Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells
Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.
- 1A cloned cDNA encoding a human 5-hydroxytryptamine3 receptor type A subunit (h5-HT3R-AS) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected.
- 2Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (Bmax = 4.49 ± 0.46 pmol mg−1 protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [3H]-granisetron with high affinity (pKD = 8.87 ± 0.08). Kinetic studies (at 37°C) revealed rapid association (k+1 = 4.76 ± 0.3 × 108 m−1 min−1) and dissociation (k-1 = 0.21 ± 0.003 min−1) of the radioligand.
- 3Selective and non-selective 5-HT3 receptor ligands competed for [3H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT> metoclopramide >> phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS.
- 4In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of −60 mV, locally applied 5-HT (10 μm) evoked transient 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).
- 5The construction of a stable cell line expressing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT3 receptor pharmacology.