Regulation Of Plasma Membrane Ca2 Research Articles

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+influx and modulates generation of reactive oxygen species in cultured rice cells

BackgroundMechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.ResultsWe here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.ConclusionsWe have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.

Regulation of plasma membrane Ca2 +-ATPase activity by acetylated tubulin: Influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca2+-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca2+ transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

Regulation of plasma membrane Ca2+-ATPase in human platelets by calpain

The plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca2+ in resting human platelets by extruding Ca2+ from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca2+ efflux in platelets, it is a key point for regulation of platelet Ca2+ metabolism. PMCA has been shown to be an excellent substrate for the Ca2+-activated cysteine protease calpain, a major platelet protein that is turned on during platelet activation. The objectives of the present work were to determine if PMCA is degraded during thrombin- and collagen-mediated platelet activation, and if calpain is responsible. The kinetics of PMCA degradation during platelet activation were analysed using SDS polyacrylamide gel electrophoresis and immunoblotting. The role of calpain was tested using the calpain inhibitors calpeptin and ALLN. Platelet activation mediated by both collagen and thrombin resulted in degradation of 60% of platelet PMCA within 18 minutes. Calpeptin and ALLN significantly inhibited the rate and extent of PMCA degradation. We conclude that calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca2+ regulation and, therefore, to platelet function.

Regulation of Plasma Membrane Ca2+‐ATPase in Human Platelets by Calpain

The plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca2+ in resting human platelets by extruding Ca2+ from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca2+ removal in platelets, it is a key point for regulation of platelet Ca2+ metabolism. The Ca2+-activated cysteine protease calpain, a major platelet protein, has been shown to excise the auto-inhibitory C-terminus of PMCA resulting in a 124 kDa calmodulin-independent species. To determine the kinetics PMCA degradation during platelet activation, the cleavage of PMCA into smaller forms (124 kDa and 100 kDa) was analyzed using SDS PAGE and immunoblotting. Platelet activation mediated by both collagen and thrombin resulted in proteolysis of PMCA (20 and 40% of total PMCA, respectively), leading to the formation of the active 124 kDa PMCA species and a 100 kDa inactive fragment. Calpeptin and ALLN, calpain inhibitors, blocked PMCA degradation and formation of the 124 kDa product. Proteolysis of PMCA to the 100 kDa species was also inhibited by calpain inhibitors but to a lesser extent indicating the participation of other proteases. Thus, calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca2+ regulation and, therefore, to platelet function.

Ca2+/Calmodulin-dependent Protein Kinase II Is an Essential Mediator in the Coordinated Regulation of Electrocyte Ca2+-ATPase by Calmodulin and Protein Kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.

Mechanisms of capacitative calcium entry.

Capacitative Ca(2+) entry involves the regulation of plasma membrane Ca(2+) channels by the filling state of intracellular Ca(2+) stores in the endoplasmic reticulum (ER). Several theories have been advanced regarding the mechanism by which the stores communicate with the plasma membrane. One such mechanism, supported by recent findings, is conformational coupling: inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptors in the ER may sense the fall in Ca(2+) levels through Ca(2+)-binding sites on their lumenal domains, and convey this conformational information directly by physically interacting with Ca(2+) channels in the plasma membrane. In support of this idea, in some cell types, store-operated channels in excised membrane patches appear to depend on the presence of both Ins(1,4,5)P(3) and Ins(1,4,5)P(3) receptors for activity; in addition, inhibitors of Ins(1,4,5)P(3) production that either block phospholipase C or inhibit phosphatidylinositol 4-kinase can block capacitative Ca(2+) entry. However, the electrophysiological current underlying capacitative Ca(2+) entry is not blocked by an Ins(1,4,5)P(3) receptor antagonist, and the blocking effects of a phospholipase C inhibitor are not reversed by the intracellular application of Ins(1,4,5)P(3). Furthermore, cells whose Ins(1,4,5)P(3) receptor genes have been disrupted can nevertheless maintain their capability to activate capacitative Ca(2+) entry channels in response to store depletion. A tentative conclusion is that multiple mechanisms for signaling capacitative Ca(2+) entry may exist, and involve conformational coupling in some cell types and perhaps a diffusible signal in others.

Regulation of Plasma Membrane Ca2+-ATPase by Small GTPases and Phosphoinositides in Human Platelets

We have investigated the restoration of [Ca(2+)](i) in human platelets following the discharge of the intracellular Ca(2+) stores. We found that the plasma membrane Ca(2+)-ATPase is the main mechanism involved in Ca(2+) extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-l-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca(2+)](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca(2+) extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca(2+)](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca(2+) extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca(2+)-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca(2+)](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca(2+) extrusion in human platelets.

Cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane Ca2+ influx

We have studied the mechanism of the regulation of plasma membrane Ca2+ permeability by the degree of filling of the intracellular Ca2+ stores. Using Mn2+ as a Ca2+ surrogate for plasma membrane Ca2+ channels, we found that Mn2+ uptake by rat thymocytes is inversely related to the degree of filling of the intracellular Ca2+ stores. This store-dependent plasma membrane permeability is inhibited by oxygen scavenging, CO, imidazole antimycotics and other cytochrome P-450 inhibitors. The pattern of inhibition is similar to that reported previously for the inhibition of microsomal cytochrome P-450-mediated aryl hydrocarbon hydroxylase activity of lymphocytes. Several calmodulin antagonists, both phenothiazinic (trifluoperazine, fluphenazine and chlorpromazine) and dibenzodiazepinic (clozapine), accelerate Mn2+ uptake by cells with Ca2(+)-filled stores, and this effect is prevented by imidazole antimycotics. Our results suggest that cytochrome P-450 may be the link between the stores and the plasma membrane Ca2+ pathway. We propose a model in which this cytochrome, sited at the stores, stimulates plasma membrane Ca2+ influx. This stimulatory effect is, in turn, prevented by the presence of Ca2+ inside the stores, possibly via a calmodulin-dependent mechanism.

27] Purification, reconstitution, and regulation of plasma membrane Ca2+-pumps

This chapter focuses on the purification, reconstitution, and regulation of plasma membrane Ca 2+ -pumps. The Ca 2+ -pumping ATPase of cell plasma membranes is a ubiquitous and critical element in the control of intracellular Ca 2+ . The concentration of free Ca 2+ within cells is kept very low (about 0.1 μM) by this and other Ca 2+ -pumps, exchangers, and channels. The low Ca 2+ concentration allows the free Ca 2+ concentration to change by a large factor with the movement of small total amounts of Ca 2+ . Thus, control of intracellular processes is accomplished with a small number of transporting systems per cell. This is favorable for the cell's function, but it makes these systems difficult to study, because of their low concentration. The first studies on purified ion pumps were carried out in specialized situations in which the pump was present in high concentration. In such cases, the application of the standard techniques of enzymology adapted for use in membranes was successful. The plasma membrane Ca 2+ -ATPase of erythrocytes resembles the other ion-transporting ATPases of the E1-E2 type in its formation of a high-energy phosphorylated intermediate and in the general mechanism by which it operates. However, it differs in size, modes of regulation, and physiological function.