Characterization of the Gene Encoding Human Sarcolipin (SLN), a Proteolipid Associated with SERCA1: Absence of Structural Mutations in Five Patients with Brody Disease

Sarcolipin (SLN) is a low-molecular-weight protein that copurifies with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca²⁺ATPase (SERCA1). Genomic DNA and cDNA encoding human sarcolipin (SLN) were isolated and characterized and theSLNgene was mapped to chromosome 11q22–q23. Human, rabbit, and mouse cDNAs encode a protein of 31 amino acids. Homology of SLN with phospholamban (PLN) suggests that the first 7 hydrophilic amino acids are cytoplasmic, the next 19 hydrophobic amino acids form a single transmembrane helix, and the last 5 hydrophilic amino acids are lumenal. The cytoplasmic and transmembrane sequences are not well conserved among the three species, but the lumenal sequence is highly conserved. Like SERCA1, SLN is highly expressed in rabbit fast-twitch skeletal muscle, but it is expressed to a lower extent in slow-twitch muscle and to an even lower extent in cardiac muscle, where SERCA2a and PLN are highly expressed. It is expressed in only trace amounts in pancreas and prostate.SLNandPLNgenes resemble each other in having two small exons, with their entire coding sequences lying in exon 2 and a large intron separating the two segments. Brody disease is an inherited disorder of skeletal muscle function, characterized by exercise-induced impairment of muscle relaxation. Mutations in theATP2A1gene encoding SERCA1 have been associated with the autosomal recessive inheritance of Brody disease in three families, but not with autosomal dominant inheritance of the disease. A search for mutations in theSLNgene in five Brody families, four of which were not linked toATP2A1,did not reveal any alterations in coding, splice junction or promoter sequences. The homozygous deletion of C438 in the coding sequence ofATP2A1in Brody disease family 3, leading to a frameshift and truncation following Pro₁₄₇in SERCA1, is the fourthATP2A1mutation to be associated with autosomal recessive Brody disease.     

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G > A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.     

Targeted disruption of the ATP2A1 gene encoding the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1) impairs diaphragm function and is lethal in neonatal mice

Mutations in the ATP2A1 gene, encoding isoform 1 of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1), are one cause of Brody disease, characterized in humans by exercise-induced contraction of fast twitch (type II) skeletal muscle fibers. In an attempt to create a model for Brody disease, the mouse ATP2A1 gene was targeted to generate a SERCA1-null mutant mouse line. In contrast to humans, term SERCA1-null mice had progressive cyanosis and gasping respiration and succumbed from respiratory failure shortly after birth. The percentage of affected homozygote SERCA1(-/-) mice was consistent with predicted Mendelian inheritance. A survey of multiple organs from 10-, 15-, and 18-day embryos revealed no morphological abnormalities, but analysis of the lungs in term mice revealed diffuse congestion and epithelial hypercellularity and studies of the diaphragm muscle revealed prominent hypercontracted regions in scattered fibers and increased fiber size variability. The V(max) of Ca(2+) transport activity in mutant diaphragm and skeletal muscle was reduced by 80% compared with wild-type muscle, and the contractile response to electrical stimulation under physiological conditions was reduced dramatically in mutant diaphragm muscle. No compensatory responses were detected in analysis of mRNAs encoding other Ca(2+) handling proteins or of protein levels. Expression of ATP2A1 is largely restricted to type II fibers, which predominate in normal mouse diaphragm. The absence of SERCA1 in type II fibers, and the absence of compensatory increases in other Ca(2+) handling proteins, coupled with the marked increase in contractile function required of the diaphragm muscle to support postnatal respiration, can account for respiratory failure in term SERCA1-null mice.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.     

Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs)

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.     

Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans)

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.     

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.     

Co-expression of slow-twitch/cardiac muscle Ca2(+)-ATPase (SERCA2) and phospholamban

Full length cDNAs encoding both slow-twitch/cardiac (SERCA2) and fast-twitch skeletal muscle (SERCA1) Ca2(+)-ATPases were expressed by transient transfection of COS-1 cells. Studies of the Ca2(+)-dependency of Ca2(+)-transport in microsomes isolated from these cells showed that both isoforms had an affinity for Ca2+ of about 0.2 microM. The Ca2(+)-affinity of SERCA2 was lowered when phospholamban was co-expressed with it, demonstrating that the two proteins interact in this expression system. These studies support the view that phospholamban inhibition accounts for the low Ca2(+)-affinity and low activity of SERCA2 in cardiac muscle sarcoplasmic reticulum.     

HSP70 binds to the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA1a) and prevents thermal inactivation

This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41 degrees C for either 30, 60, or 120 min. Maximal SERCA1a activity (micromol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41 degrees C compared with 37 degrees C. HSP70 protected against thermal inactivation of SERCA1a activity at 37 degrees C but not at 41 degrees C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys515 in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were co-transfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40 degrees C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions both in vitro and in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain.     

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

Ca2+ signalling and muscle disease.

Transient elevations of intracellular Ca2+ play a signalling role in such complex cellular functions as contraction, secretion, fertilization, proliferation, metabolism, heartbeat and memory. However, prolonged elevation of Ca2+ above about 10 microM is deleterious to a cell and can activate apoptosis. In muscle, there is a narrow window of Ca2+ dysregulation in which abnormalities in Ca2+ regulatory proteins can lead to disease, rather than apoptosis. Key proteins in the regulation of muscle Ca2+ are the voltage-dependent, dihydropyridine-sensitive, L-type Ca2+ channels located in the transverse tubule and Ca2+ release channels in the junctional terminal cisternae of the sarcoplasmic reticulum. Abnormalities in these proteins play a key role in malignant hyperthermia (MH), a toxic response to anesthetics, and in central core disease (CCD), a muscle myopathy. Sarco(endo)plasmic reticulum Ca2+ ATPases (SERCAs) return sarcoplasmic Ca2+ to the lumen of the sarcoplasmic reticulum. Loss of SERCA1a Ca2+ pump function is one cause of exercise-induced impairment of the relaxation of skeletal muscle, in Brody disease. Phospholamban expressed in cardiac muscle and sarcolipin expressed in skeletal muscle regulate SERCA activity. Studies with knockout and transgenic mice show that gain of inhibitory function of phospholamban alters cardiac contractility and could be a causal feature in some cardiomyopathies. Calsequestrin, calreticulin, and a series of other acidic, lumenal, Ca2+ binding proteins provide a buffer for Ca2+ stored in the sarcoplasmic reticulum. Overexpression of cardiac calsequestrin leads to cardiomyopathy and ablation of calreticulin alters cardiac development.     

Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca²⁺-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca²⁺-uptake through SERCA1a (more than 35%) at micromolar Ca²⁺ but not at nanomolar Ca²⁺, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.     

Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we compared the kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 microM) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and exhibited low steady-state levels of E1P. The results indicate that the kinetic differences in Ca2+ transport between skeletal and cardiac SR are due to the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences between SERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interferes with SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletal muscle SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules during catalytic cycling is obligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of the cardiac SR Ca2+-ATPase.     

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.     

The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases.

Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.     

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.     

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.     

Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum.

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.