Membrane Phosphoinositide Turnover by Voltage Sensing Phosphatases

Abstract

The PI levels can be controlled by the state of dynamic equilibrium between lipid kinase-induced phosphorylation and lipid phosphatase-induced dephosphorylation. The voltage sensing phosphatases (VSPs) gain enzymatic activity upon membrane depolarization so they can trigger temporary imbalance of PIs by electrophysiological method. Recent studies reported that VSPs have both 3- and 5- phosphatase activities upon membrane depolarization. However, its enzymatic characteristics and mechanism are still unclear. Here we examined the functional role of Dr-VSP and Ci-VSP fused with PTEN (Ci-VSPTEN) in the PI turnover through Fluorescence Resonance Energy Transfer (FRET) using Pleckstrin Homology (PH) domains. VSP-induced turnover of diverse PIs, such as PI(3,4)P2, PI(4,5)P2 and PI(3,4,5)P3 showed different kinetics in degradation and resynthesis. When the Dr-VSP expressing cells were given with a 120 mV/3 s of depolarization, the level of PI(4,5)P2, indicated by PH(PLCδ) FRET fairs, decreased rapidly (τ = 1.18 ± 0.17 sec), while the levels of PI(3,4)P2 and PI(3,4,5)P3, indicated by PH(Akt), decreased slowly (τ = 1.36 ± 0.07 sec) and incompletely. The voltage dependent enzymatic activity of phosphatases was also investigated by addressing ramp depolarization which increases from 0 mV to 120 mV for 20 s (holding potential = −80 mV). PH(PLCδ) FRET started to decrease from the voltage of 33.0 ± 6.9 mV, whereas PH(Akt) FRET began to decrease from 71.0 ± 11 mV. In addition, recovery of PI(4,5)P2 and PI(3,4)P2 /PI(3,4,5)P3 showed distinct phenomena in time constants (τrcv = 7.17 ± 1.46 sec and 57.46 ± 4.78 sec, respectively) and in absolute quantity (92.3 ± 8.0 % and 55.3 ± 2.4 %, respectively). Thus, our results suggest that VSP can act as a dual phosphatase and deplete PIs with different time constants. The data also provide the molecular properties of endogenous PI re-synthesis in living cells.