ANALYSIS OF THE LIGAND-BINDING SITE OF THE 5-HT3 RECEPTOR USING SITE-DIRECTED MUTAGENESIS - IMPORTANCE OF GLUTAMATE-106

The 5-HT3 receptor is a ligand-gated ion channel with significant stru ctural similarity to the nicotinic acetylcholine receptor. Several reg ions that form the Ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably ref lecting the conserved signal transduction mechanism. Specific amino ac id differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have repla ced one of these residues, glutamate 106 (E106), with aspartate (D), a sparagine (N), alanine (A) or glutamine (Q) and characterized the Liga nd-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the sel ective 5-HT3 receptor antagonist [H-3]GR65630 was decreased 14-fold in the mutant E106D (K-d = 3.69 +/- 0.32 nM) when compared to wildtype ( WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E1 06N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreas ed affinities for both E106D and E106N were observed for the antagonis ts 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 s even-fold in E106N (8.7 mu M) when compared to wildtype (1.2 mu M), bu t unchanged in E106D, and the potency of the antagonist ondansetron fo r both mutants was decreased. E106A and E106Q expressed poorly prevent ing a detailed characterization. These data suggest that E106 contribu tes to the Ligand-binding site of the 5-HT3 receptor and may form an i onic or hydrogen bond interaction with the primary ammonium group of 5 -HT. (C) 1997 Elsevier Science Ltd.