Fast Na+ channel inactivation is thought to occur by the binding of an intracellular inactivation gate to regions around or within the Na+ channel pore through hydrophobic interactions. Previous studies indicate that the intracellular loop between domains III and IV of the Na+ channel alpha subunit (LIII-IV) forms the inactivation gate. A three-residue hydrophobic motif (IFM) is an essential structural feature of the gate and may serve as an inactivation particle that binds within the pore. In this study, we used alanine-scanning mutagenesis to examine the functional role of amino acid residues in transmembrane segment IVS6 of the Na+ channel alpha subunit in fast inactivation. Mutant F1764A, in the center of IVS6, and mutant V1774A, near its intracellular end, exhibited substantial sustained Na+ currents at the end of 30-ms depolarizations. The double mutation F1764A/V1774A almost completely abolished fast inactivation, demonstrating a critical role for these amino acid residues in the process of inactivation. Single channel analysis of these three mutants revealed continued reopenings late in 40-ms depolarizing pulses, indicating that the stability of the inactivated state was substantially impaired compared with wild type. In addition, the cumulative first latency distribution for the V1774A mutation contained a new component arising from opening transitions from the destabilized inactivated state. Substitution of multiple amino acid residues showed that the disruption of inactivation was not correlated with the hydrophobicity of the substitution at position 1774, in contrast to the expectation if this residue interacts directly with the IFM motif. Thermodynamic cycle analysis of simultaneous mutations in the IFM motif and in IVS6 suggested that mutations in these two regions independently disrupt inactivation, consistent with the conclusion that they do not interact directly. Furthermore, a peptide containing the IFM motif (acetyl-KIFMK-amide) restored inactivation to the F1764A/V1774A IVS6 mutant, indicating that the binding site for the IFM motif remains intact in these mutants. These results suggest that the amino acid residues 1764 and 1774 in IVS6 do not directly interact with the IFM motif of the inactivation gate but instead play a novel role in fast inactivation of the Na+ channel.
McPhee et al. (Mon,) studied this question.