title: Stoichiometry of δ subunit containing GABA(A) receptors.
abstract: OBJECTIVE
  Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach.
  
  
  METHODS
  Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits.
  
  
  RESULTS
  Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits.
  
  
  CONCLUSIONS
  Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ.
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
authors.forename: B
authors.initials: B
authors.lastname: Patel
authors.forename: M
authors.initials: M
authors.lastname: Mortensen
authors.forename: T G
authors.initials: TG
authors.lastname: Smart
mesh_terms: D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology*; D011963:Receptors, GABA-A / Q000737:chemistry / Q000502:physiology*; D016415:Sequence Alignment; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D021122:Protein Subunits; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid
keywords: GABAA receptor; extrasynaptic; stoichiometry; δ subunit