The voltage-sensor-containing phosphatase Ci-VSP consists of a voltage-sensing domain homologous to voltage-gated K+ channels and a domain homologous to a phosphoinositide phosphatase. Indeed, Ci-VSP was shown to modulate PI(4,5)P2- dependent ion-channels in a voltage dependent fashion in-vivo; however, it degraded PI(3,4,5)P3 to PI(4,5)P2 in-vitro (Murata et al. 2005, Nature. 435:1239-1243). Here, we report experiments that address the question of the phosphoinositide substrate of Ci- VSP in living cells.

We used genetically encoded fluorescent probes for PI(4,5)P2 (PLC-[delta]1-PH-GFP) and PI(4)P (OSBP-PH-GFP) in combination with TIRF microscopy to dynamically monitor the PI(4,5)P2 and PI(4)P content of the cell membrane. The membrane voltage of the cell was controlled by whole-cell patch-clamping. Upon depolarization, cells expressing Ci-VSP exhibited a decrease in PI(4,5)P2 and a concomitant increase in PI(4)P. The recovery of PI(4,5)P2 and PI(4)P concentrations upon repolarization required intracellular ATP and was inhibited by the non-hydrolysable ATP analog AMP-PNP. PI4-kinase inhibition by wortmannin or phenylarsine oxide did not affect the recovery. These results indicate a voltage-controlled, specific, and fast conversion of PI(4,5)P2 to PI(4)P, making Ci-VSP a useful tool to study phosphoinositide metabolism in living cells.

Supported by DFG grant OL 240/2-1