Massive calcium-activated endocytosis without involvement of classical endocytic proteins.

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 (PIP(2)) synthesis: PIP(2) 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 PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) 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 PIP(2) 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 ß-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 A(2) 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.

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains.

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4-64, into vesicles and loss of reversible FM 4-64 binding confirm that 40-75% of the cell surface is internalized. Ongoing MEND stops in 2-4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and ß-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces.

Massive Ca-induced membrane fusion and phospholipid changes triggered by reverse Na/Ca exchange in BHK fibroblasts.

Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25-100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to approximately 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient approximately 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 influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent 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 influx 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 influx 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 influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P(2)-binding peptides, and PLD modification 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 reflect 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 fibroblasts.

Unrestricted diffusion of exogenous and endogenous PIP(2 )in baby hamster kidney and Chinese hamster ovary cell plasmalemma.

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP(2) were incorporated into plasma membrane "lawns" ( approximately 20 x 30 microm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, approximately 0.3 microm(2)/s, than on the cell surface, approximately 0.1 microm(2)/s. For membrane lawns, the fractional recoveries (75-90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP(2)-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 microm in diameter. When the agonist carbachol (0.3 mM: ) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 microm(2)/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies.

Dual control of cardiac Na+ Ca2+ exchange by PIP(2): analysis of the surface membrane fraction by extracellular cysteine PEGylation.

We describe a new assay to determine the fraction of cardiac Na(+)-Ca(2+) exchangers (NCX1) in the surface membrane of cells (F(surf)). An extracellular NCX1 disulphide bond is rapidly reduced by tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cysteines are 'PEGylated' by alkylation with an impermeable conjugate of maleimide and a 5000 MW polyethylene glycol (MPEG), and F(surf) is quantified from Western blots as the fraction of NCX1 that migrates at a higher molecular weight. F(surf) remains less than 0.1 when NCX1 is expressed via transient transfections. Values of 0.15-0.4 are obtained for cell lines with stable NCX1 expression, 0.3 for neonatal myocytes and 0.6-0.8 for adult hearts. To validate the assay, we analysed an intervention that promotes clathrin-independent endocytosis in fibroblasts. Using BHK cells, removal of extracellular potassium (K(+)) caused yellow fluorescent protein (YFP)-tagged NCX1 to redistribute diffusely into the cytoplasm within 30 min, F(surf) decreased by 35%, and whole-cell exchange currents decreased by > 50%. In both HEK 293 and BHK cell lines, expression of human hPIP5Ibeta kinase significantly decreases F(surf). In BHK cells expressing M1 receptors, a muscarinic agonist (carbachol) causes a 40% decrease of F(surf) in normal media. This decrease is blocked by a high wortmannin concentration (3 mum), suggesting that type III phosphatidylinositol-4-kinase (PI4K) activity is required. As predicted from functional studies, carbachol increases F(surf) when cytoplasmic Ca(2) is increased by removing extracellular Na(+). Phorbol esters are without effect in BHK cells. In intact hearts, interventions that change contractility have no effect within 15 min, but we have identified two long-term changes. First, we analysed the diurnal dependence of F(surf) because messages for cardiac phosphatidylinositol-4-phosphate (PIP) 5-kinases increase during the light phase in entrained mice (i.e. during sleep). Cardiac phosphatidylinositol-(4,5)-bis-phosphate (PIP(2)) levels increase during the light phase and F(surf) decreases in parallel. Second, we analysed effects of aortic banding because NCX1 currents do not mirror the increases of NCX1 message and protein that occur in this model. F(surf) decreases significantly within 10 days, and cardiac PIP and PIP(2) levels are significantly increased. In summary, multiple experimental approaches suggest that PIP(2) synthesis favours NCX1 internalization, that NCX1 internalization is probably clathrin-independent, and that significant changes of NCX1 surface expression occur physiologically and pathologically in intact myocardium.

Dual control of cardiac Na+ Ca2+ exchange by PIP(2): electrophysiological analysis of direct and indirect mechanisms.

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-bis-phosphate (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 GTPgammaS 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 (hPIP5KIbeta) and a PI4-kinase (PI4KIIalpha). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KIIalpha. This wortmannin-insensitive PI4KIIalpha 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.

Molecular control of cardiac sodium homeostasis in health and disease.

INTRODUCTION: Cardiac myocytes utilize three high-capacity Na transport processes whose precise function can determine myocyte fate and the triggering of arrhythmias in pathological settings. We present recent results on the regulation of all three transporters that may be important for an understanding of cardiac function during ischemia/reperfusion episodes. METHODS AND RESULTS: Refined ion selective electrode (ISE) techniques and giant patch methods were used to analyze the function of cardiac Na/K pumps, Na/Ca exchange (NCX1), and Na/H exchange (NHE1) in excised cardiac patches and intact myocytes. To consider results cohesively, simulations were developed that account for electroneutrality of the cytoplasm, ion homeostasis, water homeostasis (i.e., cell volume), and cytoplasmic pH. The Na/K pump determines the average life-time of Na ions (3-10 minutes) as well as K ions (>30 minutes) in the cytoplasm. The long time course of K homeostasis can determine the time course of myocyte volume changes after ion homeostasis is perturbed. In excised patches, cardiac Na/K pumps turn on slowly (-30 seconds) with millimolar ATP dependence, when activated for the first time. In steady state, however, pumps are fully active with <0.2 mM ATP and are nearly unaffected by high ADP (2 mM) and Pi (10 mM) concentrations as may occur in ischemia. NCX1s appear to operate with slippage that contributes to background Na influx and inward current in heart. Thus, myocyte Na levels may be regulated by the inactivation reactions of the exchanger which are both Na- and proton-dependent. NHE1 also undergo strong Na-dependent inactivation, whereby a brief rise of cytoplasmic Na can cause inactivation that persists for many minutes after cytoplasmic Na is removed. This mechanism is blocked by pertussis toxin, suggesting involvement of a Na-dependent G-protein. Given that maximal NCX1- and NHE1-mediated ion fluxes are much greater than maximal Na/K pump-mediated Na extrusion in myocytes, the Na-dependent inactivation mechanisms of NCX1 and NHE1 may be important determinants of cardiac Na homeostasis. CONCLUSIONS: Na/K pumps appear to be optimized to continue operation when energy reserves are compromised. Both NCX1 and NHE1 activities are regulated by accumulation of cytoplasmic Na. These principles may importantly control cardiac cytoplasmic Na and promote myocyte survival during ischemia/reperfusion episodes by preventing Ca overload.