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Ethanol Potently and Competitively Inhibits Binding of the Alcohol Antagonist Ro15-4513 to ␣4/6␤3␦ GABAA Receptors H

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Ethanol Potently and Competitively Inhibits Binding of the Alcohol Antagonist Ro15-4513 to ␣4/6␤3␦ GABAA Receptors H Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to ␣4/6␤3␦ GABAA receptors H. Jacob Hanchar*†, Panida Chutsrinopkun†‡, Pratap Meera§, Porntip Supavilai‡, Werner Sieghart¶, Martin Wallner*ʈ, and Richard W. Olsen*ʈ Departments of *Molecular and Medical Pharmacology and §Neurobiology, University of California, Los Angeles, CA 90095; ‡Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10 400, Thailand; and ¶Center for Brain Research, Division of Biochemistry and Molecular Biology and Section of Biochemical Psychiatry, Medical University of Vienna, A-1090 Vienna, Austria Edited by Floyd E. Bloom, The Scripps Research Institute, La Jolla, CA, and approved February 10, 2006 (received for review November 16, 2005) Although GABAA receptors have long been implicated in mediating (14) concluded that the EtOH-induced increase in tonic current ethanol (EtOH) actions, receptors containing the ‘‘nonsynaptic’’ ␦ is due to increased GABA release rather than a postsynaptic subunit only recently have been shown to be uniquely sensitive to effect. Also, for reasons on which we can only speculate, a recent EtOH. Here, we show that ␦ subunit-containing receptors bind the article by Borghese et al. (15) states that effects of low dose imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with EtOH on ␦ subunit-containing receptors could not be observed, high affinity (Kd < 10 nM), contrary to the widely held belief that in particular with human ␣4␤3␦ GABAAR clones expressed in these receptors are insensitive to BZs. In immunopurified native oocytes and cell lines. cerebellar and recombinant ␦ subunit-containing receptors, bind- EtOH pharmacology shares many characteristics with alloste- 3 ing of the alcohol antagonist [ H]Ro15-4513 is inhibited by low ric activators of GABAARs (loosely referred to as GABAAR concentrations of EtOH (Ki Ϸ 8 mM). Also, Ro15-4513 binding is agonists), such as benzodiazepines (BZs) (5). Additional evi- inhibited by BZ-site ligands that have been shown to reverse the dence for a link between EtOH and BZ actions on GABAAR behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, comes from the surprising finding that a mutation in the ␣6 ␤-carbolinecarboxylate ethyl ester (␤-CCE), and N-methyl-␤-carbo- subunit (␣6-R100Q), which was identified in alcohol-nontolerant line-3-carboxamide (FG7142), but not including any classical BZ and -nonpreferring rats in refs. 16 and 17, leads to receptors with agonists like diazepam). Experiments that were designed to dis- increased alcohol sensitivity in recombinant expression (in cer- tinguish between a competitive and allosteric mechanism suggest ebellar granule cell neurons) and to alcohol-hypersensitive an- that EtOH and Ro15-4513 occupy a mutually exclusive binding site. imals in behavioral studies (12). Histidine residues at positions The fact that only Ro15-4513, but not flumazenil, can inhibit the that are homologous to the ␣6-R100 residue (which affects EtOH EtOH effect, and that Ro15-4513 differs from flumazenil by only a sensitivity in ␣6␤3␦ GABAARs) are critical for high-affinity single group in the molecule (an azido group at the C7 position of binding of classical BZ agonists at the interface between ␣ and the BZ ring) suggest that this azido group in Ro15-4513 might be ␥2 subunits. the area that overlaps with the alcohol-binding site. Our findings, It has been thought that the ‘‘extrasynaptic’’ GABAAR ␦ combined with previous observations that Ro15-4513 is a behav- subunit, which presumably takes the position of the ␥2 subunit in ioral alcohol antagonist, suggest that many of the behavioral functional pentameric GABAARs, renders receptors insensitive effects of EtOH at relevant physiological concentrations are me- to BZs (18). Also, most ␦ subunits are found to be associated ͞ diated by EtOH Ro15-4513-sensitive GABAA receptors. with ␣4 and ␣6 subunits that differ from other GABAAR ␣ subunits at a critical position (a histidine in ␣1,2,3,5 replaced by an ͉ ͉ ␤ ͉ alcohol receptor flumazenil -carbolines extrasynaptic GABAA arginine in ␣4,6) that makes ␣4/6␤x␥2 receptors insensitive to receptors classical BZ agonists such as diazepam (DZ) and flunitrazepam (19, 20). However, Arg-100 (WT) in ␣4 and ␣6 receptors still lthough many proteins show changes in their function at allows high-affinity binding of the imidazo-BZs Ro15-4513 and ␣ ␤ ␥ Avery high alcohol concentrations (Ͼ50 mM), the molecular flumazenil (in x x 2 GABAARs). In functional receptor assays, basis for behavioral alcohol effects at low to moderately intox- Ro15-4513 is a weak partial inverse agonist (i.e., it leads to a icating doses experienced during social alcohol consumption slight reduction in GABAAR activity) on the most abundant remains elusive (1). GABAA receptors (GABAARs) and the GABAAR subtypes in the brain (20). Ro15-4513 is a partial inhibitory GABAergic system have long been suspected to be agonist (i.e., it enhances GABA action, but less than DZ even at ␣ ␣ ␤ targets for acute alcohol effects (2–4). For example, the saturating concentrations) on the 4 and 6 receptors (with and ␥ GABAAR agonist muscimol potentiates the sedative actions of 2 subunits), whereas flumazenil is essentially silent in functional alcohol, whereas the opposite effect, a reduction of ethanol (EtOH)-produced sedation, is detected with the GABAAR blockers picrotoxin and bicuculline (5). Although most Conflict of interest statement: M.W., R.W.O., and H.J.H. have filed a U.S. Provisional Patent Application, Serial No. 60͞693,844. GABAAR subunit combinations can be activated by high (an- This paper was submitted directly (Track II) to the PNAS office. esthetic) alcohol concentrations (6), only very specific GABAAR subunit combinations (containing the ␦ as well as the ␤ subunit) Abbreviations: GABAAR, GABAA receptor; HEK, human embryonic kidney; BZ, benzodiaz- 3 epine; EtOH, ethanol; IP, immunoprecipitated͞immunoprecipitation; ␤-CCE, ␤-carboline- exhibit dose dependencies that mirror blood alcohol levels that 3-carboxyethyl ester; DMCM, methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate; are associated with mild to moderate intoxication in humans (7, DZ, diazepam; DZ-IS, DZ-insensitive; FG7142, N-methyl-␤-carboline-3-carboxamide. 8) (Ϸ3–30 mM, because the legal drinking limit is 17 mM or See Commentary on page 8307. ␦ 0.08%). GABAARs containing the subunit are located either †H.J.H. and P.C. contributed equally to this work. outside (9) or in the perimeter of (10) synapses but not in the ʈTo whom correspondence may be addressed at: Department of Molecular and Medical subsynaptic membrane, and they give rise to a persistent (tonic) Pharmacology, University of California, Room 23-120 CHS, Charles Young Drive South, Los GABA current (11) that is enhanced by low alcohol concentra- Angeles, CA 90095-1735. E-mail: [email protected] or [email protected]. tions (12–14). However, there is controversy because Carta et al. © 2006 by The National Academy of Sciences of the USA 8546–8551 ͉ PNAS ͉ May 30, 2006 ͉ vol. 103 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509903103 Downloaded by guest on September 24, 2021 SEE COMMENTARY Fig. 1. EtOH-sensitive ␣4/6␤3␦ GABAARs have a high-affinity Ro15-4513- binding site. [3H]Ro15-4513 saturation binding in native immuno-purified (␦-IP pellet) and ␦-depleted (␦-IP supernatant) cerebellar GABAAR fractions and to recombinant ␣4␤3␦ and ␣4␤3␥2 GABAARs receptor expressed in the HEK 293T cell line. assays. Interestingly, Ro15-4513, but not other inverse agonists [like the ␤-carbolines ␤-carboline-3-carboxyethyl ester (␤-CCE) and methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate (DMCM)], has been shown to have fairly dramatic alcohol- antagonist actions, reported in various mammals; in particular, alcohol effects at lower doses can be reversed almost completely by Ro15-4513 (3, 21–25). Fig. 2. Alcohol receptor binding assay. EtOH-displaceable high-affinity 3 ␦ Here, we show that alcohol-sensitive ␣4/6␤3␦ GABAARs bind [ H]Ro15-4513 binding to native and recombinant subunit-containing 3 [3H]Ro15-4513 with high affinity, and that this binding is inhib- GABAAR. (a)[H]Ro15-4513 binding is inhibited by low concentrations of 3 ited dose-dependently by low doses of EtOH. This result is EtOH. Receptors were equilibrated with 10 nM [ H]Ro15-4513 and varying EtOH concentrations. We tested native immunopurified (␦-IP pellet) and consistent with the observation that Ro15-4513 potently inhibits ␦ ␦ ␣ ␤ ␦ -depleted ( -IP supernatant) cerebellar GABAAR fractions, and recombinant EtOH enhancement of functional 4/6 3 GABAARs (45). The ␣4␤3␦ and ␣4␤3␥2 GABAARs expressed in the HEK 293T cell line. (b) Approxi- Ro15-4513͞EtOH antagonism appears to be competitive. Im- mately one-third of the DZ-IS [3H]Ro15-4513 binding to cow cerebellar portantly, our data explain the puzzling observation that Ro15- GABAARs is antagonized by low EtOH concentrations. (Inset) Total, 10 ␮M 4513 is a behavioral alcohol antagonist and suggest that EtOH͞ DZ-sensitive, and DZ-IS 30 nM [3H]Ro15-4513 binding to cow cerebellar membranes. Ro15-4513-sensitive GABAARs are important mediators of alcohol actions that are experienced during moderate social drinking. EtOH Inhibits [3H]Ro15-4513 Binding to ␦ Subunit-Containing Results GABAARs. Inspired by the finding that these alcohol-sensitive receptors bind the alcohol antagonist Ro15-4513 with high ␦ Alcohol Antagonist Ro15-4513 Binds to Subunit-Containing affinity, we investigated whether [3H]Ro15-4513 binding to ␦ GABAARs. Based on the observations that ␦ subunit-containing PHARMACOLOGY subunit-containing GABAAR can be inhibited by EtOH. Fig. 2a receptors are uniquely sensitive to EtOH (7), and that a BZ-site shows that [3H]Ro15-4513 binding to native cerebellar ␦ subunit ␣ single-nucleotide polymorphism ( 6-R100Q) increases alcohol immunopurified receptors (␦-IP pellet), as well as to recombi- sensitivity in ␣6␤3␦ receptors (12), we decided to investigate nant ␣4␤3␦ receptors, was dose-dependently inhibited by 3–300 whether native and recombinant ␦ subunit-containing receptors 3 mM EtOH (IC50 Ϸ 12 mM).
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