Determinants of slow gating in ClC-O, the voltage-gated chloride channel of Torpedo marmorata
Determinants of slow gating in ClC-O, the voltage-gated chloride channel of Torpedo marmorata
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DOI：
10.1152/ajpcell.1998.274.4.c966
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发表时间：
1998-04-01
影响因子：
5.5
通讯作者：
Jentsch, TJ
中科院分区：
文献类型：
--
作者：
Fong, PY;Rehfeldt, A;Jentsch, TJ
膜超极化通常会激活鱼雷电压门控氯通道 (ClC-0) 的慢门。为了阐明这一过程的结构基础，构建了羧基末端截断突变体和嵌合体，在非洲爪蟾卵母细胞中表达，并使用两微电极电压钳进行评估。在跨膜结构域 12 和 13（D12 和 D13）之间的几个位置引入终止密码子没有显示出表达，而在 D13 之后的截断则产生了野生型电流。嵌合体（022）需要用 ClC-2 的相应区域取代 Lys-520 后的羧基末端细胞质尾部，缺乏慢门控，而更保守的构建体（嵌合体 002），其中 D13 被其 ClC-2 类似物取代，保留了其慢门控的能力。这些发现表明重要的结构位于 D12 和 D13 之间的域间延伸 (IDS) 内。与ClC-2不同，在ClC-2中“球”结构的移植可以恢复对组成型开放突变体的门控，而将ClC-0 IDS移植到嵌合体022的氨基末端并不能恢复门控。令人惊讶的是，用ClC-1或ClC-2的类似区域替换IDS显示缓慢的电压激活门控，尽管门控发生了改变。我们的研究结果使我们得出结论，ClC-0 的功能表达和慢电压门控都依赖于通道羧基末端的结构。
Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel(ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of "ball" structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.