Time-Dependent Block and Resurgent Tail Currents Induced by Mouse β4154–167 Peptide in Cardiac Na+ Channels

Abstract

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.