Arginine 246 of the pretransmembrane domain 1 region alters 2,2,2-trichloroethanol action in the 5-hydroxytryptamine3A receptor.
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 ( approximately 10% of maximal 5-HT response). The current activated by trichloroethanol could be blocked by the competitive 5-HT(3) 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.
Hu
Xiang-Qun
XQ
Department of Biomedical Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881, USA.
Peoples
Robert W
RW
D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence / Q000187:drug effects / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D005023:Ethylene Chlorohydrin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000187:drug effects / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics*; D058827:Serotonin 5-HT3 Receptor Agonists
D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists; D005023:Ethylene Chlorohydrin; D001120:Arginine; C005849:2,2,2-trichloroethanol