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J. Biol. Chem., Vol. 281, Issue 24, 16576-16582, June 16, 2006

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Mutagenesis and Molecular Modeling Reveal the Importance of the 5-HT₃ Receptor F-loop^*

Andrew J. Thompson, Claire L. Padgett, and Sarah C. R. Lummis¹

From the Department of Biochemistry, University of Cambridge, and the Neurobiology Division, Laboratory of Molecular Biology, Cambridge, United Kingdom

The 5-HT₃ receptor is a member of the Cys-loop family of ligandgated ion channels. The extracellular domains of these proteins contain six amino acid loops (A–F) that converge to form the ligand binding site. In this study we have mutated 21 residues in or close to the 5-HT₃ receptor F-loop (Ile¹⁹² to Gly²¹²) to Ala or to a residue with similar chemical properties. Mutant receptors were expressed in HEK293 cells, and binding affinity was measured using [³H]granisetron. Two regions displayed decreases in binding affinity when mutated to Ala (Ile¹⁹²–Arg¹⁹⁶ and Asp²⁰⁴–Ser²⁰⁶), but only one region was sensitive when mutated to chemically similar residues (Ile¹⁹²–Val²⁰¹). Homology modeling using acetylcholine-binding protein crystal structures with a variety of different bound ligands suggests there may be distinct movements of Trp¹⁹⁵ and Asp²⁰⁴ upon ligand binding, indicating that these residues and their immediate neighbors have the ability to interact differently with different ligands. The models suggest predominantly lateral movement around Asp²⁰⁴ and rotational movement around Trp¹⁹⁵, indicating the former is in a more flexible region. Overall our results are consistent with a flexible 5-HT₃ receptor F-loop with two regions that have specific but distinct roles in ligand binding.

Received for publication, February 9, 2006 , and in revised form, March 29, 2006.

^* This work was supported by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and Merck Sharp and Dohme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A Wellcome Trust Senior Research Fellow in Basic Biomedical Studies. To whom correspondence should be addressed: Dept. of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom. Tel.: 44-1223-765-950; Fax: 44-1223-333-345; E-mail: sl120{at}cam.ac.uk.

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