We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca(2+) pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca(2+) with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca(2+) concentration at the site of collagen synthesis, and a high Ca(2+) concentration may be necessary for the function of molecular chaperones involved in collagen folding.

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.

Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2(+)-ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca(2+)-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.

Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2(+)-ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca(2+)-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.

Ca(2+) mobilization from intracellular stores is mediated by Ca(2+) release channels, designated ryanodine and IP(3) receptors, and directly regulates important cellular reactions including muscle contraction, endo/exocrine secretion, and neural excitability. In order to function as an intracellular store, the endo/sarcoplasmic reticulum is equipped with cooperative Ca(2+) uptake, storage and release machineries, comprising synergic collaborations among integral-membrane, cytoplasmic and luminal proteins. Our recent studies have demonstrated that junctophilins form junctional membrane complexes between the plasma membrane and the endo/sarcoplasmic reticulum in excitable cells, and that TRIC (trimeric intracellular cation) channels act as novel monovalent cation-specific channels on intracellular membrane systems. Knockout mice have provided evidence that both junctophilins and TRIC channels support efficient ryanodine receptor-mediated Ca(2+) release in muscle cells. This review focuses on cardiac Ca(2+) release by discussing pathological defects of mutant cardiomyocytes lacking ryanodine receptors, junctophilins, or TRIC channels.

Ca2+-ATPase molecules present in the microsomal fraction from non-muscle cells were examined immunologically. Rabbit whole brain, cerebellum, liver, kidney, and COS-1 cell microsomes all displayed a polypeptide of about 110 kDa which was immunoreactive with a polyclonal antiserum against the cardiac muscle sarcoplasmic reticulum Ca2+-ATPase molecule, but was not immunoreactive with a monoclonal antibody specific for the fast-twitch muscle Ca2+-ATPase. cDNAs encoding the full length of two Ca2+-ATPase molecules were isolated from a human kidney library using a mixture of nucleotide probes derived from both rabbit fast-twitch and cardiac muscle Ca2+-ATPase cDNAs. The human kidney cDNAs, HK1 and HK2, are the products of alternative splicing. HK2 codes for a protein identical to rabbit cardiac muscle Ca2+-ATPase, with the exception of 6 scattered amino acid replacements, whereas HK1 codes for a protein identical to that encoded by HK2, but with the carboxyl-terminal 4 amino acids replaced by an extended sequence of 49 amino acids. cDNAs of the HK1 type are by far the most abundant in the library. The partial structure of a 40-kilobase genomic DNA encoding all but the 5' end of the human cardiac Ca2+-ATPase is described. The exons which give rise to the alternatively spliced products were located by Southern blotting and sequencing, and the alternative splicing patterns were determined.