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. The maximal turnover rates of the ATPase activity for SPCA1 isoforms were 4.7-6.4-fold lower than that of SERCA1a (lowest for the shortest SPCA1a isoform). The kinetic analysis traced these differences to a decreased rate of the E(1) approximately P(Ca) to E(2)-P transition. The apparent affinity for inorganic phosphate was reduced in the SPCA1 enzymes. This could be accounted for by an enhanced rate of the E(2)-P hydrolysis, which showed constitutive activation, lacking the SERCA1a-specific dependence on pH and K(+).     

Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum

Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.     

Multiple effects of SERCA2b mutations associated with Darier's disease

Darier's disease (DD) is an autosomal dominant disorder caused by mutations in the ATP2A2 gene, encoding sarco/endoplasmic reticulum Ca2+-ATPase pump type 2b isoform (SERCA2b). Although >100 mutations in the ATP2A2 gene were identified, no apparent relation between genotype/phenotype emerged. In this work, we analyzed 12 DD-associated mutations from all of the regions of SERCA2b to study the underlying pathologic mechanism of DD and to elucidate the role of dimerization in SERCA2b activity. Most mutations markedly affected protein expression, partially because of enhanced proteasome-mediated degradation. All of the mutants showed lower activity than the wild type pump. Notably, several mutants that cause relatively severe phenotype of DD inhibited the activity of the endogenous and the co-expressed wild type SERCA2b. Importantly, these effects were not attributed to changes in passive Ca2+ leak, inositol 1,4,5-trisphosphate receptor activity, or sensitivity to inositol 1,4,5-trisphosphate. Rather, co-immunoprecipitation experiments showed that SERCA2b monomers interact to influence the activity of each other. These findings reveal multiple molecular mechanisms to account for the plethora of pathologic states observed in DD and provide the first evidence for the importance of SERCA2b dimerization in pump function in vivo.     

Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle

We relate solution behavior to the crystal structure of the Ca2+ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the gamma-phosphate to reach Asp351 to form a complex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.     

Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.     

Plasticity and adaptation of Ca2+ signaling and Ca2+-dependent exocytosis in SERCA2(+/-) mice

Darier's disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2(+/-) mouse, to define the adaptation of Ca2+ signaling and Ca2+-dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca2+]i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2(+/-) mice, due to an up-regulation of specific plasma membrane Ca2+ pump isoforms. The change in cellular Ca2+ handling caused approximately 50% reduction in [Ca2+]i oscillation frequency. Nonetheless, agonist-stimulated exocytosis was identical in cells from wild-type and SERCA2(+/-) mice. This was due to adaptation in the levels of the Ca2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2(+/-) mice. Accordingly, exocytosis was approximately 10-fold more sensitive to Ca2+ in cells from SERCA2(+/-) mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signaling and Ca2+-dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.     

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. Here, the interplay was studied between the increased Ca2+ affinity and a reduced expression of the pump and its role in the cardiac remodeling was investigated. First, SERCA2b/b mice were crossed with SERCA2b transgenes to boost cardiac SERCA2b expression. However, the enforced expression of SERCA2b was spontaneously countered by an increased inhibition by phospholamban (PLB), reducing the pump's Ca2+ affinity. Moreover, the higher SERCA2 content did not prevent hypertrophy. Second, we studied heterozygous SERCA2b/WT mice, which also express lower SERCA2 levels compared to wild-type. Hypertrophy was not observed. In heterozygotes, SERCA2b expression was specifically suppressed, explaining the reduced SERCA2 content. The SERCA2b/WT model strikingly differs from the homozygote models because SERCA2a (not SERCA2b) is the major isoform and because the inhibition of the pump by PLB is decreased instead of being increased. Thus, a tight correlation exists between the SERCA2 levels and Ca2+ affinity (controlled by PLB). This compensatory response may be important to prevent cardiac remodeling.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca(2+)-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca(2+)-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca(2+) in activation of the overall reaction due to a decreased E(2) to E(1)Ca(2) transition rate and an increased rate of Ca(2+) dissociation from E(1)Ca(2). 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 Ca(2+) level (either an enhanced efflux of lumenal Ca(2+) through the pump in E(2) form or insensitivity to inhibition by lumenal Ca(2+)). 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 Ca(2+) and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E(2)P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.     

Ca2+-dependent redox modulation of SERCA 2b by ERp57

We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.     

Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.     

Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase. Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed by transient kinetic measurements. In the Ca(2+)-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation "overshoot" was observed upon reaction of the Ca(2+)-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca(2+) binding transition (E2 --> E1 --> Ca(2)E1) as well as Ca(2+) dissociation from Ca(2+) site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca(2+) interaction at Ca(2+) site II. Because mutation of Asp(813) inhibited the Ca(2+) binding transition, but not Ca(2+) dissociation, and increased the apparent affinity for vanadate, the effect on the Ca(2+) binding transition seems in this case to be exerted by slowing the E2 --> E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca(2+) concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca(2+). Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P --> E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.     

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 microm) 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 approximately PCa 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.     

Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.     

Roles of conserved P domain residues and Mg2+ in ATP binding in the ground and Ca2+-activated states of sarcoplasmic reticulum Ca2+-ATPase

Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.     

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.     

Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c)

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.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)     

Comprehensive analysis of expression and function of 51 sarco(endo)plasmic reticulum Ca2+-ATPase mutants associated with Darier disease

We examined possible defects of sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595-35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.     

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues,heart failure and atherosclerosis

The sarco(endo)plasmic reticulum Ca²⁺ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis).Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~ 99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~ 91% of total SERCA and SERCA2b for ~ 5%. Among SERCA3, SERCA3b was the most expressed (~ 3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b.In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively.In conclusion: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~ 30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation–contraction coupling in both CMs and contractile VSMCs.