ATPase Isoform 2b Research Articles

Redox states in the endoplasmic reticulum directly regulate the activity of calcium channel, inositol 1,4,5-trisphosphate receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are one of the two types of tetrameric ion channels that release calcium ion (Ca2+) from the endoplasmic reticulum (ER) into the cytosol. Ca2+ released via IP3Rs is a fundamental second messenger for numerous cell functions. Disturbances in the intracellular redox environment resulting from various diseases and aging interfere with proper calcium signaling, however, the details are unclear. Here, we elucidated the regulatory mechanisms of IP3Rs by protein disulfide isomerase family proteins localized in the ER by focusing on four cysteine residues residing in the ER lumen of IP3Rs. First, we revealed that two of the cysteine residues are essential for functional tetramer formation of IP3Rs. Two other cysteine residues, on the contrary, were revealed to be involved in the regulation of IP3Rs activity; its oxidation by ERp46 and the reduction by ERdj5 caused the activation and the inactivation of IP3Rs activity, respectively. We previously reported that ERdj5 can activate the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b) using its reducing activity [Ushioda etal., Proc. Natl. Acad. Sci. U.S.A.113, E6055-E6063 (2016)]. Thus, we here established that ERdj5 exerts the reciprocal regulatory function for IP3Rs and SERCA2b by sensing the ER luminal Ca2+ concentration, which contributes to the calcium homeostasis in the ER.

Interaction of Some Asymmetrical Porphyrins with U937 Cell Membranes-In Vitro and In Silico Studies.

The aim of the present study was to assess the effects exerted in vitro by three asymmetrical porphyrins (5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin, 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatozinc(II), and 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatocopper(II)) on the transmembrane potential and the membrane anisotropy of U937 cell lines, using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), respectively, as fluorescent probes for fluorescence spectrophotometry. The results indicate the hyperpolarizing effect of porphyrins in the concentration range of 0.5, 5, and 50 μM on the membrane of human U937 monocytic cells. Moreover, the tested porphyrins were shown to increase membrane anisotropy. Altogether, the results evidence the interaction of asymmetrical porphyrins with the membrane of U937 cells, with potential consequences on cellular homeostasis. Molecular docking simulations, and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy of binding calculations, supported the hypothesis that the investigated porphyrinic compounds could potentially bind to membrane proteins, with a critical role in regulating the transmembrane potential. Thus, both the free base porphyrins and the metalloporphyrins could bind to the SERCA2b (sarco/endoplasmic reticulum ATPase isoform 2b) calcium pump, while the metal complexes may specifically interact and modulate calcium-dependent (large conductance calcium-activated potassium channel, Slo1/KCa1.1), and ATP-sensitive (KATP), potassium channels. Further studies are required to investigate these interactions and their impact on cellular homeostasis and functionality.

Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

The molecular mechanism underlying the characteristic high apparent Ca(2+) affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca(2+) transport cycle in mutant and chimeric Ca(2+)-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca(2+) dissociation from Ca(2)E1. Addition of the SERCA2b tail to SERCA1a slowed Ca(2+) dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca(2)E1. The interaction of LE with L7/8 is also important for the low rate of the Ca(2)E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca(2)E1 and Ca(2)E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca(2+)-free E2/E2P states. Collectively these properties ensure a higher Ca(2+) affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.

1,25-Dihydroxyvitamin D3 contributes to regulating mammary calcium transport and modulates neonatal skeletal growth and turnover cooperatively with calcium

To assess the interaction of 1,25(OH)(2)D(3) and dietary calcium on mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate, female lactating 1α(OH)ase(+/-) or 1α(OH)ase(-/-) mice were fed either a high-calcium diet containing 1.5% calcium in the drinking water or a "rescue diet." Dietary effects on the expression of molecules mediating mammary calcium transport were determined in the dams, and the effects of milk calcium content were assessed on skeletal growth and turnover in 2-wk-old 1,25(OH)(2)D(3)-deficient pups. Results showed that the reduction of milk calcium levels in the 1α(OH)ase(-/-) dams and the elevation of milk calcium levels in dams fed the rescue diet were associated with the down- or upregulation of calbindin D(9k) and plasma membrane Ca(2+) ATPase isoform 2b expression, respectively, in mammary epithelial cells. The action of ambient calcium in stimulating skeletal growth in the neonates appeared to supercede the direct action of 1,25(OH)(2)D(3), and the response of chondrocytes in the neonates to elevated calcium was more sensitive in hypocalcemic animals. Osteopenia was more apparent in pups nursed by dams with lower milk calcium than in 1,25(OH)(2)D(3)-deficient pups nursed by dams with higher milk calcium. Bone formation parameters were increased significantly in all pups fed by dams on the rescue diet but were still lower in 1α(OH)ase(-/-) pups than in 1α(OH)ase(+/-) pups. Consequently, there is an important contributory role of calcium in conjunction with 1,25(OH)(2)D(3) to mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate.

Characterization of SERCA2b Ca 2+–Mg 2+ ATPase mRNA decay by nuclear proteins

Gene expression is controlled at several levels including mRNA decay. Sarco/endoplasmic reticulum Ca 2+–Mg 2+-ATPase isoform 2b (SERCA2b) is central to Ca 2+ signalling and homeostasis in several tissues. SERCA2b mRNA decay involves interactions between cis-acting elements in its 3′-region and trans-acting nuclear protein factors. In the presence of the protein factors, the synthetic capped and polyadenylated RNA fragment 2b1 (3444–3753) decays faster than other SERCA2b 3′-region fragments. Here we determined the minimum cis-acting destabilizing element in the decay and its interactions with the nuclear protein factors. The in vitro decay required ATP hydrolysis and Mg 2+ but not Ca 2+. The decay was directional from 3′ to 5′, and involved a novel 35b GC rich domain designated 2b1–4 corresponding to 3521–3555. The decay of 2b1 RNA was decreased by (a) competition with 2b1–4, (b) mutation of 2b1 to delete 2b1–4, and (c) depleting the extracts of destabilizing trans-acting factors using immobilized 2b1–4. To determine the minimal destabilizing elements 2b1–4 was divided into 7b domains A–E. Deleting AB, BC, CD or DE inactivated the destabilizing cis-acting element but deleting A, B, C, D or E had no effect. In electrophoresis mobility shift assays the nuclear protein extracts retarded the mobility of labeled uncapped 2b1 RNA without a poly A + tail. A positive co-operativity in the interactions was shown in protein concentration dependence of the shift and in the competition of 2b1–4 in inhibiting the mobility of 2b1 RNA. Based on further experiments, the domain CDE (3535–3555) was sufficient to compete with 2b1 RNA for the protein binding. Consistent with this competition, excess CDE RNA retarded the in vitro decay of 2b1 RNA. Thus the RNA decay required ATP hydrolysis and Mg 2+ but not Ca 2+, the minimum binding domain was in the sequence 3535–3555, and the decay may involve a multimeric protein complex.

Plasma Membrane Ca2+ ATPase Isoform 2b Interacts Preferentially with Na+/H+ Exchanger Regulatory Factor 2 in Apical Plasma Membranes

Spatial and temporal regulation of Ca(2+) signaling require the assembly of multiprotein complexes linking molecules involved in Ca(2+) influx, sensing, buffering, and extrusion. Recent evidence indicates that plasma membrane Ca(2+) ATPases (PMCAs) participate in the control of local Ca(2+) fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood. Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dlg, ZO-1 (PDZ) domain-containing Na(+)/H(+) exchanger regulatory factor-2 (NHERF2) as an interacting partner. Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective. PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1. Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2. Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins. NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the beta(2)-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca(2+) channel, and NHERF2 is now shown to also interact with PMCA2b. This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca(2+) signaling complex, facilitating integrated cross-talk between local Ca(2+) influx and efflux.

Plasma Membrane Ca2+-ATPase Isoforms 2b and 4b Interact Promiscuously and Selectively with Members of the Membrane-associated Guanylate Kinase Family of PDZ (PSD95/Dlg/ZO-1) Domain-containing Proteins

Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.

Distribution of the intracellular Ca(2+)-ATPase isoform 2b in pig brain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform.

The presence and distribution of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoform 2b in microsomes and other subcellular fractions isolated from pig brain has been demonstrated by the combined use of a specific antibody raised against the SERCA2b isoform and ATP phosphorylation experiments. All subcellular fractions show an approximately 110 kDa phosphorylated protein, the band intensity being stronger in microsomes. Preliminary treatment of the samples with trypsin generates two phosphorylated fragments of about 57 and 33 kDa in the presence of Ca(2+). The observed fragments are typical trypsinized products of the SERCA2b isoform. The monoclonal antibody Y/1F4 raised against the sarcoplasmic reticulum Ca(2+)-ATPase (isoform 1) binds to the 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also recognizes a clear band at around 115 kDa. This band is always stronger in plasma membrane than in synaptosomes or microsomes and is unaffected by trypsin. Phosphorylation studies in the absence of Ca(2+) suggest that the 115 kDa protein is not a Ca(2+)-ATPase.

Measurement of Resting Cytosolic Ca2+ Concentrations and Ca2+ Store Size in HEK-293 Cells Transfected with Malignant Hyperthermia or Central Core Disease Mutant Ca2+ Release Channels

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.