Abstract
Incorporation of the gamma 2 subunit into gamma-aminobutyric acid type A (GABAA) receptors is required for the expression of benzodiazepine pharmacology, but the regions of the subunit responsible for benzodiazepine actions have not been defined. Using mutagenesis, we identified a single amino acid of the gamma 2 subunit of the human GABAA receptor that profoundly alters the nature of this pharmacology. When threonine 142 was mutated to serine, the benzodiazepine receptor antagonist, flumazenil, and the weak inverse agonist, Ro 15-4513, both acted as potent partial agonists. Further, potentiation of GABA responses by diazepam, alprazolam, clonazepam, or flunitrazepam doubled in receptors containing the Ser-142 gamma 2 subunit. In contrast, responses to the Type I benzodiazepine receptor selective ligands, zolpidem, alpidem, and CL218,872, were roughly halved. This change in pharmacology appears to occur at a stage following ligand binding, i.e. the mutation affects benzodiazepine efficacy. There was no effect on GABA affinity or efficacy or pentobarbital, Ro 5-4864, or alphaxalone modulation of GABA responses. These findings demonstrate that very minor changes in receptor structure can profoundly affect the efficacy of receptor ligands. Thus, agonism is determined not only by the structure of the drug, but also by the structure of the receptor, or protein complex, with which it interacts.
Highlights
From the +Department of Pharmacology, University of Colorado Health Sciences Centeq Denver, Colorado 80262, the Ilkterans Administration Medical Center, Denver, Colorado 80262, and Werck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, CMZO 2QR United Kingdom
We identified a single amino acid o f the yz subunit of the human GABAA receptor that profoundly alters the nature of this pharmacology
When we mutated a number of our wild type yz cDNAs (Y~.~,Jw, hich contained the Thr-142 residue, to Ser-142(Y~.~~.), we found that this single amino acid mutation profoundly affected benzodiazepine pharmacology in GABA, receptor constructs expressed in Xenopus laevis oocytes
Summary
Theabbreviationsused are: GABA, y-aminobutyric acid; DMCM, methyl-6,7-dimethoxy-4-ethyl-fi-carboline-3-carboxDylZa,tdqiazepam. Sponses being observed in a,-containing receptor constructs, and type I1 responses observed in receptors possessing othea!r subunits (l’ritchettet al., 1989a). These pharmacological observations, coupled with [3Hlflunitrazepam photoaffinity labeling experiments showing incorporation into botthhe a! We isolated and sequenced a number of human and bovine yz cDNA clones and found a few amino acidsthat differed from those itnhe original human y2 sequencepublished by Pritchett et al (1989b). When we mutated a number of our wild type yz cDNAs (Y~.~,,Jw, hich contained the Thr-142 residue, to Ser-142(Y~.~~.), we found that this single amino acid mutation profoundly affected benzodiazepine pharmacology in GABA, receptor constructs expressed in Xenopus laevis oocytes
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