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

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.