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

Human embryonic kidney (HEK) 293 cells were stably transfected with the cDN A 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 propertie s were compared with those of the native 5-HT3 receptor of the mouse neurob lastoma 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 l ie) at position 52 of the amino acid sequence. Both radioligand binding stu dies with the selective 5-HT3 receptor antagonist [H-3]GR65630 (3-(5-methyl -1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) and functional e xperiments by measurement of [C-14]guanidinium influx evoked by 5-HT in the absence and presence of 10 mu M substance P were carried out. Binding of [H-3]GR65630 to the recombinant receptor in HEK 293 cells and th e native receptor in N1E-115 cells was specific and of high affinity (K-d 4 .4 and 3.0 nM, respectively) and characterized by B-max values of 875 and 1 414 fmol/mg protein, respectively. At 10 nM [H-3]GR65630, specific binding was inhibited by the selective 5-HT3 receptor antagonist ondansetron (K-i I I and 42 nM, respectively) and by 5-HT (K-i 294 and 563 nM, respectively). In the transfected HEK 293 cells, 5-HT induced an influx of [C-14]guanidini um both in the absence (pEC(50) 5.7) and presence of substance P (pEC(50) 6 .6,) which was counteracted by 0.3 mu M ondansetron; in the N1E-115 cells, 5-HT also evoked [C-14]guanidinium influx in the absence (pEC(50) 6.0) and presence of substance P (pEC(50) 6.0). Both in transfected HEK 293 cells an d in N1E-115 cells, the 5-HT receptor ligand RS-056812-198 ((R)-N-(quinucli din-3-yl)-2-( l-methyl-l H-indol-3-yl)-2-oxo-acetamide; in the presence of substance P) induced an influx of [C-14]guanidinium (pEC(50) 9.8 and 8.7, r espectively) with a maximum of about 70 and 30% of the maximum response to 5-HT, respectively. 5-HT tin the presence of substance P)-induced [C-14]gua nidinium influx was inhibited by the imidazoline BDF 6143 (4-chloro-2(2-imi dazolin-2-ylamino)-isoindoline; pIC(50) 4.9 and 5.3, respectively) and by t he zeta-site ligand (+/-)-ifenprodil (pIC(50) 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 nativ e 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 catio n 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 a gonist at the recombinant than at the native receptor. These differences ma y be due to cell-specific post-translational modifications of the 5-HT3 rec eptor 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 differe nce in the amino acid sequence at position 52 of the short splice variants of the m5-HT3 receptors expressed in the two cell lines.