Characterization of the 5-HT6 receptor coupled to Ca2+ signaling using an enabling chimeric G-protein

We examined the feasibility of coupling the 5-HT6 receptor to a Ca2+ signaling read-out using a chimeric G-protein, comprising of G(alphaq) with the C-terminal five amino acids from G(alphas) to facilitate assays on the fluorometric imaging plate reader (FLIPR). Using a transient transfection assay in human embryonic kidney (HEK) cells, Ca2+ signaling in response to serotonin (5-HT) was facilitated by co-transfection of the 5-HT6 receptor with the G(alphaq)/G(alphas) chimera, but not with the 5-HT6 receptor alone or with a similar chimera incorporating the C-terminal five amino acids of G(alphai3). A series of agonist concentration -response curves were constructed using the 5-HT6-G(alphaq)/G(alphas) signaling assay generating the following rank order of agonist potency; 5-methoxytryptamine (EC50, 9 nM)=5-HT (12 nM)=2-methyl 5-HT (13 nM)>tryptamine(86 nM)=5-carboxamidotryptamine(5-CT)(119 nM)>>Iisuride(>1 muM). In comparison, essentially identical EC50 values were observed for the stimulation of cAMP accumulation with the same compounds; 5-methoxytryptamine (EC50, 6 nM) = 5-HT (6 nM) = 2-methyl 5-HT (15 nM)>tryptamine (91 nM) = 5-CT (153 nM)>lisuride (>350 nM). Clozapine and SB 271046 both produced a concentration-dependent antagonism of the 5-HT-stimulated Ca2+ response with IC50 values of 45 and 11 nM, respectively. In contrast, aripiprazole, a recently launched atypical anti-psychotic with a novel mechanism of action described as a dopamine/serotonin stabilizer, was essentially devoid of 5-HT6 receptor antagonist activity. Our results demonstrate that a FLIPR-based Ca2+ signaling assay is a feasible approach to the functional characterization of 5-HT6 receptor ligands. Moreover, the equivalent coupling efficiency, as indexed by agonist potency, observed using this system compared with the native coupling assay to cAMP suggests that the C-terminal five amino acids of G(alphas) are the major determinant for the receptor/G-protein interaction of the 5-HT6 receptor subtype. (C) 2003 Elsevier Science B.V. All rights reserved.