PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.
PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.
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DOI：
10.1085/jgp.201311033
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发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Mori MX
中科院分区：
文献类型：
--
作者：
Itsuki K;Imai Y;Hase H;Okamura Y;Inoue R;Mori MX
磷脂酰肌醇4，5-二磷酸在调节受体操纵的TRPC通道激活和失活中具有直接作用。瞬时受体电位经典（或经典）（TRPC）3、TRPC 6和TRPC 7是由磷脂酶C（PLC）水解磷脂酰肌醇4，5-二磷酸（PI（4，5）P2）产生的二酰基甘油（DAG）激活的TRPC通道亚家族。PI（4，5）P2被异源表达的磷酸酶消耗抑制TRPC 3、TRPC 6和TRPC 7活性，与DAG无关;然而，PI（4，5）P2减少对通道活性的生理作用仍不清楚。我们使用Förster共振能量转移（FRET）来测量PI（4，5）P2或DAG动力学，同时在激动剂刺激与Gq偶联的受体后测量TRPC 6或TRPC 7电流，从而激活PLC。在不同水平的受体激活的测量揭示了PI（4，5）P2减少的动力学和受体操作的TRPC 6和TRPC 7电流激活和失活之间的相关性。与此相反，DAG生产与通道激活，但不失活;此外，通道失活的时间过程中蛋白激酶C不敏感的突变体是不变的。这些结果表明，受体操纵的TRPC电流的失活主要是由PI（4，5）P2的解离介导的。我们使用结合PI（4，5）P2的PLCδ Pleckstrin同源结构域（PHd）的FRET确定PI（4，5）P2对TRPC通道的功能解离常数，并使用该常数将我们的实验数据拟合到其中通道门控由PI（4，5）P2和DAG控制的模型。该模型预测PHd的FRET动力学与在人胚胎肾细胞或平滑肌细胞中测量的FRET相似，而缺乏PI（4，5）P2调节的模型未能再现实验数据，证实了PI（4，5）P2耗尽对TRPC电流的抑制作用。我们的模型还解释了各种PLC依赖的通道活动的特性，包括最大开放概率的限制，缩短的峰值时间，和总电流的钟形响应。总之，我们的研究表明PI（4，5）P2在调节PLC偶联受体刺激触发的TRPC 6和TRPC 7活性中的基本作用。
Phosphatidylinositol 4,5-bisphosphate has a direct role in regulating receptor-operated TRPC channel activation and inactivation. Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C–insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.
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DOI：
10.1085/jgp.200910388
发表时间：
2011-03
期刊：
The Journal of general physiology
影响因子：
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