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-HT3A 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-HT3A 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.
Footnotes
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This work was supported by institutional funds from Marquette University and by a grant from the National Institutes of Health (to R.W.P.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.131011.
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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); TM, transmembrane domain; preTM, pretransmembrane domain; HEK, human embryonic kidney; MDL 72222, tropanyl 3,5-dichlorobenzoate; TcEt, trichloroethanol; DA, dopamine; WT, wild type; EtOH, ethanol.
- Received August 30, 2007.
- Accepted December 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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