title: Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.
abstract: BACKGROUND
  Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the &amp;quot;+&amp;quot; of the β subunit, in the β-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the &amp;quot;-&amp;quot; side of the β subunit, in the α-β interface (or β-β interface, in the case of homomeric β receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the β(H267) residue lines a cavity that docks propofol with favorable interaction energy.
  
  
  METHODS
  We used two-electrode voltage clamp to determine the functional effects of mutations to the &quot;+&quot; and &quot;-&quot; sides of the β subunit on activation of the α1β3 GABAA receptor by propofol.
  
  
  RESULTS
  We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-β interface leads to strong reduction in gating efficacy for propofol.
  
  
  CONCLUSIONS
  We conclude that α1β3 GABAA receptors can be activated by propofol interactions with the β-β, α-β, and β-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.
authors.forename: Megan M
authors.initials: MM
authors.lastname: Eaton
authors.forename: Allison L
authors.initials: AL
authors.lastname: Germann
authors.forename: Ruby
authors.initials: R
authors.lastname: Arora
authors.forename: Lily Q
authors.initials: LQ
authors.lastname: Cao
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Gao
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Shin
authors.forename: Albert
authors.initials: A
authors.lastname: Wu
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
authors.forename: Joe Henry
authors.initials: JH
authors.lastname: Steinbach
authors.forename: Alex S
authors.initials: AS
authors.lastname: Evers
authors.affiliation: Department of Anesthesiology, Washington University, Campus Box 8054, 660 South Euclid Ave, St. Louis, MO 63110.
authors.forename: Gustav
authors.initials: G
authors.lastname: Akk
mesh_terms: D000818:Animals; D018682:GABA Agents / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D015742:Propofol / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*
chemical_list: D018682:GABA Agents; D011963:Receptors, GABA-A; D015742:Propofol
references.citation: BMC Pharmacol. 2003 Nov 12;3:13
references.pmid: 14613517
references.citation: J Biol Chem. 2004 May 14;279(20):20982-92
references.pmid: 15016806
references.citation: Mol Pharmacol. 2008 Dec;74(6):1687-95
references.pmid: 18805938
references.citation: Mol Pharmacol. 2004 Jan;65(1):68-76
references.pmid: 14722238
references.citation: Nat Chem Biol. 2013 Nov;9(11):715-20
references.pmid: 24056400
references.citation: Br J Pharmacol. 1991 Nov;104(3):619-28
references.pmid: 1665745
references.citation: Curr Opin Anaesthesiol. 2012 Aug;25(4):411-8
references.pmid: 22614249
references.citation: Brain Res. 1998 Feb 16;784(1-2):179-87
references.pmid: 9518600
references.citation: Trends Pharmacol Sci. 1999 Dec;20(12):496-502
references.pmid: 10603492
references.citation: Nature. 2014 Aug 21;512(7514):270-5
references.pmid: 24909990
references.citation: J Neurochem. 2000 Feb;74(2):827-38
references.pmid: 10646536
references.citation: Mol Pharmacol. 1996 Mar;49(3):494-504
references.pmid: 8643089
references.citation: J Phys Chem A. 2007 Aug 16;111(32):7987-94
references.pmid: 17645323
references.citation: Mol Pharmacol. 2008 Sep;74(3):614-27
references.pmid: 18544665
references.citation: Anesthesiology. 2012 Jan;116(1):47-55
references.pmid: 22104494
references.citation: Brain Res. 1984 Dec 10;323(2):287-92
references.pmid: 6098342
references.citation: Mol Pharmacol. 1998 Mar;53(3):530-8
references.pmid: 9495821
references.citation: Anesthesiology. 2015 Apr;122(4):787-94
references.pmid: 25575161
references.citation: J Biol Chem. 2013 Jul 5;288(27):19343-57
references.pmid: 23677991
references.citation: J Neurosci. 1994 Dec;14(12):7747-60
references.pmid: 7996209
references.citation: Biophys J. 1999 Nov;77(5):2542-51
references.pmid: 10545355
references.citation: Br J Pharmacol. 1996 Jun;118(3):563-76
references.pmid: 8762079
references.citation: Nat Rev Neurosci. 2002 Feb;3(2):102-14
references.pmid: 11836518
references.citation: Neuroreport. 1996 Mar 22;7(4):943-7
references.pmid: 8724679
references.citation: J Mol Biol. 1965 May;12 :88-118
references.pmid: 14343300
references.citation: J Med Chem. 2010 Aug 12;53(15):5667-75
references.pmid: 20597506
references.citation: Mol Pharmacol. 2015 Oct;88(4):736-45
references.pmid: 26206487
references.citation: J Neurochem. 2002 Jan;80(1):140-8
references.pmid: 11796752
references.citation: Neuropharmacology. 2001 Dec;41(8):952-64
references.pmid: 11747900
references.citation: J Pharmacol Exp Ther. 2001 Apr;297(1):338-51
references.pmid: 11259561
references.citation: J Biol Chem. 2014 Oct 3;289(40):27456-68
references.pmid: 25086038
references.citation: Nature. 1993 Dec 9;366(6455):565-9
references.pmid: 7504783
references.citation: FASEB J. 2003 Feb;17(2):250-2
references.pmid: 12475885