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Palonosetron is a 5-HT3-receptor antagonist (5-HT3-RA) that has been shown to be superior to other 5-HT3-RAs in phase III clinical trials for the prevention of acute, delayed, and overall chemotherapy-induced nausea and vomiting. The improved clinical efficacy of palonosetron may be due, in part, to its more potent binding and longer half-life. However, these attributes alone are not sufficient to explain the results with palonosetron. We sought to elucidate additional differences among 5-HT3-RAs that could help explain the observations in the clinic.Receptor site saturation binding experiments were performed with [3H] palonosetron, [3H] granisetron, and [3H] ondansetron to obtain the corresponding Scatchard analyses and Hill coefficients. Diagnostic equilibrium binding experiments and kinetic dissociation experiments were conducted to examine competitive versus potential allosteric interactions between ondansetron, granisetron and palonosetron and the 5-HT3 receptor. Finally, the long-term effect of the three antagonists on receptor function as measured by Ca2+ influx in HEK 293 cells expressing the 5-HT3-receptor was compared.Analyses of binding isotherms using both Scatchard and Hill plots suggested positive cooperativity for palonosetron and simple bimolecular binding for both granisetron and ondansetron. Equilibrium diagnostic tests discriminated differential effects of palonosetron on [3H] ligand binding indicating that palonosetron was an allosteric antagonist whereas granisetron and ondansetron were competitive antagonists. Using dissociation rate strategies, palonosetron was shown to be an allosteric modifier that accelerated the rate of dissociation from the receptor of both granisetron and ondansetron. Differences in the binding mode of palonosetron to the 5-HT3 receptor were shown to have an impact on receptor function. In these experiments, cells were incubated with each antagonist, followed by infinite dilutions and dissociation for 2.5 h; cells previously incubated with either granisetron or ondansetron showed calcium-ion influx similar to control cells that had not been exposed to a 5-HT3 receptor antagonist. In contrast, substantial inhibition of calcium-ion influx was observed in cells that had been incubated with palonosetron.Palonosetron exhibited allosteric binding and positive cooperativity when binding to the 5-HT3 receptor. Palonosetron also triggered functional effects that persisted beyond its binding to the 5-HT3 receptor at the cell surface. Differences in binding and effects on receptor function may be relevant to the unique beneficial actions of palonosetron. To our knowledge, this is the first report showing palonosetron's interaction with the 5-HT3 receptor at the molecular level, clearly differentiating it from other 5-HT3-RAs.