Elsevier

Neuropharmacology

Volume 36, Issues 4–5, April–May 1997, Pages 637-647
Neuropharmacology

Analysis of the Ligand Binding Site of the 5-HT3 Receptor Using Site Directed Mutagenesis: Importance of Glutamate 106

https://doi.org/10.1016/S0028-3908(97)00044-0Get rights and content

Abstract

The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 ± 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 ± 0.03 nM), while the affinity for E106N was unchanged (0.42 ± 0.07 nM, means ± SEM, n = 3–10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 μM) when compared to wildtype (1.2 μM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT. © 1997 Elsevier Science Ltd.

Section snippets

Site-directed mutagenesis of the 5-HT3-AL cDNA

A cDNA isolated from NG108-15 cells encoding the mouse 5-HT3-AL receptor (Werner et al., 1994) was generously provided by Dr Eric Kawashima (Glaxo Molecular Biology Institute, Geneva). The cDNA sequence was subcloned into the phagemid pAlter (Promega, Madison, WI, U.S.A.) and the eukaryotic expression vector pRC/CMV (Promega). Mutagenesis was performed using the Altered Sites Mutagenesis Kit (Promega). Glutamate (E) 106 was mutated to aspartate (D), asparagine (N), glutamine (Q), or alanine (A)

Radioligand binding characterization of mutant 5-HT3 receptors E106D and E106N

In saturation studies with membranes obtained from HEK 293 cells transiently expressing wildtype (WT), E106D or E106N mutant 5-HT3 receptors, [3H]GR65630 labelled a homogeneous population of binding sites (Fig. 2). The binding site density varied with the efficiency of both transfection and expression. Therefore, for ease of comparison, the saturation data are normalized to Bmax in the Scatchard transformations (Fig. 2B). The affinity of [3H]GR65630 was decreased by 14-fold for the mutant E106D

DISCUSSION

In the nicotinic acetylcholine receptor, amino acids involved in ligand recognition are concentrated in three “loops” of the α subunits and additional sites are present on the γ and δ subunits (reviewed in Changeux et al., 1992; Karlin and Akabas, 1995). A comparison of the amino acid sequence of the 5-HT3 receptor and nAChR α subunits shows that there is significant sequence conservation in the first of these recognition loops (Fig. 1). However, there are also major differences, for example,

Acknowledgements

The authors would like to thank Drs Tom Blackburn (SmithKline Beecham) and Gavin Kilpatrick (Glaxo) for their kind gifts of drugs. Supported by Glaxo Canada and MRC Canada. L.J.S. holds a MRC/PMAC Pfizer (Canada) Fellowship.

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    Present address: F. Hoffman-La Roche AG, Pharma Division, Preclinical Research, P.O. Box, CH-4070 Basel, Switzerland.

    Present address: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, Robertson Building, University of Glasgow, Glasgow, G11 6NU, U.K.

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