Abstracts

Rationale: While phenytoin(PHT)is one of the oldest anti-epileptic drugs, commonly termed a "sodium channel blocker", its action is still not well understood. To clarify this, we studied the effect of PHT on activation, fast inactivation (both of closed and open channels) and slow inactivation processes of human Nav1.2 channels. Enzymatic removal of fast inactivation allowed direct observation of PHT effects on slow inactivation. Methods: HEK 293 cells were transiently transfected with Nav1.2 and voltage clamped using the whole-cell patch clamp technique. By minimising series resistance with low resistance pipettes (200-800kΩ)combined with active series resistance compensation and low capacitance cells (typically <10 pF), it was possible to resolve the activation and deactivation phase of sodium currents. Fast inactivation of open channels was measured from the decay of INa, and transitions to and from closed states were examined with double pulse protocols. Slow inactivation was also measured with longer double pulse protocols, but could also be directly observed after removal of fast inactivation with intracellular trypsin (2mg/ml). Phenytoin (50-100μM) was applied via a rapid perfusion manifold. Results: Steady-state activation, time constants of activation and deactivation of INa were unaffected by phenytoin. Amplitudes and time constants of the macroscopic bi-exponential inactivation of I_Na were also unchanged. Steady-state inactivation (h_∞)with conventional 150 ms conditioning pulses was shifted ~-5mV by 50uM PHT, but was no longer well-fitted by a single Boltzman function, suggesting the conditioning pulse was too short to reach equilibrium. Longer (1s) conditioning pulses revealed a h_∞ relation again well fitted by the single Boltzman function, consistent with a steady-state channel redistribution being reached over this time-scale. At even the most negative conditioning potential (-140 mV), the amplitude of I_Na was reduced in PHT. Double pulse experiments revealed an apparent slowing of transitions between closed activatable and closed inactivated states over a a time scale commensurate with conventional "fast" inactivation. Surprisingly, with longer double pulse protocols, faster transition to the slow inactivated state occurred after phenytoin treatment. When fast inactivation was removed with trypsin, the reduction in current amplitude with PHT was indistinguishable from that with intact inactivation, and with long (10s) depolarising pulses a faster rate of decay of macroscopic I_Na was observed with PHT. Conclusions: These observations suggest that the primary effect of PHT is to accelerate transitions to the slow inactivated state of Nav 1.2, rather than to fast inactivated states. It is important to distinguish effects of PHT and other AED's on slow inactivation from fast inactivation mechanisms. It is suggested that the term sodium channel "modulator" be substituted for "blocker".