Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells.
1. A 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. 2. Membrane 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 degrees C) revealed rapid association (kappa +1 4.76 +/- 0.3 x 10(8) M-1 min-1) and dissociation (kappa -1 = 0.21 +/- 0.003 min-1) of the radioligand. 3. Selective 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. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 microM) 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). 5. The 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.
Hope
A G
AG
Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, U.K.
Peters
J A
JA
Brown
A M
AM
Lambert
J J
JJ
Blackburn
T P
TP
D001667:Binding, Competitive; D002478:Cells, Cultured / Q000378:metabolism; D004594:Electrophysiology; D017829:Granisetron / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D018733:Nicotinic Antagonists / Q000378:metabolism; D011985:Receptors, Serotonin / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism; D014403:Tubocurarine / Q000378:metabolism
D017829:Granisetron; D018733:Nicotinic Antagonists; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D014403:Tubocurarine