Acidic amino acids in the first intracellular loop contribute to voltage- and calcium- dependent gating of anoctamin1/TMEM16A.
Acidic amino acids in the first intracellular loop contribute to voltage- and calcium- dependent gating of anoctamin1/TMEM16A.
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第一个细胞内环中的酸性氨基酸有助于 Anoctamin1/TMEM16A 的电压和钙依赖性门控
DOI：
10.1371/journal.pone.0099376
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Cui Y
中科院分区：
文献类型：
--
作者：
Xiao Q;Cui Y
Anoctamin 1（Ano 1或TMEM 16 A）是一种由电压和Ca 2+门控的Ca 2+激活的氯离子通道。我们以前已经确定，含有高密度的酸性残基介导的电压和钙依赖性门控Ano 1的第一个细胞内环。突变的四个连续的谷氨酸（444 EEEE 447）抑制电压依赖性激活的Ano 1，而这些残基的删除降低表观钙敏感性。在本研究中，我们进一步发现，删除444 EEEEEAVKD 452产生了超过40倍的减少，在表观钙敏感性与改变激活动力学。然后，我们系统地将每个酸性残基突变为丙氨酸，并分析每个突变的电压和钙依赖性激活。野生型Ano 1的激活动力学由代表电压依赖性模式的快分量（τfast）和反映Ca 2+依赖性模式门控的慢分量（τslow）组成。E444 A、E445 A、E446 A、E447 A、E448 A和E457 A突变显示τfast降低，在缺乏Ca 2+的情况下显著抑制Ano 1的电压依赖性激活，并使G-V曲线大大向右移动，表明这些谷氨酸参与Ano 1的电压门控。此外，D452 A、E464 A、E470 A和E475 A突变不改变通道的电压依赖性激活，显著降低G-V曲线的Ca 2+依赖性，表现出τslow增加，并使表观Ca 2+敏感性降低2-3倍，表明这些酸性残基参与通道的Ca 2+依赖性门控。我们的数据表明，酸性残基在第一个细胞内环是重要的结构决定因素，耦合电压和钙依赖门控的Ano 1。
Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2–3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.
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影响因子：
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作者：
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影响因子：
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DOI：
10.1085/jgp.201110764
发表时间：
2012-04
期刊：
The Journal of general physiology
影响因子：
--
作者：
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通讯作者：
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