Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α4/6β3δ GABAA receptors

H. Jacob Hanchar; Panida Chutsrinopkun; Pratap Meera; Porntip Supavilai; Werner Sieghart; Martin Wallner; Richard W. Olsen

doi:10.1073/pnas.0509903103

New Research In

Edited by Floyd E. Bloom, The Scripps Research Institute, La Jolla, CA, and approved February 10, 2006
↵ ^†H.J.H. and P.C. contributed equally to this work. (received for review November 16, 2005)

Abstract

Although GABA_A 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 GABA_A 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
See Commentary on page 8307.
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.
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.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:
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.
© 2006 by The National Academy of Sciences of the USA