title: Pharmacological differences and similarities between the native mouse 5-HT3 receptor in N1E-115 cells and a cloned short splice variant of the mouse 5-HT3 receptor expressed in HEK 293 cells.
abstract: Human embryonic kidney (HEK) 293 cells were stably transfected with the cDNA encoding the short splice variant of the mouse 5-HT3 receptor (m5-HT3A(b); isolated by RT-PCR from NG108-15 cells) and its pharmacological properties were compared with those of the native 5-HT3 receptor of the mouse neuroblastoma cell line N1E-115. The m5-HT3A(b) receptor of N1E-115 cells differs from that isolated from NG108-15 cells by one amino acid (Val instead of Ile) at position 52 of the amino acid sequence. Both radioligand binding studies with the selective 5-HT3 receptor antagonist [3H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) and functional experiments by measurement of [14C]guanidinium influx evoked by 5-HT in the absence and presence of 10 microM substance P were carried out. Binding of [3H]GR65630 to the recombinant receptor in HEK 293 cells and the native receptor in N1E-115 cells was specific and of high affinity (Kd 4.4 and 3.0 nM, respectively) and characterized by Bmax values of 875 and 1414 fmol/mg protein, respectively. At 10 nM [3H]GR65630, specific binding was inhibited by the selective 5-HT3 receptor antagonist ondansetron (Ki 11 and 42 nM, respectively) and by 5-HT (Ki 294 and 563 nM, respectively). In the transfected HEK 293 cells, 5-HT induced an influx of [14C]guanidinium both in the absence (pEC50 5.7) and presence of substance P (pEC50 6.6,) which was counteracted by 0.3 microM ondansetron; in the N1E-115 cells, 5-HT also evoked [14C]guanidinium influx in the absence (pEC50 6.0) and presence of substance P (pEC50 6.0). Both in transfected HEK 293 cells and in N1E-115 cells, the 5-HT receptor ligand RS-056812-198 ((R)-N-(quinuclidin-3-yl)-2-(1-methyl-1 H-indol-3-yl)-2-oxo-acetamide; in the presence of substance P) induced an influx of [14C]guanidinium (pEC50 9.8 and 8.7, respectively) with a maximum of about 70 and 30% of the maximum response to 5-HT, respectively. 5-HT (in the presence of substance P)-induced [14C]guanidinium influx was inhibited by the imidazoline BDF 6143 (4-chloro-2(2-imidazolin-2-ylamino)-isoindoline; pIC50 4.9 and 5.3, respectively) and by the sigma-site ligand (+/-)-ifenprodil (pIC50 5.0 and 5.2, respectively). In conclusion, most of the drugs exhibited practically identical properties at both the recombinant m5-HT3A(b) receptor in HEK 293 cells and the native m5-HT3 receptor of N1E-115 cells. However, the recombinant receptor had a higher affinity for ondansetron, and the potency of 5-HT in inducing cation influx through the recombinant, but not through the native receptor, was increased by substance P. RS-056812-198 was a 10-fold more potent partial agonist at the recombinant than at the native receptor. These differences may be due to cell-specific post-translational modifications of the 5-HT3 receptor protein in the two cell lines, to the expression of other subunits in addition to the m5-HT3A(b) receptor in N1E-115 cells and/or to the difference in the amino acid sequence at position 52 of the short splice variants of the m5-HT3 receptors expressed in the two cell lines.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Universität Bonn, Germany. m.bruess@uni-bonn.de
authors.forename: M
authors.initials: M
authors.lastname: Brüss
authors.forename: G J
authors.initials: GJ
authors.lastname: Molderings
authors.forename: H
authors.initials: H
authors.lastname: Bönisch
authors.forename: M
authors.initials: M
authors.lastname: Göthert
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001667:Binding, Competitive; D002250:Carbon Radioisotopes; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D014644:Genetic Variation; D019791:Guanidine / Q000378:metabolism; D006801:Humans; D006822:Hybrid Cells; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D054833:Isoindoles; D008566:Membranes / Q000378:metabolism; D051379:Mice; D010880:Piperidines / Q000494:pharmacology; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012701:Serotonin / Q000494:pharmacology; D018490:Serotonin Agents / Q000494:pharmacology*; D013373:Substance P / Q000494:pharmacology; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D002250:Carbon Radioisotopes; D018076:DNA, Complementary; D007093:Imidazoles; D007211:Indoles; D054833:Isoindoles; D010880:Piperidines; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D018490:Serotonin Agents; D014316:Tritium; C059068:GR 65630; D012701:Serotonin; D013373:Substance P; C036966:4-chloro-2-(2-imidazolin-2-ylamino)isoindoline; D019791:Guanidine; C010739:ifenprodil