Engineering an enhanced voltage-sensing phosphatase
Engineering an enhanced voltage-sensing phosphatase
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DOI：
10.1085/jgp.201912491
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发表时间：
2020-05-01
影响因子：
3.8
通讯作者：
Okamura, Yasushi
中科院分区：
文献类型：
--
作者：
Kawanabe, Akira;Mizutani, Natsuki;Okamura, Yasushi
电压敏感磷酸酶（VSP）由跨膜电压传感器结构域和对磷酸肌醇（Pk）具有酶活性的胞质区域组成。VSP酶活性受膜电位调节，其活化导致细胞PIP水平的快速和可逆的改变。这些特性使VSP可用作研究磷脂酰肌醇-4，5-二磷酸（PI（4，5）P-2）与离子通道和转运蛋白结合的作用的工具。例如，通过简单改变膜电位，斑马鱼VSP（Dr-VSP）已被有效地用于在哺乳动物细胞中操纵PI（4，5）P-2，副作用很少（如果有的话）。在本研究中，我们报告了一种增强版的Dr-VSP作为一种改进的分子工具，用于从培养的哺乳动物细胞中消耗PI（4，5）P-2。我们以两种方式修改了Dr-VSP。它的电压依赖性磷酸酶活性的增强，通过引入一个芳香族残基的位置Leu-223内的膜相互作用区域的磷酸酶结构域称为疏水脊柱。此外，通过与玻璃海鞘肠VSP的N-末端区域融合，促进了Dr-VSP的选择性质膜靶向。这种改良的Dr-VSP（CiDr-VSPmche L223 F，或我们称之为eVSP）使用Kir2.1、KCNQ 2/3和TRPC 6通道的活性作为功能读数，诱导PI（4，5）P-2水平发生更剧烈的电压诱发变化。因此，eVSP是用于评估活细胞中离子通道的PI（4，5）P-2敏感性的改进的分子工具。
Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (Pk). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P-2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P-2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestines VSP. This modified Dr-VSP (CiDr-VSPmche L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P-2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P-2 sensitivity of ion channels in living cells.