title: Whole spectrum analysis of ligand efficacy at constitutively active human wild-type and S267K 5-HT6 receptors in HEK-293F cells.
abstract: BACKGROUND
  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 B(261)BXXB(265) CIII-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 (E(max), % 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 pK(i) values. 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratorios Dr. Esteve S.A., Av. Mare de Déu de Montserrat 221, 08041 Barcelona, Spain.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Romero
authors.forename: Marta
authors.initials: M
authors.lastname: Pujol
authors.forename: Pilar
authors.initials: P
authors.lastname: Pérez
authors.forename: Helmut
authors.initials: H
authors.lastname: Buschmann
authors.forename: Petrus J
authors.initials: PJ
authors.lastname: Pauwels
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D014363:Tryptamines / Q000494:pharmacology
chemical_list: D008024:Ligands; C419803:N1-benzenesulfonyl-5-methoxy-N,N-dimethyltryptamine; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013449:Sulfonamides; D013876:Thiophenes; D014363:Tryptamines; C097216:serotonin 6 receptor; D000242:Cyclic AMP; C117443:SB 271046