Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel–phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.

慢延迟整流钾电流（slow delayed-rectifier potassium current, IKs）是人类心脏动作电位复极过程的关键调控因子。IKs的形成依赖于KCNQ1 α亚基与KCNE1 β亚基的共同组装，这两个亚基各自发生突变时，分别会引发遗传性长QT综合征1型与5型。目前学界已达成共识：KCNQ1/KCNE1（简称Q1/E1）通道发挥功能需要结合磷脂酰肌醇-4,5-二磷酸（phosphatidylinositol-4,5-bisphosphate, PIP2）。我们此前在KCNQ1的近端C端区域发现了一组碱性残基，它们共同构成了PIP2/磷酸肌醇结合位点。当对这些残基进行电荷中和操作后，我们观察到该通道在内质网中的滞留比例显著升高，这提示通道与磷酸肌醇的相互作用可能参与调控通道的运输过程。为进一步探究这一潜在机制，我们采用化学诱导二聚化（chemically induced dimerization, CID）系统，选择性地耗尽质膜（plasma membrane, PM）或高尔基体处的PIP2和/或磷脂酰肌醇-4-磷酸（phosphatidylinositol-4-phosphate, PI(4)P），随后监测其对通道运输与功能的影响。实验结果显示，在质膜或高尔基体处耗尽PIP2和/或PI(4)P，并未改变通道的细胞表面表达水平。然而，通道功能对质膜处的PIP2耗尽表现出极强的敏感性，这与已测试的其他心脏钾通道（Kir2.1与Kv11.1）的响应特征截然不同。令人意外的是，在诱导二聚化启动前，使用该CID系统就已使IKs水平大幅降低，这凸显了该系统在研究对PIP2耗尽高度敏感的生物学过程时存在的应用局限性。综上，我们的研究表明Q1/E1通道的顺向运输并不依赖PIP2或PI(4)P，但一旦抵达质膜，其功能则高度依赖PIP2。