Molecular cloning and pharmacological characterization of serotonin 5-HT(3A) receptor subtype in dog.
In order to establish if the canine 5-hydroxytryptamine type 3A (5-HT(3A)) receptors share the pharmacological profile with human 5-HT(3A) receptors, we cloned and performed a molecular pharmacological characterization of the canine 5-HT(3A) receptor. The 5-HT(3A) cDNA was cloned from canine brain by polymerase chain reaction amplification. It encodes a 483 amino acid peptide that exhibits from 80% (mice) to 90% (ferrets) identity to other sequenced mammalian 5-HT(3A) receptors. The receptor agonists 5-hydroxytryptamine (5-HT) and meta-chlorophenylbiguanide (mCPBG) showed little differences between the two species, whereas 2-methyl-5-hydroxytryptamine (2-Me-5-HT) was ten times weaker at canine receptors than at human receptors. The potencies at the canine 5-HT(3) receptors were 9.9 microM (5-HT), 79 microM (2-Me-5-HT) and 0.8 microM (mCPBG). The selective, competitive receptor antagonist ondansetron was ten times more potent at human receptors compared to canine receptors (K(b)=0.9 nM), while (+)-tubocurarine was 1000-fold more potent at canine receptors (K(b)=3.0 nM) than at human receptors. Examination of the presumed ligand binding extracellular domain revealed one residue, where the canine receptor differs from all previously characterized 5-HT(3A) receptors, i.e. other species contain a conserved Trp(195), whereas the canine orthologue contains a Leu(195). To address the differences in potencies at the human and canine 5-HT(3A) receptors seen in this study, we introduced a L195W point mutation in the canine orthologue. Data showed that the 195 residue can affect receptor agonist potency and efficacy as well as antagonist potency, but did produce a pharmacological profile identical to the human orthologue. We therefore conclude that position 195 is strongly involved in the receptor-ligand interaction, but additional residues must contribute to the overall pharmacological profile.
Jensen
Thomas N
TN
Department of Electrophysiology, H. Lundbeck A/S, 9 Ottiliavej, DK-2500 Valby, Denmark.
Nielsen
Jacob
J
Frederiksen
Kristen
K
Ebert
Bjarke
B
D000595:Amino Acid Sequence; D000818:Animals; D001645:Biguanides / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004285:Dogs / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D017294:Ondansetron / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics* / Q000502:physiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014981:Xenopus
D001645:Biguanides; D018076:DNA, Complementary; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D012701:Serotonin; D017294:Ondansetron; C065734:1-(3-chlorophenyl)biguanide