title: Etomidate produces similar allosteric modulation in α1β3δ and α1β3γ2L GABA(A) receptors.
abstract: OBJECTIVE
  Neuronal GABA(A) receptors are pentameric chloride ion channels, which include synaptic αβγ and extrasynaptic αβδ isoforms, mediating phasic and tonic inhibition respectively. Although the subunit arrangement of αβγ receptors is established as β-α-γ-β-α, that of αβδ receptors is uncertain and possibly variable. We compared receptors formed from free α1, β3 and δ or γ2L subunits and concatenated β3-α1-δ and β3-α1 subunit assemblies (placing δ in the established γ position) by investigating the effects of R-(+)-etomidate (ETO), an allosteric modulator that selectively binds to transmembrane interfacial sites between β3 and α1.
  
  
  METHODS
  GABA-activated receptor-mediated currents in Xenopus oocytes were measured electrophysiologically, and ETO-induced allosteric shifts were quantified using an established model.
  
  
  RESULTS
  ETO (3.2 μM) similarly enhanced maximal GABA (1 mM)-evoked currents in oocytes injected with 5 ng total mRNA and varying subunit ratios, for α1β3(1:1), α1β3δ(1:1:1) and α1β3δ(1:1:3), but this potentiation by ETO was significantly greater for β3-α1-δ/β3-α1(1:1) receptors. Reducing the amount of α1β3δ(1:1:3) mRNA mixture injected (0.5 ng) increased the modulatory effect of ETO, matching that seen with β3-α1-δ/β3-α1(1:1, 1 ng). ETO similarly reduced EC₅₀s and enhanced maxima of GABA concentration-response curves for both α1β3δ and β3-α1-δ/β3-α1 receptors. Allosteric shift parameters derived from these data depended on estimates of maximal GABA efficacy, and the calculated ranges overlap with allosteric shift values for α1β3γ2L receptors.
  
  
  CONCLUSIONS
  Reducing total mRNA unexpectedly increased δ subunit incorporation into receptors on oocyte plasma membranes. Our results favour homologous locations for δ and γ2L subunits in α1β3γ2/δ GABA(A) receptors.
authors.affiliation: Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: H-J
authors.initials: HJ
authors.lastname: Feng
authors.forename: Y
authors.initials: Y
authors.lastname: Jounaidi
authors.forename: M
authors.initials: M
authors.lastname: Haburcak
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: S A
authors.initials: SA
authors.lastname: Forman
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D018681:Anesthetics, General / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D054333:Drug Partial Agonism; D005045:Etomidate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D058785:GABA-A Receptor Agonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus
chemical_list: D018681:Anesthetics, General; D058785:GABA-A Receptor Agonists; C546240:GABRA1 protein, human; C546273:GABRB3 protein, human; C546279:GABRG2 protein, human; C000592156:Gabrd protein, rat; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005045:Etomidate
keywords: GABAA receptors; allosteric modulation; concatenated subunit assemblies; electrophysiology; etomidate; general anaesthetics; δ subunits