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 char acterize hS-HT3A receptors stably transfected in HEK 293 cells and to compa re the effects of the barbiturate anaesthetics methohexital and pentobarbit al on this ligand-gated cation channel. At negative membrane potentials 5-H T induced inward currents in a concentration-dependent manner (EC50 = 8.6 m u 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 abol ished the response. Methohexital and pentobarbital inhibited 5-HT-induced ( 30 mu M) currents in a concentration-dependent manner. The maximal inhibiti on with a given methohexital or pentobarbital concentration was reached whe n the respective drug was applied 45 s prior to and during the 2-s 5-HT pul se (IC50 values = 95 mu M and 127 mu M, Hill coefficient = -1.0 and -1.6, r espectively). Although the barbiturates were, thus, equipotent, their effec ts differed substantially with respect to the dependence on the time schedu le of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist puls e, but its inhibitory potency decreased considerably when it was exclusivel y applied during the 2-s 5-HT pulse (IC50 = 380 mu M) Conversely, pentobarb ital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was con siderably lower (IC50 similar to 500 mu M) when applied exclusively 45 s be fore 5-HT. Another difference between both barbiturates involves the rate o f inactivation of 5-HT3 receptor-mediated currents: whereas high concentrat ions of methohexital (greater than or equal to 300 mu M) were necessary to induce moderate (less than or equal to twofold) acceleration of this parame ter, pentobarbital produced such an effect at all concentrations and the ex tent of acceleration increased with increasing concentration (1.5- to fivef old). In conclusion, two barbiturates, chemically closely related but of di fferent 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 hydrophob ic pathway. Pentobarbital, in contrast to methohexital, inhibits h5-HT3A re ceptor-mediated currents at anaesthetic concentrations (similar to 90 mu M) .