A critical role for the S4-S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation.

Na+ channel fast inactivation is thought to involve the closure of an intracellular inactivation gate over the channel pore. Previous studies have implicated the intracellular loop connecting domains III and IV and a critical IFM motif within it as the inactivation gate, but amino acid residues at the intracellular mouth of the pore required for gate closure and binding have not been positively identified. The short intracellular loops connecting the S4 and S5 segments in each domain of the Na+ channel alpha-subunit are good candidates for this role in the Na+ channel inactivation process. In this study, we used scanning mutagenesis to examine the role of the IVS4-S5 region in fast inactivation. Mutations F1651A, near the middle of the loop, and L1660A and N1662A, near the COOH-terminal end, substantially disrupted Na+ channel fast inactivation. The mutant F1651A conducted Na+ currents that decayed very slowly, while L1660A and N1662A had large sustained Na+ currents at the end of 30-ms depolarizing pulses. Inactivation of macroscopic Na+ currents was nearly abolished by the N1662A mutation and the combination of the F1651A/L1660A mutations. Single channel analysis revealed frequent reopenings for all three mutants during 40-ms depolarizing pulses, indicating a substantial impairment of the stability of the inactivated state compared with wild type (WT). The F1651A and N1662A mutants also had increased mean open times relative to WT, indicating a slowed rate of entry into the inactivated state. In addition to these effects on inactivation of open Na+ channels, mutants F1651A, L1660A, and N1662A also impaired fast inactivation of closed Na+ channels, as assessed from measurements of the maximum open probability of single channels. The peptide KIFMK mimics the IFM motif of the inactivation gate and provides a test of the effect of mutations on the hydrophobic interaction of this motif with the inactivation gate receptor. KIFMK restores fast inactivation of open channels to the F1651A/L1660A mutant but does not restore fast inactivation of closed F1651A/L1660A channels, suggesting that these residues interact with the IFM motif during inactivation of closed channels. Our results implicate F1651, L1660, and N1662 of the IVS4-S5 loop in inactivation of both closed and open Na+ channels and suggest that the IFM motif of the inactivation gate interacts with F1651 and/or L1660 in the IVS4-S5 loop during inactivation of closed channels.