Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes

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Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP2 levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP2 levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP2. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca2+ influx driven by the increased driving force of Ca2+. High Ca2+ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca2+-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP2-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca2+-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP2 levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP2 levels, (2) PIP2 levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP2 levels were reduced by Ca2+ administration. Therefore, PLCζ effectively supports understanding how PIP2 regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP2 and the PLC pathway in Xenopus oocytes.