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The function of the 5-hydroxytryptamine3 (5-HT3) receptor is enhanced by ethanol, but the amino acid residue(s) that confers sensitivity to ethanol remains to be identified. Phosphorylation of the related GABAA receptor has been implicated in conferring its sensitivity to ethanol. In common with the GABAA receptor, the 5-HT3 receptor contains multiple consensus sites for protein kinases. To evaluate the possibility that phosphotylation of the 5-HT3 receptor underlies its ethanol sensitivity, we examined the ability of ethanol to enhance 5-HT-mediated currents in a mutant 5-HT3 receptor containing no intracellular serines, threonines, or tyrosines. Mutation of these 13 residues in the intracellular loops produced a modest leftward shift in the 5-HT concentration response curve, with the EC50's for 5-HT decreasing from 0.839 ± 0.03 μM in the wild-type receptor to 0.713 ± 0.03 μM in the mutant receptor. Cooperativity of the 5-HT binding sites was enhanced by the mutations, with Hill coefficients of 2.92 for the wild-type receptor and 3.74 for the mutant receptor, respectively. In oocytes expressing mutant receptors, ethanol (50 to 200 mM) enhanced the currents produced by low concentrations of 5-HT by ∼5 to 45%, which was not statistically different from the potentiation produced by ethanol in wild-type receptors. These results suggest that ethanol enhancement of the 5-HT3 receptor function does not require receptor phosphorylation. [ABSTRACT FROM AUTHOR]