CHARACTERIZATION OF A HUMAN 5-HYDROXYTRYPTAMINE(3) RECEPTOR-TYPE-A (H5-HT(3)R-A(S)) SUBUNIT STABLY EXPRESSED IN HEK-293 CELLS

1 A cloned cDNA encoding a human 5-hydroxytryptamine(3) receptor type A subunit (h5-HT(3)R-A(s)) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line exp ressing a high density of the recombinant receptor was selected. 2 Mem brane homogenates prepared from transfected, but not untransfected, ce lls exhibited a homogeneous and saturable population (B-max = 4.49 +/- 0.46 pmol mg(-1) protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [H-3]-granisetron with high affinity (pK(D) = 8. 87 +/- 0.08). Kinetic studies (at 37 degrees C) revealed rapid associa tion (k(+ 1) = 4.76 +/- 0.3 x 10(8) M(-1) min(-1)) and dissociation (k (- 1) = 0.21 +/- 0.003 min(-1)) of the radioligand. 3 Selective and no n-selective 5-HT3 receptor ligands competed for [H-3]-granisetron bind ing with a rank order of potency (granisetron > ondansetron > meta-chl orophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide > > phenylb iguanide > cocaine>(+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS. 4 In electrophysiologica l recordings performed on transfected cells, voltage-clamped at a hold ing potential of -60 mV, locally applied 5-HT (10 mu M) evoked transie nt inward current responses that reversed in sign at a potential of -1 .0 +/- 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5 The construction of a stable cell line expres sing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further char acterization of this receptor subtype and the exploration of species d ifferences in 5-HT3 receptor pharmacology.