title: Multiple roles for the first transmembrane domain of GABAA receptor subunits in neurosteroid modulation and spontaneous channel activity.
abstract: Neurosteroids exert potent physiological effects by allosterically modulating synaptic and extrasynaptic GABA(A) receptors. Some endogenous neurosteroids, such as 3alpha, 21-dihydroxy-5beta-pregnan-20-one (5alpha, 3alpha-THDOC), potentiate GABA(A) receptor function by interacting with a binding pocket defined by conserved residues in the first and fourth transmembrane (TM) domains of alpha subunits. Others, such as pregnenolone sulfate (PS), inhibit GABA(A) receptor function through as-yet unidentified binding sites. Here we investigate the mechanisms of PS inhibition of mammalian GABA(A) receptors, based on studies of PS inhibition of the UNC-49 GABA receptor, a GABA(A)-like receptor from Caenorhabditis elegans. In UNC-49, a 19 residue segment of TM1 can be mutated to increase or decrease PS sensitivity over a 20-fold range. Surprisingly, substituting these UNC-49 sequences into mammalian alpha(1), beta(2), and gamma(2) subunits did not produce the corresponding effects on PS sensitivity of the resulting chimeric receptors. Therefore, it is unlikely that a conserved PS binding pocket is formed at this site. However we observed several interesting unexpected effects. First, chimeric gamma2 subunits caused increased efficacy of 5alpha, 3alpha-THDOC potentiation; second, spontaneous gating of alpha(6)beta(2)delta receptors was blocked by PS, and reduced by chimeric beta(2) subunits; and third, direct activation of alpha(6)beta(2)delta receptors by 5alpha, 3alpha-THDOC was reduced by chimeric beta(2) subunits. These results reveal novel roles for non-alpha subunits in neurosteroid modulation and direct activation, and show that the beta subunit TM1 domain is important for spontaneous activity of extrasynaptic GABA(A) receptors.
authors.affiliation: Department of Bioengineering, University of Toledo, 2801 W Bancroft Street, Toledo, OH 43606, United States.
authors.forename: Carrie
authors.initials: C
authors.lastname: Baker
authors.forename: Brianne L
authors.initials: BL
authors.lastname: Sturt
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Bamber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000502:physiology; D003900:Desoxycorticosterone / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D011284:Pregnenolone / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000502:physiology*
chemical_list: D029742:Caenorhabditis elegans Proteins; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D021122:Protein Subunits; D011963:Receptors, GABA-A; C018370:pregnenolone sulfate; D003900:Desoxycorticosterone; C009413:tetrahydrodeoxycorticosterone; D011284:Pregnenolone