Vol. 57, Issue 6, 1249-1255, June 2000

Importance of Phenylalanine 107 in Agonist Recognition by the 5-Hydroxytryptamine_3A Receptor

Lucinda J. Steward, Frank G. Boess, Joy A. Steele, Dan Liu, Norris Wong, and Ian L. Martin

Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada (J.A.S., D.L., N.W.); Yoshitomi Research Institute of Neuroscience in Glasgow, University of Glasgow, Glasgow, United Kingdom (L.J.S.); Bayer AG, Business Group Pharma, Pharma Research CNS, Wuppertal, Germany (F.G.B.); and Pharmaceutical Sciences Research Institute, School of Life and Health Sciences, Aston University, Birmingham, United Kingdom (I.L.M.)

The 5-hydroxytryptamine (5-HT)₃ receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, -aminobutyric acid_A, and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT_3AL subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K_i = 203 versus 15.6 nM) and an increase of similar magnitude in the EC₅₀ value (10.6 versus 1.2 µM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC₅₀ value for this agonist (0.2 versus 1.2 µM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC₅₀ = 260 µM). The response to acetylcholine was blocked by the 5-HT₃ receptor antagonist renzapride with a similar IC₅₀ value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT₃ receptor.