev Neurosci. 2008 May;9(5):370-86
references.pmid: 18425091
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6275-80
references.pmid: 20308576
references.citation: Nature. 2003 Jun 26;423(6943):949-55
references.pmid: 12827192
references.citation: J Biol Chem. 2008 May 30;283(22):15250-7
references.pmid: 18387955
references.citation: FASEB J. 2003 Feb;17(2):250-2
references.pmid: 12475885
references.citation: Nature. 1997 Sep 25;389(6649):385-9
references.pmid: 9311780
references.citation: Anesth Analg. 2005 Aug;101(2):412-418
references.pmid: 16037154
references.citation: J Mol Graph Model. 2004 Sep;23(1):39-49
references.pmid: 15331052
references.citation: J Pharmacol Exp Ther. 2003 Oct;307(1):42-52
references.pmid: 14500778
references.citation: Nature. 2009 Jan 1;457(7225):111-4
references.pmid: 18987633
references.citation: J Biol Chem. 2004 Aug 6;279(32):33919-27
references.pmid: 15169788
references.citation: J Neurochem. 2007 Sep;102(6):2097-2109
references.pmid: 17561937
references.citation: Mol Pharmacol. 2004 Jan;65(1):68-76
references.pmid: 14722238
references.citation: J Neurosci. 2003 Sep 17;23(24):8608-17
references.pmid: 13679430
references.citation: J Gen Physiol. 1996 Feb;107(2):195-205
references.pmid: 8833341
references.citation: J Neurosci. 2001 Mar 15;21(6):RC136
references.pmid: 11245705
references.citation: Pharmacol Rev. 2008 Sep;60(3):243-60
references.pmid: 18790874
references.citation: J Biol Chem. 2003 Apr 11;278(15):13166-72
references.pmid: 12556472
references.citation: Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9305-10
references.pmid: 10908659