Depolarization activates the phosphoinositide phosphatase GNP, as detected in Xenopus oocytes coexpressing sensors of PIP2
Depolarization activates the phosphoinositide phosphatase GNP, as detected in Xenopus oocytes coexpressing sensors of PIP2
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DOI：
10.1113/jphysiol.2007.134775
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发表时间：
2007-09-15
影响因子：
5.5
通讯作者：
Okamura, Yasushi
中科院分区：
文献类型：
--
作者：
Murata, Yoshimichi;Okamura, Yasushi
电压诱发信号在神经活动、肌肉收缩和胞吐作用中发挥着关键作用。含有磷酸酶的 Ciona 电压传感器 (Ci-VSP) 由跨膜电压传感器结构域 (VSD) 和磷酸肌醇磷酸酶的胞质结构域组成，与 10 号染色体上缺失的磷酸酶和张力蛋白同源物 (PTEN) 同源。先前利用钾通道作为磷酸肌醇传感器的实验表明，Ci-VSP 的磷酸酶活性是电压依赖性的。然而，目前尚不清楚酶活性是通过去极化还是超极化激活的。此外，VSD 的电荷运动和钾通道报告磷酸酶活性之间存在很大的电压依赖性差距。在这项研究中，通过非洲爪蟾卵母细胞中的共聚焦成像和电测量检查了 Ci-VSP 介导的磷酸肌醇的电压依赖性动态。使用来自磷脂酶 C delta 亚基 (PLC-delta) 的绿色荧光蛋白 (GFP) 标记的 pleckstrin 同源 (PH) 结构域对 4,5-二磷酸磷脂酰肌醇 (PtdIns(4,5)P-2) 进行成像，结果表明 PtdIns(4,5)P-2 浓度在去极化过程中降低。在存在 Ci-VSP 的情况下，对磷酸肌醇的敏感性高于 GIRK2 通道的 IRK1 通道在去极化超过 0 mV 期间降低其幅度，表明 PtdIns(4,5) P-2 水平在去极化时降低。与 Ci-VSP 共表达的 KCNQ2/3 通道表现出外向电流的电压依赖性衰减，在高达 100 mV 的电压范围内，随着去极化的增加，外向电流的衰减变得更加尖锐。这些结果表明，与 PTEN 不同，Ci-VSP 具有耗尽 PtdIns(4,5)P-2 的活性，并且去极化激活的电压传感器运动转化为磷酸酶活性的激活。
Voltage-evoked signals play critical roles in neural activities, muscle contraction and exocytosis. Ciona voltage-sensor containing phosphatase (Ci-VSP) consists of the transmembrane voltage sensor domain (VSD) and a cytoplasmic domain of phosphoinositide phosphatase, homologous to phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Previous experiments utilizing potassium channels as the sensor for phosphoinositides have demonstrated that phosphatase activities of Ci-VSP are voltage dependent. However, it still remained unclear whether enzyme activity is activated by depolarization or hyperpolarization. Further, a large gap in voltage dependency was found between the charge movement of the VSD and potassium channel-reporting phosphatase activities. In this study, voltage-dependent dynamics of phosphoinositides mediated by Ci-VSP were examined by confocal imaging and electrical measurements in Xenopus oocytes. Imaging of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P-2) using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains from phospholipase C delta subunit (PLC-delta) showed that PtdIns(4,5)P-2 concentration is reduced during depolarization. In the presence of Ci-VSP, IRK1 channels with higher sensitivity to phosphoinositide than GIRK2 channels decreased their magnitude during depolarization over 0 mV, indicating that the PtdIns(4,5) P-2 level is reduced upon depolarization. KCNQ2/3 channels coexpressed with Ci-VSP exhibited voltage-dependent decay of the outward current that became sharper with higher depolarization in a voltage range up to 100 mV These results indicate that Ci-VSP has an activity that depletes PtdIns(4,5)P-2 unlike PTEN and that depolarization-activated voltage sensor movement is translated into activation of phosphatase activity.