McPhee J C, Ragsdale D S, Scheuer T, Catterall W A
Department of Pharmacology SJ-30, University of Washington, Seattle 98195, USA.
J Biol Chem. 1995 May 19;270(20):12025-34. doi: 10.1074/jbc.270.20.12025.
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.
快速钠通道失活被认为是通过细胞内失活门与钠通道孔周围或内部区域通过疏水相互作用结合而发生的。先前的研究表明，钠通道α亚基结构域III和IV之间的细胞内环（LIII-IV）形成失活门。一个三残基疏水基序（IFM）是该门的一个基本结构特征，可能作为一个失活颗粒结合在孔内。在本研究中，我们使用丙氨酸扫描诱变来研究钠通道α亚基跨膜片段IVS6中氨基酸残基在快速失活中的功能作用。位于IVS6中心的突变体F1764A和靠近其细胞内末端处的突变体V1774A在30毫秒去极化结束时表现出大量持续的钠电流。双突变体F1764A/V1774A几乎完全消除了快速失活，表明这些氨基酸残基在失活过程中起关键作用。对这三个突变体的单通道分析显示，在40毫秒去极化脉冲后期持续重新开放，表明与野生型相比，失活状态的稳定性显著受损。此外，V1774A突变的累积首次潜伏期分布包含一个新成分，该成分源于从不稳定失活状态的开放转变。多个氨基酸残基的取代表明，失活的破坏与1774位取代的疏水性无关，这与如果该残基直接与IFM基序相互作用的预期相反。对IFM基序和IVS6中同时发生的突变进行的热力学循环分析表明，这两个区域的突变独立地破坏失活，这与它们不直接相互作用的结论一致。此外，含有IFM基序的肽（乙酰基-KIFMK-酰胺）恢复了F1764A/V1774A IVS6突变体的失活，表明这些突变体中IFM基序的结合位点保持完整。这些结果表明，IVS6中的氨基酸残基1764和1774不直接与失活门的IFM基序相互作用，而是在钠通道快速失活中发挥新的作用。
