Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells:: basic properties and barbiturate effects

The patch-clamp technique was used on excised (outside-out) patches to characterize hS-HT3A 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 mu M, Hill coefficient = 1.5). The mean peak current induced by 30 mu M 5-HT was -110 pA at -100 mV. The 5-HT3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 mu M) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 mu M) 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 mu M and 127 mu M, 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 mu M) 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 similar to 500 mu M) 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 (greater than or equal to 300 mu M) were necessary to induce moderate (less than or equal to 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-HT3A receptor-mediated currents at anaesthetic concentrations (similar to 90 mu M).