McPhee J C, Ragsdale D S, Scheuer T, Catterall W A
Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195-7280, USA.
J Biol Chem. 1998 Jan 9;273(2):1121-9. doi: 10.1074/jbc.273.2.1121.
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.
钠通道快速失活被认为涉及通道孔上方细胞内失活门的关闭。先前的研究表明，连接结构域III和IV的细胞内环及其内部的关键IFM基序是失活门，但尚未明确确定门关闭和结合所需的孔细胞内口处的氨基酸残基。连接钠通道α亚基每个结构域中S4和S5段的短细胞内环在钠通道失活过程中很可能起到这一作用。在本研究中，我们使用扫描诱变来研究IVS4-S5区域在快速失活中的作用。位于环中部附近的F1651A突变以及靠近COOH末端的L1660A和N1662A突变，严重破坏了钠通道的快速失活。突变体F1651A传导的钠电流衰减非常缓慢，而L1660A和N166将30毫秒去极化脉冲结束时存在大量持续的钠电流。N1662A突变以及F1651A/L1660A突变组合几乎消除了宏观钠电流的失活。单通道分析显示，在40毫秒去极化脉冲期间，所有三个突变体都频繁重新开放，这表明与野生型（WT）相比，失活状态的稳定性受到了严重损害。相对于WT，F1651A和N1662A突变体的平均开放时间也增加，这表明进入失活状态的速率减慢。除了对开放钠通道失活的这些影响外，从单通道最大开放概率的测量评估，F1651A、L1660A和N1662A突变体也损害了关闭钠通道的快速失活。肽KIFMK模拟失活门的IFM基序，并提供了一个测试突变对该基序与失活门受体疏水相互作用影响的方法。KIFMK可恢复F1651A/L1660A突变体开放通道的快速失活，但不能恢复关闭的F1651A/L1660A通道的快速失活，这表明这些残基在关闭通道失活过程中与IFM基序相互作用。我们的结果表明，IVS4-S5环的F1651、L1660和N1662参与了关闭和开放钠通道的失活，并表明在关闭通道失活过程中，失活门的IFM基序与IVS4-S5环中的F1651和/或L1660相互作用。
