THE ACTIVATION GATE OF CARDIAC NA+ CHANNEL MODULATES VOLTAGE-DEPENDENT AND PH-DEPENDENT UNBINDING OF DISOPYRAMIDE

To assess the drug unbinding process from receptor sites in cardiac Na+ channels, we examined the recovery kinetics of disopyramide-blocked Na+ current (I-Na) in isolated guinea-pig ventricular myocytes using the whole-cell variation of the patch-clamp technique. In the presence of disopyramide (20 mu M), the time course of I-Na recovery from use-dependent block (unbinding) was described by a double exponential function. Although the time constant for the fast phase (tau(f)) of recovery was unchanged at different membrane voltages, the slow phase (tau(s)) increased with hyperpolarizing membrane potential: 4.4 +/- 0.2 s at a holding potential of -90 mV and 6.4 +/- 0.3 s at -140 mV (n = 10, P < 0.01). The slow time constant of I-Na recovery was also increased by acidification. These findings suggest that disopyramide molecules can escape from the receptor site through the hydrophobic pathway after deprotonation, because slowing of recovery from use-dependent block by acidification is caused by a decreased deprotonation rate of receptor-bound drug molecules. In addition to the hydrophobic escape, the roles of the fast inactivation gate and activation gate (m-gate) were evaluated during the recovery process. After inhibition of the fast inactivation process of I-Na by pretreatment with chloramine-T (2 mM), the fast phase of recovery from use-dependent block by disopyramide was abolished. However, the slow time constant (tau), the voltage- and pH-dependent nature of the recovery time course remained almost unchanged after inhibition of the fast inactivation process (tau: 5.3-7.5 s, n = 6), suggesting that some population of drug molecules can escape directly from the channel when the m-gate is open. We conclude that the unbinding process of disopyramide molecules from the receptor site of Na+ channels may be composed of both the hydrophobic pathway after deprotonation and the m-gate untrapping,