Abstract

Ligand-gated ion channels participate in synaptic transmission, and they are involved in neurotransmitter release. The functions of the channels are regulated by a variety of modulators. The interaction of 2,2,2-trichloroethanol, the active hypnotic metabolite of chloral hydrate, with the 5-hydroxytryptamine (5-HT) (serotonin) type 3 receptor results in a positive allosteric modulation. We have demonstrated previously that arginine 246 (R246) located in the pretransmembrane domain 1 is critical for coupling agonist binding to gating. In this study, we examined the role of R246 in the action of trichloroethanol with a combination of mutagenesis and whole-cell patch-clamp techniques. The R246A mutation converted the partial agonist dopamine into a full agonist at the 5-HT_3A receptor, and it facilitated activation of the mutant receptor by dopamine, suggesting an enhanced gating process due to the mutation. The positive modulation of the 5-HT_3A receptor by trichloroethanol was dramatically reduced by the R246A mutation. Trichloroethanol had little agonist activity in the wild-type receptor (<1% of maximal 5-HT response). However, the R246A mutation significantly increased the direct activation of the receptor by trichloroethanol in the absence of agonist (∼10% of maximal 5-HT response). The current activated by trichloroethanol could be blocked by the competitive 5-HT₃ receptor antagonist tropanyl 3,5-dichlorobenzoate (MDL 72222), and it had a similar reversal potential to those of current activated by 5-HT. In addition, predesensitization of the mutant receptor by trichloroethanol prevented 5-HT from activating the receptor. These data suggest that R246 is a crucial site for mediating the actions of both agonists and modulators.