A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition.
A class of bicuculline-insensitive gamma-aminobutyric acid (GABA) receptors, GABAC, has been identified in retina. Several lines of evidence indicate that GABAC receptors are formed partially or wholly of GABA rho subunits. These receptors generate a Cl- current in response to GABA but differ from GABAA receptors in a number of ways. Picrotoxin, widely accepted as a noncompetitive antagonist of GABAA receptors, displays competitive and noncompetitive antagonism of GABAC receptors in perch and bovine retina and GABA rho 1 receptors expressed in Xenopus oocytes. The aim of this study was to identify the molecular basis of the two components of picrotoxin inhibition of GABA rho 1 receptors. By using a domain-swapping and mutagenesis strategy, a difference in picrotoxin sensitivity between rho 1 and rho 2 receptors was localized to a single amino acid in the putative second transmembrane domain. Substitution of this amino acid with residues found in the analogous position in highly picrotoxin-sensitive glycine alpha and GABAA subunits increased the sensitivity of rho 1 mutants 10- to 500-fold. Importantly, the competitive component of picrotoxin inhibition of the rho 1 mutant receptors was almost eliminated. These findings demonstrate that an amino acid in the putative channel domain of GABA rho 1 receptors influences picrotoxin sensitivity and mediates agonist binding by an allosteric mechanism.
Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA.
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Wang
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Hackam
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Guggino
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Cutting
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D018756:GABA Antagonists / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes; D010852:Picrotoxin / Q000494:pharmacology*; D011487:Protein Conformation; D018079:Receptors, GABA / Q000187:drug effects* / Q000235:genetics; D011963:Receptors, GABA-A / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects; D013329:Structure-Activity Relationship
D018756:GABA Antagonists; D010852:Picrotoxin; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; C087200:GABA-C receptor