Abstract
Voltage-sensitive phosphatases (VSPs) have a voltage sensor linked to a phosphoinositide (PI) 5-phosphatase, which hydrolyzes plasma membrane PI-(4,5)-bisphosphate (PIP2) to PI(4)P (Iwasaki, PNAS 105, 7970). We used PIP2 hydrolysis by VSP from Ciona intestinalis (ci-VSP) and zebrafish (dr-VSP) to better understand PIP2 binding and resynthesis. PIP2 was monitored using the PIP2-sensitive M-current (KCNQ2/3) and FRET between a pair of PIP2-binding probes (PH-PLCδ1-CFP & PH-PLCδ1-YFP). Depolarizations to +100 mV lasting >50 ms reduced M-current and PH-probe FRET. PIP2 depletion was saturated by depolarizations lasting 500-1000 ms. Evidently PH-probe FRET and M-current respond quickly to changes in plasma membrane PIP2.After repolarization, PH-probe FRET and M-current relaxed to baseline values with time constants of ~10 s in a wortmannin-insensitive manner. This reflects endogenous PIP 5-kinase converting PI(4)P back to PI(4,5)P2. Overexpression of PIP 5-kinase increased the length of depolarization required to deplete PIP2, and speeded PIP2 recovery after repolarization.Recovery of PIP2 after VSP activation is ~10x faster than after PLC activation. However, it only requires PIP 5-kinase, whereas recovery after PLC activation requires PI 4-kinase and PIP 5-kinase in series. Thus PI 4-kinase must be the slower enzyme. To estimate the rate of PI 4-kinase, we compared translocation of a fluorescent probe that reports plasma membrane PI(4)P (PH-OSH2, T.Balla) to translocation of the PIP2-binding probe in confocal time-lapse imaging. Upon PLC activation through M1 receptors, the plasma membrane PI(4)P signal decreased 20 s later than the PIP2 signal. Both probes recovered with similar time courses. FRET photometry between either PIP2-probes or PI(4)P-probes showed comparable results. The simultaneous recovery of both probes is consistent with the hypothesis that recovery of PIP2 is governed by rate-limiting synthesis of PI(4)P by the PI 4-kinase, followed by rapid conversion of PI(4)P into PI(4,5)P2 by the PIP 5-kinase.NIH-NS008174&HFSP
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