We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4–64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP2) synthesis: PIP2 substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5′-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP2 induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP2 when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP2 and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A2 inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Large Ca transients cause massive endocytosis (MEND) in BHK fibroblasts by nonclassical mechanisms. We present evidence that MEND depends on mitochondrial permeability transition pore (PTP) openings, followed by coenzyme A (CoA) release, acyl CoA synthesis, and membrane protein palmitoylation. MEND is blocked by inhibiting mitochondrial Ca uptake or PTP openings, depleting fatty acids, blocking acyl CoA synthesis, metabolizing CoA, or inhibiting palmitoylation. It is triggered by depolarizing mitochondria or promoting PTP openings. After mitochondrial MEND blockade, MEND is restored by cytoplasmic acyl CoA or CoA. MEND is blocked by siRNA knockdown of the plasmalemmal acyl transferase, DHHC5. When acyl CoA is abundant, transient H2O2 oxidative stress or PKC activation initiates MEND, but the immediate presence of H2O2 prevents MEND. The PTP inhibitor, NIM811, significantly increases plasmalemma in normally growing cells. Thus, the MEND pathway may contribute to constitutive as well as pathological plasmalemma turnover in dependence on mitochondrial stress signaling.DOI: http://dx.doi.org/10.7554/eLife.01293.001

In fibroblasts, large Ca transients activate massive endocytosis (MEND) that involves membrane protein palmitoylation subsequent to mitochondrial permeability transition pore (PTP) openings. Here, we characterize this pathway in cardiac muscle. Myocytes with increased expression of the acyl transferase, DHHC5, have decreased Na/K pump activity. In DHHC5-deficient myocytes, Na/K pump activity and surface area/volume ratios are increased, the palmitoylated regulatory protein, phospholemman (PLM), and the cardiac Na/Ca exchanger (NCX1) show greater surface membrane localization, and MEND is inhibited in four protocols. Both electrical and optical methods demonstrate that PTP-dependent MEND occurs during reoxygenation of anoxic hearts. Post-anoxia MEND is ablated in DHHC5-deficient hearts, inhibited by cyclosporine A (CsA) and adenosine, promoted by staurosporine (STS), reduced in hearts lacking PLM, and correlates with impaired post-anoxia contractile function. Thus, the MEND pathway appears to be deleterious in severe oxidative stress but may constitutively contribute to cardiac sarcolemma turnover in dependence on metabolic stress.DOI: http://dx.doi.org/10.7554/eLife.01295.001

Cardiac Na + -Ca 2+ exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca 2+ is removed or cytoplasmic Na + is increased. Exogenous phosphatidylinositol-4,5-bisphosphate (PIP 2 ) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na + . To probe PIP 2 effects in intact cells, we manipulated PIP 2 metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C's (PLCs). As expected, outward NCX1 current (i.e. Ca 2+ influx) can be strongly inhibited when M1 agonists induce PIP 2 depletion. However, inward currents (i.e. Ca 2+ extrusion) without cytoplasmic Na + can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTPγS or the actin cytoskeleton disruptor latrunculin, even in the presence of non-hydrolysable ATP (AMP-PNP). Thus, G-protein signalling may increase NCX1 currents by destabilizing membrane cytoskeleton-PIP 2 interactions. Second, to increase PIP 2 we directly perfused PIP 2 into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP 2 by transient expression of a phosphatidylinositol-4-phosphate-5-kinase (hPIP5KIβ) and a PI4-kinase (PI4KIIα). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KIIα. This wortmannin-insensitive PI4KIIα was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4-kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both phosphatidylinositol-4-phosphate (PIP) and PIP 2 were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunohistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased 3-fold, suggesting that clathrin-independent endocytosis is enhanced. We conclude that direct effects of PIP 2 to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodelling and membrane trafficking.

Baby hamster kidney (BHK) fi broblasts increase their cell capacitance by 25 -100% within 5 s upon activating maximal Ca infl ux via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fl uo-5N, transiently exceeds 0.2 mM with total Ca infl ux amounting to ‫ف‬ 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coeffi cient ≈ 2). Capacitance can return to baseline in 1-3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca infl ux episode. Given recent interest in signaling lipids in membrane fusion, we used green fl uorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P 2 is rapidly cleaved upon activating Ca infl ux and recovers within 2 min. However, PI(4,5)P 2 depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca infl ux remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P 2 is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca infl ux. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P 2 -binding peptides, and PLD modifi cation by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might refl ect phosphatidylserine (PS) " scrambling " between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fi broblasts.on May 9, 2018 jgp.rupress.org Downloaded from