CRITICAL ROLE FOR TRANSMEMBRANE SEGMENT IVS6 OF THE SODIUM-CHANNEL ALPHA-SUBUNIT IN FAST INACTIVATION
CRITICAL ROLE FOR TRANSMEMBRANE SEGMENT IVS6 OF THE SODIUM-CHANNEL ALPHA-SUBUNIT IN FAST INACTIVATION
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DOI：
10.1074/jbc.270.20.12025
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发表时间：
1995-05-19
影响因子：
4.8
通讯作者：
CATTERALL, WA
中科院分区：
文献类型：
--
作者：
MCPHEE, JC;RAGSDALE, DS;CATTERALL, WA
Na+通道的快速失活被认为是通过疏水相互作用将细胞内失活门与Na+通道孔周围或内部的区域结合而发生的。先前的研究表明，Na+通道Lu亚基的结构域III和IV之间的细胞内环（L（III-IV））形成失活门，三残基疏水基序（IFM）是门的基本结构特征，并可作为结合在孔内的失活颗粒。在这项研究中，我们使用丙氨酸扫描诱变来检测Na+通道cu亚单位跨膜段IVS 6中氨基酸残基在快速失活中的功能作用。IVS 6中心的突变体F1764 A和靠近其胞内末端的突变体V1774 A在30-ms去极化结束时表现出实质性的持续Na+电流。F1764 A/V1774 A双突变几乎完全消除了快速失活，证明了这些氨基酸残基在失活过程中的关键作用。对这三种突变体的单通道分析显示，在40 ms去极化脉冲后期持续重新开放，表明失活状态的稳定性与野生型相比显著受损。V1774 A突变的累积第一潜伏期分布包含由从去稳定的失活状态的开放转变产生的新成分。多个氨基酸残基的取代显示失活的破坏与位置1774处取代的疏水性无关，与如果该残基直接与IFM基序相互作用的预期相反，IFM基序和IVS 6中同时突变的热力学循环分析表明，这两个区域中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F1764 A/V1774 A IVS 6突变体的失活，表明IFM基序的结合位点在这些突变体中保持完整，这些结果表明，IVS 6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Na+通道的快速失活中发挥新的作用。
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 Lu subunit (L(III-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 cu sub unit 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.