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Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α4/6β3δ GABAA receptors
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Edited by Floyd E. Bloom, The Scripps Research Institute, La Jolla, CA, and approved February 10, 2006
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↵ †H.J.H. and P.C. contributed equally to this work. (received for review November 16, 2005)
Related Article
- Alcohol-sensitive GABA receptors and alcohol antagonists- May 22, 2006

Abstract
Although GABAA receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the “nonsynaptic” δ subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that δ subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (K d < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant δ subunit-containing receptors, binding of the alcohol antagonist [3H]Ro15-4513 is inhibited by low concentrations of EtOH (K i ≈ 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, β-carbolinecarboxylate ethyl ester (β-CCE), and N-methyl-β-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABAA receptors.
Footnotes
- ‖To whom correspondence may be addressed at: Department of Molecular and Medical Pharmacology, University of California, Room 23-120 CHS, Charles Young Drive South, Los Angeles, CA 90095-1735. E-mail: mwallner{at}mednet.ucla.edu or rolsen{at}mednet.ucla.edu
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See Commentary on page 8307.
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Author contributions: H.J.H., M.W., and R.W.O. designed research; H.J.H., P.C., P.M., and M.W. performed research; P.S. and W.S. contributed new reagents/analytic tools; H.J.H., P.C., M.W., and R.W.O. analyzed data; and W.S., M.W., and R.W.O. wrote the paper.
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Conflict of interest statement: M.W., R.W.O., and H.J.H. have filed a U.S. Provisional Patent Application, Serial No. 60/693,844.
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This paper was submitted directly (Track II) to the PNAS office.
- Abbreviations:
- GABAAR,
- GABAA receptor;
- HEK,
- human embryonic kidney;
- BZ,
- benzodiazepine;
- EtOH,
- ethanol;
- IP,
- immunoprecipitated/immunoprecipitation;
- β-CCE,
- β-carboline-3-carboxyethyl ester;
- DMCM,
- methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate;
- DZ,
- diazepam;
- DZ-IS,
- DZ-insensitive;
- FG7142,
- N-methyl-β-carboline-3-carboxamide.
Abbreviations:
- © 2006 by The National Academy of Sciences of the USA