Pochynyuk Oleh, Kucher Volodymyr, Boiko Nina, Mironova Elena, Staruschenko Alexander, Karpushev Alexey V, Tong Qiusheng, Hendron Eunan, Stockand James
Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
J Biol Chem. 2009 Sep 18;284(38):25512-21. doi: 10.1074/jbc.M109.015917. Epub 2009 Jul 20.
Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.
对功能至关重要的色氨酸残基通常位于跨膜结构域的脂水界面处。上皮钠离子通道（ENaC）/退化素（Deg）通道超家族的所有成员在其第一个跨膜结构域的基部都含有一个绝对保守的色氨酸。在此，我们测试了这个保守色氨酸对ENaC/Deg功能的重要性。在小鼠ENaC和大鼠ASIC亚基中对这个色氨酸进行靶向替换会降低通道活性。在α-mENaC中用不同氨基酸进行差异替换表明，是这个关键色氨酸的缺失而非引入具有新特性的残基改变了通道活性。令人惊讶的是，色氨酸替换揭示出电压敏感性。与分别与瞬时激活和失活时间相关的去极化电位相比，突变型ENaC在超极化时具有增加的稳态活性。激活和失活时间分别以1毫秒/毫伏的线性方式随着上升和下降斜率变化。超极化电位下宏观电流的增加是由于开放概率的电压依赖性增加。电压敏感性不受二价阳离子影响；然而，它依赖于钠离子，每对数增加[Na+]达到半最大活性所需的电压降低63毫伏。突变通道对细胞内[Na+]特别敏感，因为去除这种钠离子会消除电压依赖性。我们得出结论，ENaC/Deg通道TM1基部的保守色氨酸通过掩盖一种抑制性变构或孔道阻断机制来防止电压影响，该机制会响应细胞内钠离子而降低活性。
