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.缓慢延迟整流钾电流（IKs）对人类心脏动作电位复极至关重要。IKs的形成需要KCNQ1 α亚基与KCNE1 β亚基共同组装，而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。学界普遍认为，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（PIP2）。我们此前已在KCNQ1的近端C端鉴定出一组碱性残基，它们构成了PIP2/磷酸肌醇结合位点。当对这些残基进行电荷中和操作后，我们发现该通道在内质网中的滞留量显著增加，这提示通道与磷酸肌醇的相互作用可能在通道转运过程中发挥作用。为进一步探究这一可能性，我们采用化学诱导二聚化（CID）系统，在质膜（PM）或高尔基体处选择性消耗PIP2和/或磷脂酰肌醇-4-磷酸（PI(4)P），随后监测其对通道转运与功能的影响。实验结果显示，在质膜或高尔基体处消耗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消耗极为敏感，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。值得注意的是，即便尚未诱导二聚化，使用CID系统时IKs水平就已显著降低，这凸显出该系统在研究对PIP2消耗高度敏感的生物学过程时存在局限性。综上，我们证实Q1/E1通道的顺向转运无需PIP2或PI(4)P，但当通道抵达质膜后，其功能高度依赖PIP2。