Resurgent tail Na⁺ currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse β4 subunit, β4_154–167, acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons ( Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005 . Neuron. 45:233–244). To explore these phenotypes in vitro, we characterized β4_154–167 actions in inactivation-deficient cardiac hNav1.5 Na⁺ channels expressed in human embryonic kidney 293t cells. Intracellular β4_154–167 from 25–250 μM elicited a conspicuous time-dependent block of inactivation-deficient Na⁺ currents at 50 mV in a concentration-dependent manner. On and off rates for β4_154–167 binding were estimated at 10.1 μM⁻¹s⁻¹ and 49.1 s⁻¹, respectively. Upon repolarization, large tail currents emerged with a slight delay at −140 mV, probably as a result of the rapid unblocking of β4_154–167. Near the activation threshold (approximately −70 mV), resurgent tail currents were robust and long lasting. Likewise, β4_154–167 induces resurgent currents in wild-type hNav1.5 Na⁺ channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na⁺ channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na⁺ channels opened persistently but became resistant to β4_154–167 and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na⁺ channels resistant to β4_154–167. Together, our in vitro studies identify a putative S6-binding site for β4_154–167 within the inner cavity of hNav1.5 Na⁺ channels. Such an S6 receptor readily explains (1) why β4_154–167 gains access to its receptor as an open-channel blocker, (2), why bound β4_154–167 briefly prevents the activation gate from closing by a “foot-in-the-door” mechanism during deactivation, (3) why BTX inhibits β4_154–167 binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates β4_154–167 binding.

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na⁺ channels with higher affinities. However, an alternative view suggests that activation of Na⁺ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na⁺ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC₅₀) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC₅₀ values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na⁺ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na⁺ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/ΔKPQ syndrome. Both diseases manifest small but persistent late Na⁺ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na⁺ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na⁺ channels (L435W/L437C/A438W) expressed with β1 subunits in Hek293t cells. The flecainide-resting block at −140 mV was weak for wild-type Na⁺ channels, with an estimated 50% inhibitory concentration (IC₅₀) of 365 μM when the cell was not stimulated for 1,000 s. At 100 μM flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an ∼70% use-dependent block of peak Na⁺ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at −140 mV. Inactivated wild-type Na⁺ channels interacted with flecainide also slowly at −50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na⁺ channels potently as revealed by its rapid time-dependent block of late Na⁺ currents. The IC₅₀ for flecainide open-channel block at +30 mV was 0.61 μM, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 μM⁻¹s⁻¹ and 12.2 s⁻¹, respectively. Upon repolarization to −140 mV, flecainide block of inactivation-deficient Na⁺ channels recovered, with a time constant of 11.2 s, which was ∼20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na⁺ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na⁺ channels.