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.

Vesicularization of the endoplasmic reticulum is a fast response to plasma membrane injury.

The endoplasmic reticulum of most cell types mainly consists of an extensive network of narrow sheets and tubules. It is well known that an excessive increase of the cytosolic Ca(2+) concentration induces a slow but extensive swelling of the endoplasmic reticulum into a vesicular morphology. We observed that a similar extensive transition to a vesicular morphology may also occur independently of a change of cytosolic Ca(2+) and that the change may occur at a time scale of seconds.

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.

Modeling Ca2+ dynamics of mouse cardiac cells points to a critical role of SERCA's affinity for Ca2+.

The SERCA2a isoform of the sarco/endoplasmic reticulum Ca(2+) pumps is specifically expressed in the heart, whereas SERCA2b is the ubiquitously expressed variant. It has been shown previously that replacement of SERCA2a by SERCA2b in mice (SERCA2(b/b) mice) results in only a moderate functional impairment, whereas SERCA activity is decreased by a 40% lower SERCA protein expression and by increased inhibition by phospholamban. To find out whether the documented kinetic differences in SERCA2b relative to SERCA2a (i.

Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases.

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein.

The secretory pathway Ca(2+)-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells.

Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation.

Factors controlling the activity of the SERCA2a pump in the normal and failing heart.

Heart failure is the leading cause of death in western countries and is often associated with impaired Ca(2+) handling in the cardiomyocyte. In fact, cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (ER/SR) Ca(2+) pump SERCA2a, pumping Ca(2+) from the cytosol into the lumen of the ER/SR. This review addresses three important facets that control the SERCA2 activity in the heart.

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

Sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) Ca(2+) transporters pump cytosolic Ca(2+) into the endoplasmic reticulum, maintaining a Ca(2+) 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 Ca(2+) ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca(2+) affinity because of a unique C-terminal extension (2b-tail).

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 Ca(2+) ions and the secretory pathway play pivotal roles. The secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca(2+) or Mn(2+) 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.

Contribution of intracellular Ca2+ stores to Ca2+ signaling during chemokinesis of human neutrophil granulocytes.

Extracellular agonists increase the cytosolic free Ca2+ concentration ([Ca2+]c) by Ca2+ influx and by stimulating Ca2+ release from intracellular stores, mainly the endoplasmic reticulum and to a lesser extent also later compartments of the secretory pathway, particularly the Golgi. The Golgi takes up Ca2+ via Sarco/Endoplasmic Reticulum Ca2+ATPases (SERCAs) and the Secretory-Pathway Ca2+ATPases (SPCAs). The endogenous expression of SERCAs and SPCAs neutrophils was demonstrated by Western blotting and immunocytochemistry.

Activity and localization of the secretory pathway Ca2+-ATPase isoform 1 (SPCA1) in different areas of the mouse brain during postnatal development.

Ca2+ and Mn2+ play an important role in many events in the nervous system, ranging from neural morphogenesis to neurodegeneration. As part of the homeostatic control of these ions, the Secretory Pathway Ca2+-ATPase isoform 1 (SPCA1) mediates the accumulation of Ca2+ or Mn2+ with high affinity into Golgi reservoirs. This SPCA1 represents a relatively recently characterized P-type pump that is highly expressed in nervous tissue, but information on its involvement in neural maturation is currently lacking.

Tight interplay between the Ca2+ affinity of the cardiac SERCA2 Ca2+ pump and the SERCA2 expression level.

A reduced activity of the sarcoplasmic reticulum Ca2+ pump SERCA2a is a hallmark of cardiac dysfunction in heart failure. In SERCA2b/b mice, the normal SERCA2a isoform is replaced by SERCA2b, displaying a higher Ca2+ affinity. This elicited decreased cardiac SERCA2 expression and cardiac hypertrophy.

Calcium in the Golgi apparatus.

The secretory-pathway Ca2+-ATPases (SPCAs) represent a recently recognized family of phosphorylation-type ATPases that supply the lumen of the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this structure. Mutations of the human SPCA1 gene (ATP2C1) cause Hailey-Hailey disease, an autosomal dominant skin disorder in which keratinocytes in the suprabasal layer of the epidermis detach. We will first review the physiology of the SPCAs and then discuss how mutated SPCA1 proteins can lead to an epidermal disorder.

A SERCA2 pump with an increased Ca2+ affinity can lead to severe cardiac hypertrophy, stress intolerance and reduced life span.

Abnormal Ca(2+) cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca(2+) pump, the sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca(2+) by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca(2+) affinity, results in cardiac hypertrophy and dysfunction.

Distribution of secretory pathway Ca2+ ATPase (SPCA1) in neuronal and glial cell cultures.

1. Secretory pathway Ca(2+) ATPase type 1 (SPCA1) is a newly recognized Ca(2+)/Mn(2+)-transporting pump localized in membranes of the Golgi apparatus. 2.

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.

Functional comparison between secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses.

Steady-state and transient kinetic studies were performed to functionally analyze the overall and partial reactions of the Ca(2+) transport cycle of the human secretory pathway Ca(2+)/Mn(2+)-ATPase 1 (SPCA1) isoforms: SPCA1a, SPCA1b, SPCA1c, and SPCA1d (encoded by ATP2C1, the gene defective in Hailey-Hailey disease) upon heterologous expression in mammalian cells. The expression levels of SPCA1 isoforms were 200-350-fold higher than in control cells except for SPCA1c, whose low expression level appears to be the effect of rapid degradation because of protein misfolding. Relative to SERCA1a, the active SPCA1a, SPCA1b, and SPCA1d enzymes displayed extremely high apparent affinities for cytosolic Ca(2+) in activation of the overall ATPase and phosphorylation activities.

Modulating sarco(endo)plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: cell biological implications.

Of the three mammalian members belonging to the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) family, SERCA2 is evolutionary the oldest and shows the most wide tissue-expression pattern. Two major SERCA2 splice variants are well-characterized: the muscle-specific isoform SERCA2a and the housekeeping isoform SERCA2b. Recently, several interacting proteins and post-translational modifications of SERCA2 were identified which may modulate the activity of the Ca2+ pump.

The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions.

Accumulation of Ca(2+) into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and by secretory pathway Ca(2+)-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.

Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species.

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN.

The Ca2+/Mn2+ pumps in the Golgi apparatus.

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis.

SPCA1 pumps and Hailey-Hailey disease.

Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis.

Ca2+ transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart.

Adult SERCA2(b/b) mice expressing the non-muscle Ca2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838].

The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference.

The secretory-pathway Ca(2+)-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca(2+) pump found mostly in the Golgi compartment. We have explored the contribution of this Ca(2+) pump to cytosolic Ca(2+) signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies.