Regulation of voltage-gated potassium channels by PI(4,5)P2.
Regulation of voltage-gated potassium channels by PI(4,5)P2.
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DOI：
10.1085/jgp.201210806
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发表时间：
2012-08
期刊：
影响因子：
--
通讯作者：
Hille B
中科院分区：
文献类型：
--
作者：
Kruse M;Hammond GR;Hille B
磷脂酰肌醇4，5-二磷酸(PI(4，5)P2)调节多种离子通道的活动，包括内向整流钾通道、KCNQ通道、色氨酸通道和电压门控性钙通道。一些研究表明电压门控性钾(KV)通道可能受PI(4，5)P2调节。KV通道在不同细胞中的广泛表达表明，这种调节可能具有广泛的生理后果。为了研究PI(4，5)P2对KV通道的调节，我们在TSA-201细胞中与G蛋白偶联受体(M1R)、电压敏感的脂类5-磷酸酶(DR-VSP)或同时携带脂类4-磷酸酶和5-磷酸酶活性的工程融合蛋白(伪简单宁)共表达了其中的几个KV通道。这些工具分别通过应用毒碱激动剂、去极化或雷帕霉素来耗尽PI(4，5)P2。用Förster共振能量转移(FRET)技术，在全细胞记录的同时，用δ-1的PH探针监测质膜上的PI(4，5)P2。激活DR-VSP或再灌流Pluojanin可抑制KV7.1、KV7.2/7.3和Kir2.1通道电流90-95%。激活M1R同样抑制KV7.2/7.3电流。使用这些工具，我们测试了PI(4，5)P2对Kv1.1/Kvβ1.1、Kv1.3、Kv1.4和Kv1.5/Kvβ1.3、Kv2.1、Kv3.4、Kv4.2、Kv4.3(具有不同的KChIP和DPP6-S)和HERG/KCNE2.有趣的是，我们发现KV1.1/KV VSP1.1和KV3.4的失活大大减少，导致激活M1R时电流密度上调，但仅激活β或Pluojanin时活性没有变化。除了可能的HERG之外，其他被测试的通道在我们使用的任何一种检测中都没有显示出活性变化。总之，酶耗尽质膜上的PI(4，5)P2似乎不会影响大多数被测试的KV通道的活性，而它确实强烈地抑制KV7和KIR家族的成员。
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) regulates activities of numerous ion channels including inwardly rectifying potassium (Kir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (KV) channels might be regulated by PI(4,5)P2. Wide expression of KV channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of KV channels by PI(4,5)P2, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M1R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P2 with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P2 at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited KV7.1, KV7.2/7.3, and Kir2.1 channel current by 90–95%. Activation of M1R inhibited KV7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P2 regulation of activity of KV1.1/KVβ1.1, KV1.3, KV1.4, and KV1.5/KVβ1.3, KV2.1, KV3.4, KV4.2, KV4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for KV1.1/KVβ1.1 and KV3.4, resulting in up-regulation of current density upon activation of M1R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P2 at the plasma membrane by enzymes does not seem to influence activity of most tested KV channels, whereas it does strongly inhibit members of the KV7 and Kir families.
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