Introduction: Modulation of constitutive activity by the recombinant wild-type human 5-HT6 receptor was investigated with a series of 5-HT6 receptor ligands by monitoring the cAMP signalling pathway. The impact of the mutation S267K near the B261BXXB265CIII-loop motif was analyzed on the magnitude of constitutive receptor activity as previously conflicting results have been reported. Methods: The wild-type 5-HT6 receptor plasmid was obtained by PCR and the mutant S267K5-HT6 receptor was constructed by site-directed mutagenesis and stably transfected in HEK-293F cells by electroporation. The cAMP signalling pathway was monitored as a functional read-out to investigate ligands' responses using homogeneous time resolved fluorescence. Results: Constitutive activity was present both at wild-type and mutant S267K 5-HT6 receptors. Negative efficacy (Emax, % versus basal) as observed at nanomolar concentrations with SB-271046 was larger for mutant (- 92 ± 1%) than wild-type 5-HT6 receptor (- 45 ± 1%). Ro 04-6790 also demonstrated negative efficacy at the wild-type 5-HT6 receptor with a magnitude similar to SB-271046 but with a 36-fold lower potency. MS-245 demonstrated at nanomolar concentrations intermediate negative efficacy; - 48 ± 3% and - 16 ± 2% at mutant and wild-type 5-HT6 receptor, respectively. The 5-HT-mediated cAMP response was blocked by SB-271046, MS-245 and Ro 04-6790 to their respective level of negative efficacy with pKB values fitting with their binding pKivalues. E-6801 was a highly potent (pEC50: 10.17 to 10.19) and efficacious agonist (+ 98 to + 102% versus 5-HT) at both wild-type and mutant 5-HT6 receptors. Discussion: The recombinant wild-type human 5-HT6 receptor is constitutively active in HEK-293F cells and displays a high resolution to monitor efficacy properties of 5-HT6 receptor ligands. The resolution capacity to differentiate between efficacy properties of 5-HT6 receptor ligands, in particular for negative efficacy, can be further enhanced by monitoring the mutant S267K 5-HT6 receptor. © 2006 Elsevier Inc. All rights reserved.
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