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 amosomal 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. (c) 2006 Elsevier Ltd. All rights reserved.

All compartments of the secretory pathway require a sufficiently high luminal Ca2+ (and Mn2+) concentration ([Ca2+](l)) to carry out their respective functions. The Ca2+ -uptake mechanism of the endoplasmic reticulum (ER) consists of the well-characterized sarco(endo) plasmic reticulum Ca2+ -transport ATPases (SERCAs), whereas the Golgi complex depends on both SERCAs and on the much less characterized secretory pathway Ca2+ -transport ATPases (SPCAs). SPCAs (encoded by ATP2C1 and ATP2C2 in humans) are Golgi-localized pumps that transport both Ca2+ and Mn2+ with high affinity. The role of intra-Golgi Ca2+/Mn2+ homeostasis in cellular function and its dependence on SPCAs is discussed, with special emphasis on the expression, catalytic properties, and the link between the defective ATP2C1 gene and Hailey-Hailey disease (HHD) (an autosomal dominant skin disorder).

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. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB(-/-) mice. The affinity for Ca(2+) of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca(2+)-affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca(2+) is physiologically important for normal cardiac function. An improved Ca(2+) transport in the sarcoplasmic reticulum may correct Ca(2+) mishandling in heart failure, but a SERCA pump with a much higher Ca(2+) affinity may be detrimental.

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. This review aims to give an overview of the vast literature concerning the cell biological implications of the SERCA2 isoform diversity and the factors regulating SERCA2. Proteins reported to interact with SERCA2 from the cytosolic domain involve the anti-apoptotic Bcl-2, the insulin receptor substrates IRS1/2, the EF-hand Ca2+-binding protein S100A1 and acylphosphatase. We will focus on the very particular position of SERCA2 as an enzyme functioning in a thin, highly fluid, leaky and cholesterol-poor membrane. Possible differential interactions of SERCA2b and SERCA2a with calreticulin, calnexin and ERp57, which could occur within the lumen of the endoplasmic reticulum will be discussed. Reported post-translational modifications possibly affecting pump activity involve N-glycosylation, glutathionylation and Ca2+/calmodulin kinase II-dependent phosphorylation. Finally, the pronounced vulnerability to oxidative damage of SERCA2 appears to be pivotal in the etiology of various pathologies.

Protein folding in the endoplasmic reticulum (ER) depends on Ca2+; uptake of Ca2+ into the ER is mediated by sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3). The 5′-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors. Here we determined whether biotin affects the expression of the SERCA3 gene and, thus, protein folding in human lymphoid cells. Jurkat cells were cultured in media containing 0.025 nmol/L biotin (denoted “deficient”) or 10 nmol/L biotin (“supplemented”). The transcriptional activity of the full-length human SERCA3 promoter was 50% lower in biotin-supplemented cells compared to biotin-deficient cells. Biotin-dependent repressors bind to elements located 731–1312 bp upstream from the transcription start site in the SERCA3 gene. The following suggest that low expression of SERCA3 in biotin-supplemented cells impaired folding of secretory proteins in the ER, triggering unfolded protein response: (i) sequestration of Ca2+ in the ER decreased by 14–24% in response to biotin supplementation; (ii) secretion of interleukin-2 into the extracellular space decreased by 75% in response to biotin supplementation; (iii) the nuclear abundance of stress-induced transcription factors increased in response to biotin supplementation; and (iv) the abundance of stress-related proteins such ubiquitin activating enzyme 1, growth arrest and DNA damage 153 gene, X-box binding protein 1 and phosphorylated eukaryotic translation initiation factor 2α increased in response to biotin supplementation. Collectively, this study suggests that supplements containing pharmacological doses of biotin may cause cell stress by impairing protein folding in the ER.

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca2+ store. Besides Ca2+-release channels and Ca2+-binding proteins, the Golgi complex contains Ca2+-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca2+-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca2+-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca2+ and Mn2+ and, therefore, plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey–Hailey disease, an autosomal dominant skin disorder, are discussed.

ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E1 approximately P --> E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca2+-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca2+ in activation of the overall reaction due to a decreased E-2 to E1Ca2 transition rate and an increased rate of Ca2+ dissociation from E1Ca2. At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca2+ level (either an enhanced efflux of lumenal Ca2+ through the pump in E-2 form or insensitivity to inhibition by lumenal Ca2+). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca2+ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E2P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.

The gene family of organellar-type Ca2+ transport ATPases consists of three members. SERCA1 is expressed exclusively in fast skeletal muscle; SERCA2 is ubiquitously expressed, whereas SERCA3 is considered to be mainly expressed in cells of the hematopoietic lineage and in some epithelial cells. In the brain, the organellar-type Ca2+ transport ATPases are almost exclusively transcribed from the SERCA2 gene. Four different SERCA2 mRNAs have been described (classes 1–4). However, unlike in nonneuronal cells, which express the class 1, 2, and 3 splice variants, the main SERCA2 mRNA in the brain is the class 4 messenger. Similar to classes 2 and 3, the class 4 codes for the ubiquitously expressed SERCA2b protein. Recently, we have reported the distribution of the SERCA isoforms in the brain (Baba-Aissa et al., 1996a,b). SERCA2b was present in most neurons of all investigated brain regions. The highest levels were found in the Purkinje neurons of the cerebellum and in the pyramidal cells of the hippocampus. Interestingly, SERCA3 and SERCA2a are coexpressed along with SERCA2b in the Purkinje neurons, but are weakly expressed in the other brain regions if present at all. Since these three protein isoforms have a different affinity for Ca2+, their possible roles in relation to Ca2+ stores in neurons are discussed.

Of all the SERCA pumps, SERCA3 was the latest to be described and the least well known. Its primary structure deviates more than usual from the other members of the SERCA family. It is not known whether its remarkably low affinity for Ca2+ (K0.5 > 1μM) observed upon expression in the COS cell system occurs also in its normal cellular context. SERCA3 is particularly expressed at high levels in different types of blood cells and related cells like platelets, lymphocytes, mast cells and arterial endothelial cells. It is also found in cerebellar Purkinje neurons. The physiological significance of this expression pattern remains unknown.

We have determined that one of the site-directed mutants reported was incorrectly identified as E113NA117EE122Q.The correct genotype is E113NA117E.The results and conclusions presented are unaffected by this error.Interestingly,