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• W2005205779 abstract "Ethanol can potentiate serotonin type 3 (5-HT3) receptor-mediated responses in various neurons and in cells expressing 5-HT3A receptors. However, the molecular basis for alcohol modulation of 5-HT3receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT3A receptor can alter the EC50 value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT3A receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC50values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC50 value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT3Areceptors to ethanol in the absence and presence of agonist. Ethanol can potentiate serotonin type 3 (5-HT3) receptor-mediated responses in various neurons and in cells expressing 5-HT3A receptors. However, the molecular basis for alcohol modulation of 5-HT3receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT3A receptor can alter the EC50 value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT3A receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC50values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC50 value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT3Areceptors to ethanol in the absence and presence of agonist. The serotonin type 3 (5-HT3) receptor is a member of a superfamily of ligand-gated ion channels that includes γ-aminobutyric acid type A (GABAA), 1The abbreviations used for: GABA, γ-aminobutyric acid; WT, wild type; TM, transmembrane domain; DA, dopamine; ANOVA, analysis of variance; SR, Spearman Rank; 2-Met-5-HT, 2-methyl-serotonin; MDL, MDL-72222; LY, LY 278,584. 1The abbreviations used for: GABA, γ-aminobutyric acid; WT, wild type; TM, transmembrane domain; DA, dopamine; ANOVA, analysis of variance; SR, Spearman Rank; 2-Met-5-HT, 2-methyl-serotonin; MDL, MDL-72222; LY, LY 278,584.glycine, and nicotinic acetylcholine receptors (1Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (882) Google Scholar). Initial molecular cloning studies identified a subunit, the 5-HT3A receptor, from different mammalian species (1Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (882) Google Scholar, 2Belelli D. Balcarek J.M. Hope A.G. Peters J.A. Lambert J.J. Blackburn T.P. Mol. Pharmacol. 1995; 48: 1054-1062PubMed Google Scholar). Like other members of this superfamily, the 5-HT3A receptor consists of a large N-terminal domain, four transmembrane domains (TM), and a large intracellular domain (1Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (882) Google Scholar). In situ hybridization studies have detected the expression at high levels of the 5-HT3Areceptor subunit in the hindbrain, especially in the nucleus tractus solitarius, area postrema, substantia nigra, and ventral tegmental area (3Tecott L.H. Maricq A.V. Julius D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1430-1434Crossref PubMed Scopus (316) Google Scholar, 4Spier A.D. Wotherspoon G. Nayak S.V. Nichols R.A. Priestley J.V. Lummis S.C. Brain Res. Mol. Brain Res. 1999; 67: 221-230Crossref PubMed Scopus (51) Google Scholar, 5Morales M. McCollum N. Kirkness E.F. J. Comp. Neurol. 2001; 438: 163-172Crossref PubMed Scopus (54) Google Scholar). In some of these brain areas, activation of 5-HT3receptors appears to increase the release of neurotransmitters such as glutamate, GABA, and dopamine (DA) (6Campbell A.D. McBride W.J. Pharmacol. Biochem. Behav. 1995; 51: 835-842Crossref PubMed Scopus (122) Google Scholar, 7Koyama S. Matsumoto N. Kubo C. Akaike N. J. Physiol. 2000; 529: 373-383Crossref PubMed Scopus (85) Google Scholar, 8van Hooft J.A. Vijverberg H.P. Trends Neurosci. 2000; 23: 605-610Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Because DA is thought to play an important role in brain reward and reinforcement mechanisms, stimulation of DA release by activation of 5-HT3 receptors may be of significance in the mechanisms involved in anxiety, psychosis, cognitive processes, and addiction (9Grant K.A. Drug Alcohol Depend. 1995; 38: 155-171Crossref PubMed Scopus (109) Google Scholar, 10Zeitz K.P. Guy N. Malmberg A.B. Dirajlal S. Martin W.J. Sun L. Bonhaus D.W. Stucky C.L. Julius D. Basbaum A.I. J. Neurosci. 2002; 22: 1010-1019Crossref PubMed Google Scholar). Accumulating evidence has indicated that the 5-HT3 receptor is an important target for alcohol action in the central nervous system (9Grant K.A. Drug Alcohol Depend. 1995; 38: 155-171Crossref PubMed Scopus (109) Google Scholar, 11Higgins G.A. Tomkins D.M. Fletcher P.J. Sellers E.M. Neurosci. Biobehav. Rev. 1992; 16: 535-552Crossref PubMed Scopus (89) Google Scholar, 12Lovinger D.M. Neurochem. Int. 1999; 35: 125-130Crossref PubMed Scopus (123) Google Scholar). Ethanol has been found to potentiate 5-HT3receptor-mediated currents in various neuronal cell lines (13Lovinger D.M. White G. Mol. Pharmacol. 1991; 40: 263-270PubMed Google Scholar), mammalian cell lines (14Lovinger D.M. Zhou Q. Neuropharmacology. 1994; 33: 1567-1572Crossref PubMed Scopus (69) Google Scholar), and Xenopus oocytes (15Machu T.K. Harris R.A. J. Pharmacol. Exp. Ther. 1994; 271: 898-905PubMed Google Scholar, 16Yu D. Zhang L. Eisele J.L. Bertrand D. Changeux J.P. Weight F.F. Mol. Pharmacol. 1996; 50: 1010-1016PubMed Google Scholar) expressing recombinant 5-HT3A receptors. Several lines of evidence suggest that 5-HT3 receptors may play an important role in alcohol preference and reward mechanisms (9Grant K.A. Drug Alcohol Depend. 1995; 38: 155-171Crossref PubMed Scopus (109) Google Scholar, 17Sellers E.M. Higgins G.A. Sobell M.B. Trends Pharmacol. Sci. 1992; 13: 69-75Abstract Full Text PDF PubMed Scopus (300) Google Scholar). Recent clinical studies have provided evidence that ondansetron, a selective 5-HT3 receptor antagonist, can reduce alcohol intake in early onset alcoholics (18Johnson B.A. Ait-Daoud N. Psychopharmacology. 2000; 149: 327-344Crossref PubMed Scopus (147) Google Scholar, 19Johnson B.A. Roache J.D. Ait-Daoud N. Zanca N.A. Velazquez M. Psychopharmacology. 2002; 160: 408-413Crossref PubMed Scopus (89) Google Scholar). However, the cellular and molecular mechanisms of ethanol action on 5-HT3 receptor function are not fully understood. On the cellular level, the potentiation of 5-HT3 receptor-mediated responses by ethanol was found to be inversely dependent on agonist concentration. The potentiation increased with decreasing agonist concentrations and was not observed in the presence of high agonist concentrations (16Yu D. Zhang L. Eisele J.L. Bertrand D. Changeux J.P. Weight F.F. Mol. Pharmacol. 1996; 50: 1010-1016PubMed Google Scholar, 20Lovinger D.M. Neurosci. Lett. 1991; 122: 57-60Crossref PubMed Scopus (94) Google Scholar). Recent studies showed that ethanol slowed the desensitization rate of the current activated by 5-HT and increased maximal amplitude of current activated by DA, a partial agonist of the 5-HT3 receptor, suggesting that ethanol may act on channel gating through increasing the probability of channel opening (21Zhou Q. Verdoorn T.A. Lovinger D.M. J. Physiol. 1998; 507: 335-352Crossref PubMed Scopus (67) Google Scholar, 22Lovinger D.M. Sung K.W. Zhou Q. Neuropharmacology. 2000; 39: 561-570Crossref PubMed Scopus (53) Google Scholar). Many recent investigations into the molecular mechanism of alcohol action have focused on GABAA and glycine receptors (23Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1097) Google Scholar, 24Ye Q. Koltchine V.V. Mihic S.J. Mascia M.P. Wick M.J. Finn S.E. Harrison N.L. Harris R.A. J. Biol. Chem. 1998; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Ueno S. Lin A. Nikolaeva N. Trudell J.R. Mihic S.J. Harris R.A. Harrison N.L. Br. J. Pharmacol. 2000; 131: 296-302Crossref PubMed Scopus (63) Google Scholar). Point mutations of several specific amino acids located in the second and third TMs of glycine and GABAA receptors have been shown to alter the sensitivity of the receptor to ethanol, suggesting that these residues may be crucial for the allosteric modulation of receptor function by ethanol. However, mechanistic studies of ethanol action in the absence of agonist have not been reported for ligand-gated ion channels. In addition, although some of the point mutations that alter the sensitivity of GABAA and glycine receptors to ethanol have been also found to be sensitive to ethanol in the absence of agonist (23Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1097) Google Scholar, 25Ueno S. Lin A. Nikolaeva N. Trudell J.R. Mihic S.J. Harris R.A. Harrison N.L. Br. J. Pharmacol. 2000; 131: 296-302Crossref PubMed Scopus (63) Google Scholar), the interrelationship between ethanol responses in the absence of agonist and ethanol potentiation of agonist-activated responses of these receptors remains unclear. Nevertheless, such studies have not been reported for 5-HT3 receptors. A previous study in our laboratory suggested that the N terminus of a chimeric nicotinic-serotonergic receptor may be involved in the mediation of ethanol sensitivity of that protein (16Yu D. Zhang L. Eisele J.L. Bertrand D. Changeux J.P. Weight F.F. Mol. Pharmacol. 1996; 50: 1010-1016PubMed Google Scholar). However, that study did not provide information on the molecular sites that alter the sensitivity of the receptor to ethanol. Here, we report that substitutions of an arginine (Arg) with a series of amino acid residues at 222 in the N-terminal domain, immediately preceding TM1 of the 5-HT3A receptor, can alter receptor sensitivity to ethanol and the EC50 value of the 5-HT concentration-response curve. Further, detailed analyses suggest that the sensitivity of the receptor to ethanol in the absence and presence of agonist may be mediated through different molecular processes. Some of this work has been presented previously in preliminary forms (26Zhang L. Hosoi M. Fukuzawa M. Sun H. Weight F.F. Alc. Clin. Exp. Res. 2001; 25: 58AGoogle Scholar, 27Hosoi M. Moradel E.M. Hu X.-Q. Fukuzawa M. Sun H. Weight F.F. Zhang L. Soc. Neurosci. Abstr. 2001; 27: 600Google Scholar). Point mutations of a cloned mouse 5-HT3A receptor were introduced using a QuikChange site-directed mutagenesis kit (Stratagene). The authenticity of the DNA sequence through the mutation sites was confirmed by double strand DNA sequencing using an ABI Prism 377 automatic DNA sequencer (Applied Biosystems). Complementary RNA (cRNA) was synthesized in vitro from a linearized template cDNA with a mMACHINE RNA transcription kit (Ambion Inc.). The oocytes of matureXenopus laevis frogs were isolated as described previously (28Zhang L. Oz M. Weight F.F. Neuroreport. 1995; 6: 1464-1468Crossref PubMed Scopus (34) Google Scholar). Each oocyte was injected with a total of 20 ng of RNA in 20 nl of diethylpyrocarbonate-treated water and was incubated at 19 °C in modified Barth's solution (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 2.0 mm CaCl2, 0.8 mm MgSO4, 10 mm HEPES, pH 7.4). After incubation for 2–5 days, the oocytes were studied at 20–22 °C in a 90-μl chamber. The oocytes were superfused with modified Barth's solution at a rate of ∼6 ml/min. Agonists and antagonists were diluted in the bathing solution and applied to the oocytes for a specified time, using a solenoid valve-controlled superfusion system (Automate Scientific). Membrane currents were recorded by two-electrode voltage-clamp at a holding potential of −70 mV, using a Gene Clamp 500 amplifier (Axon Instruments, Inc.). Data were routinely recorded on a chart recorder (Gould 2300S). Values are expressed as mean ± S.E. To determine [3H]5-HT binding, we used the single-oocyte binding method (29Chang Y. Weiss D.S. Nat. Neurosci. 1999; 2: 219-225Crossref PubMed Scopus (103) Google Scholar) with modifications. Briefly, oocytes injected with the cRNA of the wild type (WT) and mutant 5-HT3A receptors were prescreened by two-electrode voltage-clamp at −70 mV. Oocytes with current amplitudes from 4 to 6 μA in response to 100 μm5-HT were selected and held individually by suction at the end of a Pasteur pipette tip. The oocyte was first incubated at 20–22 °C for 30 s in 300 nm [3H]5-HT solution (specific activity = 20 Ci/mmol; Amersham Biosciences), and then rinsed for 6 s in 150 ml of ice-cold modified Barth's bathing solution to remove free [3H] ligands from the oocyte surface. The nonspecific binding was determined by incubation in 300 μm unlabeled 5-HT. The specific binding was determined by subtracting the nonspecific binding from the total binding. Each sample is the average from three or four oocytes, and each data point is the average from at least three or four separate experiments. The radioactivity (cpm) of each sample was determined in a 1409 DSA Wallac liquid scintillation counter (PerkinElmer Life Sciences). The association and dissociation rate constants were calculated using the following equation. kon=kobs−koff/[C]Equation 1 k obs is the observed rate constant (s−1), determined from fitting a one-phase exponential association equation: Y = Y max(1 − e −kX), [C] is the concentration of radioligand used, and k off is the dissociation rate constant. K d(BC50), B max, andk off were determined by equilibrium and competitive binding data using Prism Software (GraphPad). Statistical analysis of concentration-response curves was performed using the following form of the Hill equation. I=Imax1+(EC50/[A])nEquation 2 I is the peak current at a given concentration of agonist A, I max is the maximal response, EC50 is the half-maximal concentration, andn is the slope factor (apparent Hill coefficient). Data were statistically compared by the unpaired t test or ANOVA analysis. Correlation analysis was carried out using nonparametric regression or linear regression (Statistica, StatSoft). Although molecular cloning has identified two subtypes of 5-HT3 receptors, 5-HT3A and 5-HT3B (1Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (882) Google Scholar, 30Davies P.A. Pistis M. Hanna M.C. Peters J.A. Lambert J.J. Hales T.G. Kirkness E.F. Nature. 1999; 397: 359-363Crossref PubMed Scopus (488) Google Scholar), the 5-HT3A receptor is thought to be an essential component of all serotonin-gated ion channels. Fig.1 A shows three consecutive arginine residues at 220, 221, and 222 of the 5-HT3Areceptor. These arginines, which are highly conserved across 5-HT3A receptors from different species, were predicted by computational modeling to be important structural elements for activation of the 5-HT3 receptor (31Aprison M.H. Galvez-Ruano E. Lipkowitz K.B. J. Neurosci. Res. 1996; 43: 127-136Crossref PubMed Scopus (24) Google Scholar). To examine the functional role of these arginines, each of the residues (positions 220–222) was replaced with alanine. The R221A mutant receptor was not functional (data not shown), indicating that the arginine at 221 is critical for activation of the 5-HT3 receptor. In contrast, the R220A and R222A mutant receptors were functional when expressed inXenopus oocytes. The maximal amplitudes of currents activated by 5-HT were not significantly different among the WT, R220A, and R222A mutant receptors (p > 0.05; TableI). However, the alanine substitution at amino acid 222, but not at 220, significantly decreased the EC50 value of the 5-HT concentration-response curve (Fig.1 B; Table I). Next, we examined whether the R222A mutation alters the sensitivity of the receptor to 2-methyl-5-HT (2-Met-5-HT), a partial agonist of the 5-HT3 receptor. Fig. 1 Bshows that the R222A mutation also significantly shifted the 2-Met-5-HT concentration-response curve to the left. The EC50 values of the 5-HT and 2-Met-5-HT concentration-response curves for the R222A mutant receptors were approximately 70- and 14-fold lower, respectively, than those of the WT 5-HT3A receptor (n = 6; p < 0.01). The EC50 value and the Hill coefficient for 2-Met-5-HT were 13 ± 0.4 μm and 1.8, respectively, for the WT receptors, and 0.9 ± 0.2 μm and 1.6, respectively, for the R222A receptors. The R222A mutation increased the efficacy of 2-Met-5-HT from 45 ± 3% to 99 ± 6% of the maximal 5-HT-activated response; these values were significantly different (unpaired t test, p < 0.001,n = 5–7). To determine whether the R222A mutation affects receptor binding, we conducted receptor-binding experiments using a single-oocyte ligand-binding method described previously (29Chang Y. Weiss D.S. Nat. Neurosci. 1999; 2: 219-225Crossref PubMed Scopus (103) Google Scholar). The time constants of [3H]5-HT association were 9.1 ± 1.3 s for the WT receptors and 7.2 ± 1.1 s for the R222A receptors, and the dissociation rates were 0.9 ± 0.3 s for the WT receptors and 0.8 ± 0.1 s for the R222A receptors; these values were not significantly different (unpairedt test, p > 0.6). Fig. 1 Cillustrates receptor-binding data for the WT and R222A mutant 5-HT3A receptors. The 5-HT concentration-response curves of 0.3 μm [3H]5-HT binding for the WT receptors (open circles) and for the R222A mutants (solid circles) are superimposed. The values of BC50 and the Hill coefficient were 0.67 ± 0.07 μm and 1.8 ± 0.1, respectively, for the WT 5-HT3A receptors and 0.52 ± 0.06 μm and 1.7 ± 0.1, respectively, for the R222A mutant receptors; these values were not significantly different (unpaired t test,p > 0.1). These results suggest that the R222A mutation decreases the EC50 of 5-HT3A receptor through modulation of receptor gating. To understand the structure-function role of the amino acid residue at 222 of the 5-HT3A receptor, the arginine at 222 was replaced by various amino acids and the function of each mutant receptor was examined by a two-electrode voltage-clamp in Xenopusoocytes. Except for R222K (Lys), a positively charged amino acid residue, the other point mutations at 222 significantly decreased the EC50 value of the 5-HT concentration-response curve by 5–100-fold (p < 0.01) (Fig. 1 D). The values of EC50, Hill coefficient, andI max for 5-HT are given in Table I. It should be noted that the effect of the R222A mutation appeared to be site-specific because replacing an arginine with an alanine at 220 (R220A) did not significantly alter the sensitivity of the receptor to 5-HT (Fig. 1 D; Table I).Table ISummary of the properties of the WT and mutant 5-HT3A receptors expressed in Xenopus oocytesReceptor5-HT EC50Hill slopeI maxμmμmWT1.58 ± 0.11.6 ± 0.24.2 ± 0.5R220A1.30 ± 0.11.7 ± 0.23.8 ± 0.4R222A0.02 ± 0.01*2.1 ± 0.2*4.5 ± 0.6R222E0.04 ± 0.02*1.5 ± 0.14.6 ± 0.4R222I0.06 ± 0.01*1.4 ± 0.42.5 ± 0.1*R222Q0.07 ± 0.01*1.2 ± 0.35.6 ± 1.3R222G0.09 ± 0.01*1.2 ± 0.13.9 ± 0.2R222F0.09 ± 0.01*0.9 ± 0.1*1.2 ± 0.3*R222D0.13 ± 0.01*1.9 ± 0.22.7 ± 0.4R222T0.30 ± 0.01*1.6 ± 0.12.8 ± 0.5*R222N0.32 ± 0.01*1.5 ± 0.13.1 ± 0.5R222H0.60 ± 0.3*1.3 ± 0.13.4 ± 0.3R222K2.10 ± 0.4*1.7 ± 0.13.5 ± 0.6The average values of EC50, Hill coefficient (n), and I max of the 5-HT concentration-response curves are given for each receptor as mean ± S.E. These values were obtained from fitting the data to the equation given under “Experimental Procedures.” The values for each given receptor were compared with those of the WT receptor, and the statistical significance was calculated using ANOVA. * p< 0.01, compared with WT. Open table in a new tab The average values of EC50, Hill coefficient (n), and I max of the 5-HT concentration-response curves are given for each receptor as mean ± S.E. These values were obtained from fitting the data to the equation given under “Experimental Procedures.” The values for each given receptor were compared with those of the WT receptor, and the statistical significance was calculated using ANOVA. * p< 0.01, compared with WT. Whereas ethanol at 100 mm did not induce detectable current (Fig. 2 A) in cells injected with cRNAs of the WT or R222K 5-HT3Areceptors, 100 mm ethanol activated an inward current in cells expressing mutant receptors replaced with Gly and Phe at position 222. In Fig. 2 B, the inward current activated by 200 mm ethanol for the mutant receptors was normalized and presented as percentage of the maximal response activated by 5-HT (Fig.2 B). In the cells expressing the mutant receptors replaced with Phe, Ile, Gly, Gln, and Asp at 222, 200 mm ethanol activated an inward current in the absence of agonist; however, R220A, R222A/T/E/N/H/K, and WT receptors were less sensitive or insensitive to this concentration of ethanol in the absence of agonist. On average, the order of magnitude of the agonist-independent effect by 200 mm ethanol for the mutant receptors was: Phe (13 ± 1.1%) > Ile (9.5 ± 0.6%) > Gly (7.3 ± 1.2%) > Gln (5.0 ± 1.1%) > Asp (4.9 ± 1.1%) > Ala (1.0 ± 0.4%) > Thr (0.53 ± 0.2%) > Glu (0.43 ± 0.1%) = Asn (0.2 ± 0.2%) = His (0.1 ± 0.05%) = Lys (0 ± 0%) = WT (0 ± 0%). The inward currents induced by ethanol appeared to be mediated through the mutant 5-HT3A receptors because MDL-72222 (MDL), a selective 5-HT3 receptor antagonist, inhibited the currents activated by ethanol in cells expressing R222G receptors (Fig. 2 C). In these cells, both MDL and another selective 5-HT3 receptor antagonist, LY 278,584 (LY), potently reduced the amplitude of the inward current activated by ethanol in the absence of agonist in a concentration-dependent manner over a concentration range from 0.1 to 300 nm (Fig. 2 D). The IC50 values of MDL and LY inhibition were 6.7 ± 0.3 and 11 ± 1 nm, respectively; the slope factors were 1.3 ± 0.2 and 1.2 ± 0.1, respectively; and the maximal values of inhibition were 86 ± 6 and 79 ± 5%, respectively. To study further the mechanism of MDL inhibition, we examined the effect of 10 nm MDL on the inward current activated by various concentrations of ethanol (Fig. 2 E). In cells expressing R222G receptors, MDL at 10 nm reduced the amplitude of inward current induced by ethanol at concentrations of 30, 60, 100, and 200 mm by 63 ± 8, 59 ± 5, 65 ± 8, and 60 ± 6%, respectively. These values were not significantly different (p > 0.2, ANOVA,n = 5), suggesting that the inhibition by MDL is independent of ethanol concentration. These results indicate that some point mutations at 222 of the 5-HT3A receptor can increase the sensitivity of the receptor to ethanol in the absence of agonist. Previous studies have reported that point mutations in the TM domains of nicotinic acetylcholine α7 and GABAA receptors can result in spontaneously opening or constitutively active channels (32Changeux J.P. Edelstein S.J. Neuron. 1998; 21: 959-980Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 33Chang Y. Weiss D.S. Biophys. J. 1999; 77: 2542-2551Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). To determine whether point mutations at 222 of 5-HT3Areceptors can induce spontaneously active channels, we applied MDL, a 5-HT3 receptor antagonist, to cells expressing WT and mutant 5-HT3A receptors. Fig.3 A shows that, as a result of MDL inhibiting spontaneous channel opening, 300 nm MDL (300 nm) induced outward current in cells expressing R222G or R222F mutants, but not in cells expressing WT or R222K receptors. The order (Fig. 3 B) of the average amplitude of the outward current activated by MDL (300 nm) for those mutant receptors was: R222F (Phe) > R222I (Ile) > R222Q (Gln) > R222G (Gly) > R222D (Asp) > R222A (Ala) > R222N (Asn) = R222T (Thr) = R222E (Glu). In cells expressing WT (Arg-222), R222H, R222K, and R220A receptors, MDL (300 nm) did not activate detectable outward current. Because the amino acid residues at 222 of the WT (Arg-222), R222K, and R222H are positively charged, this result suggests that the positive charge at 222 may be critical for stabilizing the receptor channels in a closed state. The amplitude of the outward current activated by 300 nm MDL (Fig. 3 C) was significantly correlated with the amplitude of the current activated by 200 mm ethanol (SR = 0.96;p < 0.003, nonparametric regression, Statistica), indicating that the magnitude of the ethanol-induced inward current in the absence of agonist correlates with the magnitude of the spontaneous openings of the mutant ion channels. Next, we examined whether or not point mutations of Arg-222 can affect the ethanol sensitivity of 5-HT responses of 5-HT3A receptors. The trace records in Fig. 4 illustrate the effect of ethanol at 100 and 200 mm on the 5-HT responses of WT, R222K, R222E, or R222A mutant receptors. The inward currents were activated by 5-HT at the EC5 concentration for that receptor. In cells expressing the WT receptors, ethanol potentiated the inward currents activated by 5-HT. The magnitude of the potentiation by ethanol decreased in cells expressing the R222K receptors. On the other hand, the magnitude of the potentiation by ethanol increased in cells expressing the R222E or R222A receptors. Thus, the 5-HT responses of these receptors are differentially sensitive to ethanol. As shown above, the WT and mutant 222 receptors could be differentially sensitive to ethanol in the absence and presence of agonist. To determine the effects of mutations at 222 on ethanol modulation of the 5-HT3A receptors, we compared the ethanol-induced inward current in the absence of agonist with ethanol potentiation of 5-HT responses for the WT and mutant 5-HT3Areceptors. As shown in Fig.5 A, ethanol-activated current for the mutant R222F/I/G/Q/D receptors was concentration-dependent over a concentration range of 10–200 mm. In contrast, Fig. 5 B shows that the mutant R220A, R222A/E/N/T/H/K, and WT receptors were insensitive or relatively insensitive to ethanol concentrations up to 200 mm in the absence of agonist. In view of these results, we divided the WT and mutant receptors into two groups based on their sensitivity to ethanol in the absence of agonist. Group 1 included the receptors in which ethanol activated inward current in the absence of agonist. Group 2 included receptors in which ethanol did not activate significant inward current in the absence of agonist. On the other hand, we also found that the ethanol sensitivity of 5-HT-activated responses differed for the group 1 and group 2 receptors. Fig.5 C shows that ethanol, at concentrations from 10 to 200 mm, did not significantly affect the amplitude of current activated by low concentrations (EC5) of 5-HT in cells expressing the group 1 receptors, whereas Fig. 5 D shows that ethanol significantly enhanced responses activated by 5-HT at EC5 concentrations in cells expressing the group 2 receptors (p < 0.01). To determine whether the ethanol-induced inward current in the absence of agonist correlates with ethanol potentiation of agonist responses, we compared the sensitivity of the WT and mutant 5-HT3A receptors to ethanol in the absence or presence of 5-HT (Fig. 5 E). The result in Fig. 5 E indicates that these receptors clearly differed in their sensitivity to ethanol in the absence and presence of agonist. There was no correlation between ethanol-induced inward current in the absence of agonist and ethanol potentiation of agonist responses (Fig. 5 E; R = −0.41,p = 0.1, linear regression, n = 13). To gain insight into the structure-function relationship of the point mutations at 222, we used correlation analysis to compare the magnitudes of the direct action of ethanol and the ethanol potentiation with isoelectric point (pI) (34Lehninger A.L. Nelson D.L. Cox M.M. Principles of Biochemistry. 2nd Ed. Worth Publishers, Inc., New York1993Google Scholar), polarity (35Zimmerman J.M. Eliezer N. Simha R. J. Theor. Biol. 1" @default.
• W2005205779 created "2016-06-24" @default.
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• W2005205779 creator A5039942691 @default.
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• W2005205779 date "2002-11-01" @default.
• W2005205779 modified "2023-10-04" @default.
• W2005205779 title "Distinct Molecular Basis for Differential Sensitivity of the Serotonin Type 3A Receptor to Ethanol in the Absence and Presence of Agonist" @default.
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