Dynamic Contacts between the Endoplasmic Reticulum and the Plasma Membrane Regulate Phosphoinositide Metabolism and Control Cellular Excitability

Abstract

Contacts between the endoplasmic reticulum and the plasma membrane (ER-PM junctions) facilitate intracellular communication and are essential to excitation-contraction coupling and store-operated calcium entry (SOCE). We find that they also regulate membrane phosphoinositides, lipids that control ion channels and electrical excitability. We examined how Sac1, an ER-integral 4-phosphatase, regulates PM phosphoinositides via ER-PM junctions. Through the use of HPLC mass spectrometry to quantitatively monitor phosphoinositides, and super resolution microscopy to determine the localization of Sac1, we have discovered that at steady state the cortical ER tethering protein extended synaptotagmin 2 (E-Syt2) positions the ER and Sac1 in discrete ER-PM junctions. Here, the enzyme's phosphatase activity participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G-protein coupled receptors that deplete PM PI(4,5)P2 also dynamically redistributes the ER, and as a consequence Sac1, away from the PM. Conversely, physiological depletion of ER luminal calcium and consequent activation of SOCE leads to increased contact between the ER and the PM and enhanced Sac1 depletion of PM PI(4)P. Further, Sac1 overexpression increases the number of STIM1 puncta at steady-state and during SOCE, resulting in higher cytoplasm calcium levels at rest and during calcium entry. Thus, the dynamic presence of Sac1 at ER-PM junctions allows it to act as a cellular sensor and controller of PM phosphoinositides and thereby influence many PM processes including electrical excitability. Supporting NIH grant R37NS08174.