Negatively charged residues adjacent to IFM motif in the DIII–DIV linker of hNaV1.4 differentially affect slow inactivation

The effects on slow inactivation (SI) of charge substitutions, neutralizations, and reversals were studied for the negatively charged residues D1309 and EE1314,15 surrounding the IFM motif in the DIII-DIV cytoplasmic linker - the putative fast inactivation particle - of human skeletal muscle voltage-gated sodium channel (hNa(V)1.4). Changing aspartate (D) at position 1309 to glutamate (E) (substitution) did not strongly affect SI, whereas charge neutralization to glutamine (Q) and charge reversal to arginine (R) right-shifted the midpoint of the steady-state SI curve. Charge neutralization (D-->Q) at position 1309 also reduced the apparent valence associated with SI. Glutamates (E) at positions 1314 and 1315 were similarly mutated. Charge reversal (EE-->RR) right-shifted the steady-state SI curve and both reversal and substitution (EE-->DD) reduced its apparent valence. Charge neutralization (EE-->QQ) and reversal decreased the maximum probability of SI. These mutations also had differential effects on the rate of SI onset and recovery. These results suggest that charged residues in the DIII-DIV linker may interact with structures that control SI.