Cooperative effect of S4-S5 loops in domains D3 and D4 on fast inactivation of the Na+ channel

Cytoplasmic S4-S5 loops have been shown to be involved in fast inactivation of voltage-gated ion channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (NI151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na+ channel alpha-subunit (hNav][A) using expression in tsA201 cells and the whole cell patch-clamp technique. All cysteine mutations were accessible to intraceflularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/FI473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu-phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady-state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge-dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na+ channel involves both S4-S5 loops in D3 and D4 in a cooperative manner. D3/S4-S5 also plays an important role in activation and deactivation.