The voltage dependence of recovery from use-dependent block by QX-222 separates mechanisms for drug egress in the cardiac sodium channel

In neuronal sodium channels of squid giant axons, recovery from QX-222 block is slowed by hyperpolarization. However, in ventricular cells, hyperpolarization speeds recovery. Previously, we showed that isoform-specific residues in the external side of the cardiac sodium channel isoform (D1P-loop C373 and D4S6 T1752) influence use-dependent block (UDB) by lidocaine. To determine whether these isoformspecific residues contribute to the contrasting voltage-dependent recovery observed in ventricular myocytes, we measured recovery rates from UDB by QX-222 at holding potentials of 120, 140, 160 and 180 mV for wild-type cardiac channel (WT), the mutants C373Y (CY) and T1752V (TV), and C373Y/T1752V (CY/TV). Unlike neuronal channels, cardiac sodium channels recovered from QX block faster at hyperpolarized potentials. All mutations slowed QX-222 recovery, with the greatest rate reduction observed for the double mutant, indicating that the isoform-specific residues define external drug paths. The recovery rates varied linearly with voltage over the range tested, and we used the slopes of rate versus voltage plots to quantify voltage dependence. The TV mutation caused reduction in recovery rates without changing the slope, indicating that the mutation closed a voltage-independent egress path. The CY mutation, however, flattened the slope and reduced the voltage dependence of recovery. In addition, the reduction in rate caused by CY/TV is less than the sum of those for CY and TV, suggesting that the impacts of these two residues are interrelated. Therefore, we propose that the isoform-specific residues C373 and T1752 change recovery from UDB by distinct mechanisms but determine a common drug egress path.