title: Agonist-induced closure of constitutively open gamma-aminobutyric acid channels with mutated M2 domains.
abstract: Ligand-gated ion channels display a fundamental property-channels remain virtually closed at rest and open upon agonist binding. Here we show that substituting alanines for either of two amino acid residues (T314 or L317) in the M2 region of the gamma-aminobutyric acid (GABA) rho1 subunit results in spontaneous channel opening in the absence of ligand. Surprisingly, for two single point mutants (T314A or L317A), application of very low concentrations of agonist partially suppressed this spontaneous current, while higher concentrations re-activated the receptors. When both of these sites were mutated (T314A/L317A), GABA nearly completely suppressed the constitutive current and did not re-activate the current even at very high concentrations. This study provides important new insights into the structure-function relationship of ligand-gated ion channels, where modification of the structure of the channel pore region not only alters the allosteric transition of the receptor protein but also reverses the polarity of agonist regulation of channel gating. Our results suggest that the sites where these two residues are located are structurally critical for channel gating.
authors.affiliation: Laboratory of Cellular and Molecular Neuroscience, Children's Hospital, and Department of Neurology, Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Z H
authors.initials: ZH
authors.lastname: Pan
authors.forename: D
authors.initials: D
authors.lastname: Zhang
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.forename: S A
authors.initials: SA
authors.lastname: Lipton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005260:Female; D018755:GABA Agonists / Q000494:pharmacology*; D018756:GABA Antagonists / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D017354:Point Mutation; D051381:Rats; D018079:Receptors, GABA / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D018009:Receptors, Glycine / Q000737:chemistry / Q000502:physiology; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D018755:GABA Agonists; D018756:GABA Antagonists; D007473:Ion Channels; D046911:Macromolecular Substances; D009943:Organophosphorus Compounds; D018079:Receptors, GABA; D018009:Receptors, Glycine; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C068457:3-aminopropyl(methyl)phosphinic acid; D005680:gamma-Aminobutyric Acid