The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain*
- From the ‡Neurobiology Division, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH and the ¶Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, United Kingdom
Abstract
Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligand-gated ion channels. To examine the role of tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine3(5-HT3) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT3A receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT3A receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Trp90, Trp183, and Trp195 to tyrosine resulted in functional receptors, although with increased EC50 values (2–92-fold) to 5-HT3 receptor agonists. Changing these residues to serine either ablated function (Trp90 and Trp183) or resulted in a further increase in EC50(Trp195). Mutation of residue Trp60 had no effect on ligand binding or receptor function, whereas mutation of Trp95, Trp102, Trp121, and Trp214 ablated ligand binding and receptor function, and all but one of the receptors containing these mutations were not expressed at the plasma membrane. We propose that Trp90, Trp183, and Trp195 are intimately involved in ligand binding, whereas Trp95, Trp102, Trp121, and Trp214 have a critical role in receptor structure or assembly.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Recipient of a Medical Research Council studentship. Present address: Dept. of Molecular Biology MB10, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
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↵‖ Wellcome Senior Research Fellow in Basic Biomedical Science. To whom correspondence should be addressed. E-mail: s.lummis@mole.bio.cam.ac.uk.
- Abbreviations:
- 5-HT3
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5-hydroxytryptamine3
- nACh
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nicotinic acetylcholine
- GABA
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γ-aminobutyric acid
- WT
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wild type
- mCPBG
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meta-chlorophenylbiguanide
- LGIC
-
ligand-gated ion channel
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- Received July 16, 1999.
- Revision received November 22, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
