Tryptophan Substitution of a Putative D4S6 Gating Hinge Alters Slow Inactivation in Cardiac Sodium Channels

Voltage-gated Na+ channels display rapid activation gating (opening) as well as fast and slow inactivation gating (closing) during depolarization. We substituted residue S1759 (serine), a putative D4S6 gating hinge of human cardiac hNav1.5 Na+ channels with A (alanine), D (aspartate), K (lysine), L (leucine), P (proline), and W (tryptophan). Significant shifts in gating parameters for activation and steady-state fast inactivation were observed in A-, D-, K-, and W-substituted mutant Na+ channels. No gating shifts occurred in the L-substituted mutant, whereas the P-substituted mutant did not yield sufficient Na+ currents. Wild-type, A-, D-, and L-substituted mutant Na+ channels showed little or no slow inactivation with a 10-s conditioning pulse ranging from −180 to 0 mV. Unexpectedly, W- and K-substituted mutant Na+ channels displayed profound maximal slow inactivation around −100 mV (∼85% and ∼70%, respectively). However, slow inactivation was progressively reversed in magnitude from −70 to 0 mV. This regression was minimized in inactivation-deficient hNav1.5-S1759W/L409C/A410W Na+ channels, indicating that the intracellular fast-inactivation gate caused such a reversal. Our data suggest that the hNav1.5-S1759 residue plays a critical role in slow inactivation. Possible mechanisms for S1759 involvement in slow inactivation and for antagonism between fast and slow inactivation are discussed.