Frank Wuytack

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

The various splice variants of the three SERCA- and the two SPCA-pump genes in higher vertebrates encode P-type ATPases of the P(2A) group found respectively in the membranes of the endoplasmic reticulum and the secretory pathway. Of these, SERCA2b and SPCA1a represent the housekeeping isoforms. The SERCA2b form is characterized by a luminal carboxy terminus imposing a higher affinity for cytosolic Ca(2+) compared to the other SERCAs. This is mediated by intramembrane and luminal interactions of this extension with the pump. Other known affinity modulators like phospholamban and sarcolipin decrease the affinity for Ca(2+). The number of proteins reported to interact with SERCA is rapidly growing. Here, we limit the discussion to those for which the interaction site with the ATPase is specified: HAX-1, calumenin, histidine-rich Ca(2+)-binding protein, and indirectly calreticulin, calnexin, and ERp57. The role of the phylogenetically older and structurally simpler SPCAs as transporters of Ca(2+), but also of Mn(2+), is also addressed.

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) Ca2+ transporters pump cytosolic Ca2+ into the endoplasmic reticulum, maintaining a Ca2+ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca2+ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca2+ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca2+ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca2+-bound E1 conformation and alters Ca2+-transport kinetics, which provides the rationale for the higher apparent Ca2+ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na+,K+-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca2+ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.

Silencing the SPCA1 (Secretory Pathway Ca2+-ATPase Isoform 1) Impairs Ca2+ Homeostasis in the Golgi and Disturbs Neural Polarity

Neural cell differentiation involves a complex regulatory signal transduction network in which Ca2+ ions and the secretory pathway play pivotal roles. The secretory pathway Ca2+-ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca2+ or Mn2+ from the cytosol to the Golgi lumen. Its expression during brain development in different types of neurons has been documented recently, which raises the possibility that SPCA1 contributes to neuronal differentiation. In the present study, we investigated the potential impact of SPCA1 on neuronal polarization both in a cell line and in primary neuronal culture. In N2a neuroblastoma cells, SPCA1 was immunocytochemically localized in the juxtanuclear Golgi. Knockdown of SPCA1 by RNA interference markedly delayed the differentiation in these cells. The cells retarded in differentiation showed increased numbers of neurites of reduced length compared with control cells. Ca2+ imaging assays showed that the lack of SPCA1 impaired Golgi Ca2+ homeostasis and resulted in disturbed trafficking of different classes of proteins including normally Golgi-localized cameleon GT-YC3.3, bearing a Golgi-specific galactosyltransferase N terminus, and a normally plasma membrane-targeted, glycosyl phosphatidyl inositol-anchored cyan fluorescent protein construct. Also in hippocampal primary neurons, which showed a differential distribution of SPCA1 expression in Golgi stacks depending on differentiation stage, partial silencing of SPCA1 resulted in delayed differentiation, whereas total suppression drastically affected the cell survival. The disturbed overall cellular Ca2+ homeostasis and/or the altered targeting of organellar proteins under conditions of SPCA1 knockdown highlight the importance of SPCA1 function for normal neural differentiation.

Dissection of the Functional Differences between Human Secretory Pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 Isoenzymes by Steady-state and Transient Kinetic Analyses

Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 μm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1∼P(Ca) to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.

Aerobic and anaerobic metabolism in smooth muscle cells of taenia coli in relation to active ion transport.

1. The O2 consumption and lactic acid production of the guinea‐pigs taenia coli have been studied in relation to the active Na‐K transport, in order to estimate the ratio: active Na extrusion/active K uptake/ATP hydrolysis. 2. By applying different procedures of partial metabolic ingibition, it was found that a reactivation of the active Na‐K transport in K‐depleted tissues could occur in an anaerobic medium, provided glucose was present and in an aerobic medium free of added metabolizable substrate. The active Na‐K transport was rapidly blocked in an anaerobic‐substrate free medium. 3. Readmission of K to K‐depleted tissues under aerobic conditions stimulates both O2 consumption and lactic acid production. While the O2 consumption creeps up slowly and requires 50 min to reach control values, the aerobic lactic acid production increases to a maximum within 10 min and decreases again during the next 50 min to its steady‐state value. 4. A reactivation of the Na‐pump in K‐depleted cells in a N2‐glucose medium causes an immediate increase of the lactic acid production, which decreases to its control value after 60 min. The maximal increase in anaerobic lactic acid production during reactivation of the Na‐K pump is a function of [K]O. The system can be cescribed with first order kinetics having a Vmax = 0–72 mumole.g‐1 f. wt. min‐1 and a Km = 1‐1 mM. 5. By varying the glucose concentration of [K]O during reactivation of the Na‐K pump, different Na‐K pumping rates can be obtained. The ratios net Na extrusion/ATP or net K accumulation/ATP amount to −1–32 +/− 0–19 (36) and 1‐02 +/− 0–11 (36), in the experiments with different glucose concentrations. Taking into account the interference by net passive fluxes, one can estimate a ratio:active Na transport/active K transport/ATP, of 1‐7/0–8/1. This ratio is not very different from the values observed in other tissues.

Stimulation of the catalytic cycle of the Ca2+ pump of porcine plasma-membranes by negatively charged phospholipids

The (Ca(2+)+Mg2+)-ATPase of the plasma membrane is activated by negatively charged phospholipids. The mechanism of this activation was investigated by studying the effect of negatively charged phospholipids on the steady-state phosphointermediate level and on the p-nitrophenylphosphatase activity. Both parameters were differentially affected by different acidic phospholipids. The level of phosphoprotein intermediate was not affected by phosphatidylserine (20% of total phospholipid), but it was increased by 60% by phosphatidylinositol 4-phosphate. Phosphatidylserine increased the p-nitrophenylphosphatase activity, whereas phosphatidylinositol 4-phosphate had no significant effect. It is suggested that phosphatidylinositol 4-phosphate mainly affects a reaction step which leads to accelerated formation of the phosphointermediate, whereas the action of phosphatidylserine would affect two reaction steps, one upstream and one downstream of the phosphointermediate.

Na+-K+ ATPase, Na-Ca exchange, and excitation-contraction coupling in smooth muscle.

The evidence in favor of a direct role of active Na transport in the regulation of excitation-contraction coupling in vascular smooth muscle has been examined. The observations in vivo and those obtained in isolated tissues do not always lead to the same conclusions. The changes of the membrane potential obtained in vitro by slight reductions in, or increases of, [K]o do not modify the resting potential of the cells sufficiently to make them contract. Applying K-free or Na-free medium on isolated tissues is a much more vigorous procedure than the limited changes of [K]o that can occur in vascular beds in situ. The Na-Ca exchange-mechanism does not seem to play a major role in those smooth-muscle cells that have been analyzed in detail, but even here the experimental procedures have neither given precise information about the composition of the intracellular compartment nor allowed sufficient control of the parameters studied. The comparison of membrane vesicles from smooth muscle and from cardiac muscle indicates that important differences exist in Na-Ca exchange and in activities of Na+-K+ ATPase and Ca2+-Mg2+ ATPase. These findings suggest a poor development of Na-Ca exchange in smooth muscle as compared to cardiac muscle. Finally, the changes in the Na metabolism of erythrocytes from hypertensives are mentioned, and the present difficulties of linking those changes to an increased reactivity of vascular smooth-muscle cells are briefly discussed.

Expression of Ca(2+) Transport Genes in Platelets and Endothelial Cells in Hypertension.

Altered Ca2+ handling is observed in different cells in essential hypertension. We investigated the expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms in platelets and aortic endothelial cells (EC) isolated from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats by ratio reverse-transcriptase—polymerase chain reaction (RT-PCR) analysis and Western blotting. SERCA2b and SERCA3 were assessed at mRNA (EC and platelets) and at protein level (platelets). IP3R1, IP3R2, and IP3R3 mRNAs were demonstrated in both cell types, but only IP3R1 and IP3R2 proteins were detected in platelets. Compared with WKY, SHR EC and platelets showed higher SERCA3 and IP3R2 expression and lower IP3R1 expression. We then investigated the effect of lisinopril (20 mg · kg−1 · d−1; 10-week treatment of 4-week-old rats or 2-week treatment of adult rats) and captopril (100 mg · kg−1 · d−1; 2-week treatment of adult rats). Consequently, expression patterns of SERCAs and IP3Rs were significantly modified. Except for SERCAs mRNA in platelets, all differences between SHR and WKY disappeared. However, SERCA3 remained the predominant isoform. Both EC and platelets demonstrated a high equal expression of IP3R2 mRNA. IP3R1 was the predominant platelet protein isoform, as it was in untreated WKY. mRNA was also isolated from pancreatic islets of WKY and SHR, but no effect of either rat strain or of lisinopril treatment was observed on the expression of the studied genes. We hypothesize that the identical expression pattern of SERCAs and IP3Rs after treatment with ACE inhibitors represents a different nonhypertensive configuration, which, through changes in intracellular Ca2+ handling, improves endothelial and platelet dysfunction in SHR but has no effect in WKY.

The (Ca2+-Mg2+)-ATPases of the plasma membrane and of the endoplasmic reticulum in smooth muscle cells and their regulation

Smooth muscle cells contain two distinct Ca2+ -transport ATPases with a different subcellular localization. The plasmalemmal Ca2+- pump has a relative molecular weight (Mr) of 140k and its phospho-intermediate level is increased by La3+. Its resemblance to the erythrocyte Ca2+ pump is further confirmed by its calmodulin-binding capacity and its antigenic properties. A 100k Ca2+ -transport ATPase is localized in the endoplasmic reticulum. Its phospho-intermediate level is de-creased by La3+, and it is antigenically related to the cardiac sarcoplasmic reticulum Ca2+ -transport ATPase. These two different Ca2+-transport ATPases are present in both visceral and vascular smooth muscle, but tissue-and species-dependent differences in their relative amount have been observed. The endoplasmic-reticulum Ca2+ -transport ATPase is regulated via phospholamban. Phosphorylation of this regulatory protein by cAMP-dependent as well as by cGMP-dependent protein kinase stimulates the endoplasmic-reticulum Ca2+ pump. The activity of the plasmalemmal Ca2+-transport ATPase can be modulated by calmodulin, negatively charged phospholipids, and by receptor-binding agonists. cGMP-dependent protein kinase also exerts a stimulatory effect on the plasmalemmal Ca2+ pump, but this effect is not mediated via a direct phosphorylation of the Ca2+ pump.

Phospholamban ablation in hearts expressing the high affinity SERCA2b isoform normalizes global Ca2+ homeostasis but not Ca2+-dependent hypertrophic signaling

Cardiomyocytes from failing hearts exhibit reduced levels of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) and/or increased activity of the endogenous SERCA inhibitor phospholamban. The resulting reduction in the Ca(2+) affinity of SERCA impairs SR Ca(2+) cycling in this condition. We have previously investigated the physiological impact of increasing the Ca(2+) affinity of SERCA by substituting SERCA2a with the higher affinity SERCA2b pump. When phospholamban was also ablated, these double knockouts (DKO) exhibited a dramatic reduction in total SERCA levels, severe hypertrophy, and diastolic dysfunction. We presently examined the role of cardiomyocyte Ca(2+) homeostasis in both functional and structural remodeling in these hearts. Despite the low SERCA levels in DKO, we observed near-normal Ca(2+) homeostasis with rapid Ca(2+) reuptake even at high Ca(2+) loads and stimulation frequencies. Well-preserved global Ca(2+) homeostasis in DKO was paradoxically associated with marked activation of the Ca(2+)-dependent nuclear factor of activated T-cell-calcineurin pathway known to trigger hypertrophy. No activation of the MAP kinase signaling pathway was detected. These findings suggest that local changes in Ca(2+) homeostasis may play an important signaling role in DKO, perhaps due to reduced microdomain Ca(2+) buffering by SERCA2b. Furthermore, alterations in global Ca(2+) homeostasis can also not explain impaired in vivo diastolic function in DKO. Taken together, our results suggest that normalizing global cardiomyocyte Ca(2+) homeostasis does not necessarily protect against hypertrophy and heart failure development and that excessively increasing SERCA Ca(2+) affinity may be detrimental.