Relative Contributions of PI(4)P Pools of the Plasma Membrane and the Golgi for Maintaining the PI(4,5)P2 of the Plasma Membrane

Abstract

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is required for KCNQ2/3 channel activity. To determine precursor sources of the plasma membrane (PM) PI(4,5)P2 pool in tsA-201 cells, we monitored KCNQ tail currents and YFP-PH(PLC-delta) translocation as real-time indicators of PM PI(4,5)P2, and GFP-OSH1-PH as an indicator of Golgi PI(4)P. We selectively depleted PI(4)P pools at the PM, or the Golgi, or both using rapamycin-translocatable enzymes, pseudojanin, an engineered tandem 4- and 5-phosphatase (SAC1 and INPP5E). Selectively depleting PI(4)P at the PM with pseudojanin-SAC (PJ-SAC; only SAC1 is active) results in a secondary decrease of PI(4,5)P2 measured by KCNQ channels or by PH-PLC domains. Compared to control pseudojanin (PJ), the decrease in current with PJ-SAC is only partial (∼60% vs ∼95%) and slower (140-s vs 14-s). Likewise, translocation of PH-PLC is slower (61-s vs 20-s) than with control PJ and smaller (∼60% that of PJ). Depleting PI(4)P at the Golgi with PJ-SAC also induces a secondary decline of PM PI(4,5)P2 measured by KCNQ channels. The decrease is partial (35%), smaller than with PJ at the PM, and slower (60 s). Depleting PI(4)P simultaneously at the Golgi and PM with PJ-SAC recruited to both membranes also induces a slow secondary decrease of PI(4,5)P2 measured by KCNQ channels (100-s, 75%). Recruiting the ER towards the Golgi using rapamycin-induced dimerization, mimics the effects of depleting PI(4)P at the Golgi. In conclusion, the PM pool of PI(4,5)P2 depends on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P2 when SAC1 is active at the Golgi suggests that the Golgi contribution is on-going and does not wait until the PM is depleted. (NIH grants NS08174, GM83913 and RR025429).