Optogenetic dephosphorylation of phosphatidylinositol 4,5 bisphosphate in Xenopus laevis oocytes.
Optogenetic dephosphorylation of phosphatidylinositol 4,5 bisphosphate in Xenopus laevis oocytes.
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DOI：
10.1016/j.xpro.2022.102003
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Plant, Leigh D.
中科院分区：
文献类型：
--
作者：
Gada, Kirin D.;Xu, Yu;Winn, Brenda T.;Masotti, Meghan;Kawano, Takeharu;Vaananen, Heikki;Plant, Leigh D.
在这里，我们提出了一个协议的磷脂酰肌醇PI（4，5）P2在非洲爪蟾卵母细胞质膜的光遗传去磷酸化。我们首先描述了共注射卵母细胞与cRNA编码（1）光激活PI（4，5）P2 5-磷酸酶融合蛋白，（2）其二聚配偶体融合到质膜，和（3）的钾通道报告PI（4，5）P2去磷酸化。然后，我们详细的蓝光照射诱导PI（4，5）P2去磷酸化，结合同时双电极电压钳电生理记录，以评估钾通道电流响应。有关本方案使用和执行的完整详细信息，请参见Xu et al.（2022年）。本方案描述了爪蟾卵母细胞中PI（4，5）P2的光遗传学去磷酸化将cRNA混合物显微注射到卵母细胞中的步骤蓝光照射诱导PI（4，5）P2的快速去磷酸化同时双电极电压钳（TEVC）记录以评估离子通道反应出版者说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。在这里，我们提出了一个协议的磷脂酰肌醇PI（4，5）P2在非洲爪蟾卵母细胞质膜的光遗传去磷酸化。我们首先描述了共注射卵母细胞与cRNA编码（1）光激活PI（4，5）P2 5-磷酸酶融合蛋白，（2）其二聚配偶体融合到质膜，和（3）的钾通道报告PI（4，5）P2去磷酸化。然后，我们详细的蓝光照射诱导PI（4，5）P2去磷酸化，结合同时双电极电压钳电生理记录，以评估钾通道电流响应。
Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P2 at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P2 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P2 dephosphorylation. We then detail blue light illumination to induce PI(4,5)P2 dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses. For complete details on the use and execution of this protocol, please refer to Xu et al. (2022). This protocol describes optogenetic dephosphorylation of PI(4,5)P2 in Xenopus oocytes Steps to microinject a cRNA mix into oocytes Blue light illumination to induce rapid dephosphorylation of PI(4,5)P2 Simultaneous two-electrode voltage clamp (TEVC) recording to assess ion channel responses Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P2 at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P2 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P2 dephosphorylation. We then detail blue light illumination to induce PI(4,5)P2 dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses.
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DOI：
10.1073/pnas.1211305109
发表时间：
2012-08-28
影响因子：
11.1
作者：
Idevall-Hagren, Olof;Dickson, Eamonn J.;De Camilli, Pietro
通讯作者：
De Camilli, Pietro
影响因子：
5.8
作者：
Xu, Yu;Yang, Yuchen;Chandrashekar, Aishwarya;Gada, Kirin D.;Masotti, Meghan;Baggetta, Austin M.;Connolly, Jenna G.;Kawano, Takeharu;Plant, Leigh D.
通讯作者：
Plant, Leigh D.
影响因子：
4.8
作者：
Gada, Kirin D.;Logothetis, Diomedes E.
通讯作者：
Logothetis, Diomedes E.
影响因子：
4.5
作者：
SCHREIBMAYER, W;LESTER, HA;DASCAL, N
通讯作者：
DASCAL, N
影响因子：
21.3
作者：
Zhang, HL;He, C;Logothetis, DE
通讯作者：
Logothetis, DE