Endoplasmic Reticulum Sac1 4-Phosphatase Reduces Plasma Membrane KCNQ2/3 Currents

Abstract

Sac1 is an integral membrane lipid 4-phosphatase enzyme localized to the endoplasmic reticulum. Sac1 dephosphorylates phosphatidylinositol 4-phosphate (PI(4)P) and has been reported to visit both the Golgi and the plasma membrane transiently. We wanted to determine whether Sac1 influences the supply of plasma membrane (PM) phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) available to ion channels. PI(4,5)P2 is dynamically regulated by Gq protein-coupled receptors that activate phospholipase C. We monitored PM PI(4)P with fluorescent OSH probes, and monitored PM PI(4,5)P2 with PHPLCδ1 FRET probes and KCNQ2/3 currents. To test the influence of Sac1 on PM PI(4,5)P2, we recruited the cortical endoplasmic reticulum to the plasma membrane using rapamycin-induced dimerization. Inducing contact between the cortical endoplasmic reticulum and the plasma membrane reduced PM PI(4)P as measured by OSH probes (20%) and PI(4,5)P2, as measured both by PHPLCδ1 (41%) and KCNQ2/3 current (30%). Confocal experiments revealed punctate depletion of PM PI(4)P and PI(4,5)P2. With moderate activation of M1 muscarinic receptors (200 nM oxotremorine-M) to stimulate phospholipase C, we found that overexpression of wildtype Sac1 augmented PI(4,5)P2 depletion, and overexpression of inactive Sac1 reduced depletion (16% and 3% decrease in PHPLCδ1 probe FRET, respectively, compared to 8% in control). We explored the reported regulation of Sac1 by an allosteric binding site that recognizes phosphatidyl serine and phosphatidylinositol. We could reduce phosphatidyl inositol with overexpression of a phosphatidylinositol-specific phospholipase C localized to the endoplasmic reticulum. This prevented Sac1-induced depletion of plasma membrane PI(4,5)P2 in a manner similar to overexpression of inactive Sac1. Thus recruitment of Sac1 (either overexpressed or endogenous) can reduce plasma membrane PI(4)P and PI(4,5)P2. This change is sensed by KCNQ2/3 channels. (NIH grant NS08174).