Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P₂ pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PH_PLCδ1 domains as real-time indicators of PM PI(4,5)P₂, and translocation of PH_OSH2×2, and PH_OSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P₂ measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P₂. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P₂. The decline of PI(4,5)P₂ following 4-phosphatase recruitment takes 1–2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P₂ appears to depend 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 recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P₂ production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity.This paper concerns the dynamics of cellular pools of phosphoinositides, a family of phospholipids located on the cytoplasmic leaflet of cellular membranes, that maintain cell structure, cell motility, membrane identity, and membrane trafficking; they also play key roles in signal transduction (1). Phosphatidylinositol (PI) can be phosphorylated at three positions to generate seven additional species. The subcellular localization of each phosphoinositide is tightly governed by the concurrent presence of lipid kinases and lipid phosphatases (2, 3), giving each membrane within the cell a unique and dynamic phosphoinositide signature (4). Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is localized to the inner leaflet of the plasma membrane (PM) and is the major substrate of phospholipase C (PLC). As a consequence, PI(4,5)P₂ levels are dynamically regulated by G_q-coupled receptors activating PLC. The activity of lipid kinases and phosphatases also can be modulated by signaling; for example, a PI 4-kinase, when associated with neuronal calcium sensor-1, is accelerated in response to elevated calcium that occurs with PI(4,5)P₂ cleavage (5). In addition, transient apposition between organelles can alter phosphoinositide levels by presenting membrane-bound phosphatases in trans. For example, the endoplasmic reticulum (ER) can make contacts with the Golgi, allowing 4-phosphatases of the ER to dephosphorylate Golgi phosphatidylinositol 4-phosphate [PI(4)P] (6–8).PI(4,5)P₂ is a dynamically regulated positive cofactor required for the activity of many plasma membrane ion channels (9). Current in KCNQ2/3 channels (the molecular correlate of neuronal M-current) can be turned off in a few seconds by depletion of PI(4,5)P₂ following activation of PLC through G_q-coupled M₁ muscarinic receptors (M₁Rs) (10–12). Given the importance of PI(4,5)P₂, we wanted to understand better how it is sourced from its precursor PI(4)P within the cell. How do subcellular compartments influence PI(4,5)P₂ abundance at the plasma membrane? PI(4,5)P₂ is derived from PI in two steps: PI 4-kinases make PI(4)P, and PI(4)P 5-kinases make PI(4,5)P₂. Thus, PI(4)P is the immediate precursor of PI(4,5)P₂. Mammalian cells express at least four distinct isoforms of PI 4-kinase that phosphorylate PI on the 4 position to generate PI(4)P and are commonly referred to as PI4K types II (α and β) and III (α and β) (1, 13). PI 4-kinase type IIIα generates PI(4)P at both the Golgi and the PM (14–16). Originally thought to be localized to an ER/Golgi compartment (17, 18), recent experiments show that it is targeted to the plasma membrane by a palmitoylated peripheral membrane protein (16). PI 4-kinase IIIβ is said to be localized to the Golgi and nucleus and contributes to the biosynthesis of Golgi PI(4)P through its association with Arf1 and neuronal calcium sensor 1 (19–21). Inhibition of PI 4-kinase IIIα and -β with micromolar concentrations of wortmannin prevents the replenishment of PM PI(4,5)P₂ following PLC activation (10, 22, 23). Type IIα and IIβ PI4Ks are membrane-bound proteins due to the palmitoylation of a conserved stretch of cysteines in their catalytic domains (24). Immunocytochemical analysis has revealed that they are mostly associated with trans-Golgi, endoplasmic reticulum, and endosomal membranes (25–27). These type IIα and IIβ enzymes are blocked by adenosine and calcium, but are resistant to wortmannin. Therefore, they are not thought to contribute to the recovery of PM PI(4,5)P₂ following G_q-receptor activation (10, 24). Our understanding of the contribution of the PI 4-kinase isoforms is undergoing refinement by accumulating information concerning the unique localization, trafficking, and activity of each PI 4-kinase.Although PI 4-kinase isoforms are present in the membranes of several organelles, the most abundant pools of PI(4)P appear to be those of the PM, Golgi, and secretory vesicles (13–15, 28). A need for Golgi PI(4)P in the maintenance of PM PI(4,5)P₂ was indirectly revealed when plasma membrane PI(4,5)P₂ recovery was slowed following recruitment of a 4-phosphatase to the trans-Golgi network (28). Depletion of PM PI(4)P has been shown to result in small changes to PM PI(4,5)P₂ (15, 22), and knockout of the PM-bound PI 4-kinase IIIα resulted in loss of PI(4)P and a relocation of PI(4,5)P₂ biosensors to intracellular membranes (16). Nevertheless, others have proposed that PM PI(4)P is redundant for the synthesis of PM PI(4,5)P₂ (29–31) and may not serve as its immediate precursor because treatment with the type III PI 4-kinase inhibitor wortmannin or recruiting a 4-phosphatase to the PM had little effect on the PM localization of the PI(4,5)P₂ reporter, the pleckstrin homology (PH) domain from PLCδ1 (PH_PLCδ1).Here, we revisit the relative contributions of PI(4)P pools to PM PI(4,5)P₂. We find that the majority of PM PI(4,5)P₂ needed for maintenance of KCNQ currents comes from two precursor pools of PI(4)P in the cell, one in the PM and the other in the Golgi. The PM pool makes the larger contribution, but the contribution from both locations is significant and ongoing.