Insights on the Mechanisms of the Fast Blockade of TTX-R Na+ Channels by Eugenol

Abstract

OBJECTIVES. It was previously shown that eugenol, a phenylpropene, blocks fast and reversibly voltage-gated Na+ channels (NaV), but little concern was given to the blocker binding to different conformational states of channel molecule. Here we reported a detailed analysis of state-dependent effects of eugenol on tetrodotoxin-resistant (TTX-R) NaV isoforms, comparing them to those of lidocaine, a reference blocker.METHODS. TTX-R Na+ currents were recorded in dorsal root ganglia neurons from newborn Wistar rats with whole-cell configuration of patch clamp technique. Tetrodotoxin-sensitive Na+ currents were blocked by TTX 100nM in the extracellular solution.RESULTS and CONCLUSIONS. A dose-dependent fast blockade due to eugenol was observed in 0.2Hz time series depolarizations from a holding potential of −110 mV to a 0 mV pulse. This tonic blockage is due to eugenol binding to the closed state. The IC50 was 2.28±0.10mM for eugenol compared to 0.44±0.08mM for lidocaine. The tonic NaV blockade was more effective when the membrane was held at more depolarized, still sublimiar, holding potentials. This observation indicates a higher affinity of eugenol for closed substates dwelled at less hyperpolarized potentials. No consistent evidences for additional binding to open state were observed. A displacement of steady-state inactivation curve to more negative potentials, associated with a slower recovery from fast inactivation under eugenol indicates that this molecule also binds to fast inactivated state. For currents undergoing slow inactivation, a consistent reduction by eugenol indicates that the phenylpropene additionally binds to the slow inactivated state. A frequency-dependent blocking effect of eugenol on NaV was observed, but the effect is smaller than that induced by lidocaine. In conclusion, eugenol binds to several isoforms of TTX-R NaV and to the different states of the proteins, leading to a channel blockage.