Recombinant human 5-HT_3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects

Abstract.

The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT_3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC₅₀=8.6 µM, Hill coefficient =1.5). The mean peak current induced by 30 µM 5-HT was –110 pA at –100 mV. The 5-HT_3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 µM) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 µM) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45 s prior to and during the 2-s 5-HT pulse (IC₅₀ values=95 µM and 127 µM, Hill coefficient =–1.0 and –1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC₅₀=380 µM). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC₅₀ ~500 µM) when applied exclusively 45 s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT₃ receptor-mediated currents: whereas high concentrations of methohexital (≥300 µM) were necessary to induce moderate (≤ twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT₃ receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits h5-HT_3A receptor-mediated currents at anaesthetic concentrations (~90 µM).