Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells

Authors

  • Anthony G. Hope,

    1. Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.
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  • John A. Peters,

    Corresponding author
    1. Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.
      Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.
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  • Angus M. Brown,

    1. Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.
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  • Jeremy J. Lambert,

    1. Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.
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  • Thomas P. Blackburn

    1. Psychiatry Research Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Third Avenue, Harlow, Essex CM19 5AW
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Neurosciences Institute, Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital and Medical School, Dundee University, Dundee DD1 9SY, U.K.

Abstract

  • 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.

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