Jo Vanoevelen

The Secretory Pathway Ca2+/Mn2+-ATPase 2 Is a Golgi-localized Pump with High Affinity for Ca2+ Ions

Accumulation of Ca2+ into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) and by secretory pathway Ca2+-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 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca2+, demonstrated its Mn2+-transporting activity, showed its Ca2+- and Mn2+-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of 45Ca2+ (K0.5 = 0.27 μm) or 54Mn2+ transport into an A23187-releasable compartment.

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.

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. Measurements of the free Ca(2+) concentration in the lumen of the Golgi apparatus by specifically targeting the Ca(2+)-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca(2+) uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca(2+) spiking when stimulated with histamine, indicating that the SPCA1-containing Ca(2+) store was not absolutely needed to set up these oscillations. However, baseline Ca(2+) oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca(2+) signal in HeLa cells.

Intracellular Ca2+- and Mn2+-Transport ATPases

3.4.3. Artemisinin L 4. SPCAs L 4.1. Genes Encoding SPCAs L 4.2. Structure of SPCAs M 4.2.1. Mn2+ and Ca2+ Binding M 4.2.2. Countertransport M 4.3. Expression of SPCAs N 4.3.1. SPCA1 N 4.3.2. SPCA2 N 4.4. Kinetic Properties of SPCAs N 4.5. SPCA Inhibitors O 4.6. Function of SPCAs O 4.6.1. pmr1 Mutants in Yeast O 4.6.2. RNA Interference O 4.6.3. SPCA1 Mouse Models P 5. Other Ca2+and Mn2+-Transporting P-type ATPases P

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1.

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.

Trpv5/6 is vital for epithelial calcium uptake and bone formation

Calcium is an essential ion serving a multitude of physiological roles. Aside from its role as a second messenger, it is an essential component of the vertebrate bone matrix. Efficient uptake and storage of calcium are therefore indispensable for all vertebrates. Transient receptor potential family, vanilloid type (TRPV)5 and TRPV6 channels are known players in transcellular calcium uptake, but the exact contribution of this pathway is unclear. We used forward genetic screening in zebrafish (Danio rerio) to identify genes essential in bone formation and identified a lethal zebrafish mutant (matt‐und‐schlapp) with severe defects in bone formation, including lack of ossification of the vertebral column and craniofacial structures. Mutant embryos show a 68% reduction in calcium content, and systemic calcium homeostasis is disturbed when compared with siblings. The phenotype can be partially rescued by increasing ambient calcium levels to 25 mM. We identified the mutation as a loss‐of‐function mutation in the single orthologue of TRPV5 and 6, trpv5/6. Expression in HEK293 cells showed that Trpv5/6 is a calcium‐selective channel capable of inward calcium transport at physiological concentrations whereas the mutant channel is not. Taken together, this study provides both genetic and functional evidence that transcellular epithelial calcium uptake is vital to sustain life and enable bone formation.—Vanoevelen, J., Janssens, A., Huitema, L. F. A., Hammond, C. L., Metz, J. R., Flik, G., Voets, T., Schulte‐Merker, S. Trpv5/6 is vital for epithelial calcium uptake and bone formation. FASEB J. 25, 3197‐3207 (2011). www.fasebj.org

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.

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. In this study the microdomain niche of the Golgi-resident SPCA Ca(2+)/Mn(2+) pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.

Ca2+ signals in Pmr1-GFP-expressing COS-1 cells with functional endoplasmic reticulum

We studied the role of the Pmr1-containing Ca(2+) store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100 microM ATP with baseline Ca(2+) spiking, while non-transfected cells produced an initial Ca(2+) peak followed by a long-lasting plateau. The Ca(2+) signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca(2+) with a regenerative Ca(2+) signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca(2+) concentration. These results demonstrate the importance of the Pmr1-containing Ca(2+) store in generating or modifying cellular Ca(2+) signals.