Abstract

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, γ-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.