Capsaicin stimulates uncoupled ATP hydrolysis by the sarcoplasmic reticulum calcium pump

In muscle cells the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) couples the free energy of ATP hydrolysis to pump Ca(2+) ions from the cytoplasm to the SR lumen. In addition, SERCA plays a key role in non-shivering thermogenesis through uncoupled reactions, where ATP hydrolysis takes place without active Ca(2+) translocation. Capsaicin (CPS) is a naturally occurring vanilloid, the consumption of which is linked with increased metabolic rate and core body temperature. Here we document the stimulation by CPS of the Ca(2+)-dependent ATP hydrolysis by SERCA without effects on Ca(2+) accumulation. The stimulation by CPS was significantly dependent on the presence of a Ca(2+) gradient across the SR membrane. ATP activation assays showed that the drug reduced the nucleotide affinity at the catalytic site, whereas the affinity at the regulatory site increased. Several biochemical analyses indicated that CPS stabilizes an ADP-insensitive E(2)P-related conformation that dephosphorylates at a higher rate than the control enzyme. Under conditions where uncoupled SERCA was specifically inhibited by the treatment with fluoride, low temperatures, or dimethyl sulfoxide, CPS had no stimulatory effect on ATP hydrolysis by SERCA. It is concluded that CPS stabilizes a SERCA sub-conformation where Ca(2+) is released from the phosphorylated intermediate to the cytoplasm instead of the SR lumen, increasing ATP hydrolysis not coupled with Ca(2+) transport. To the best of our knowledge CPS is the first natural drug that augments uncoupled SERCA, presumably resulting in thermogenesis. The role of CPS as a SERCA modulator is discussed.     

Thermogenic activity of Ca2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: the role of ryanodine Ca2+ channel

The sarcoplasmic reticulum Ca(2+) ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca(2+) transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca(2+) channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (DeltaH(cal)) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg(2+) or ruthenium red, conditions that close the ryanodine Ca(2+) channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the DeltaH(cal) values of ATP hydrolysis increased significantly. Neither Mg(2+) nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the DeltaH(cal) of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca(2+) channel is opened or closed.     

A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.     

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.     

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.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Temperature dependence of cardiac sarcoplasmic reticulum Ca²⁺-ATPase from rainbow trout Oncorhynchus mykiss

In this work, the temperature dependence of the sarco-endoplasmic reticulum Ca(2+) -ATPase (SERCA2) activity from rainbow trout Oncorhynchus mykiss cardiac ventricles was measured and compared with the mammalian SERCA2 isoform. The rate of ATP-dependent Ca(2+) transport catalysed by O. mykiss vesicles was totally abolished by thapsigargin and the Ca(2+) ionophore A(23187) . At warm temperatures (25 and 30° C), the SERCA2 from O. mykiss ventricles displayed the same rate of Ca(2+) uptake. At 35° C, the activity of the O. mykiss enzyme decreased after 20 min of reaction time. The rate of Ca(2+) uptake catalysed by the mammalian SERCA2 was temperature dependent exhibiting its maximal activity at 35° C. In contrast to the rate of Ca(2+) uptake, the rate of ATP hydrolysis catalysed by O. mykiss SERCA2 was not significantly different at 25 and 35° C, but the rate of ATP hydrolysis catalysed by the rat Rattus norvegicus SERCA2 isoform at 35° C was two-fold higher than at 25° C. At low temperatures (5 to 20° C), the rate of Ca(2+) uptake from O. mykiss SR was less temperature dependent than the R. norvegicus isoform, being able to sustain a high activity even at 5° C. The mean ±s.e. Q(10) values calculated from 25 to 35° C for ATP hydrolysis were 1·112 ± 0·026 (n = 3) and 2·759 ± 0·240 (n = 5) for O. mykiss and R. norvegicus, respectively. Taken together, the results show that the O. mykiss SERCA2 was not temperature dependent over the 10 to 25° C temperature interval commonly experienced by the animal in vivo. The Q(10) value of SERCA2 was significantly lower in O. mykiss than R. norvegicus which may be key for cardiac function over the wide environmental temperatures experienced in this eurythermal fish.     

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.     

Low temperature molecular adaptation of the skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA 1) in the wood frog (Rana sylvatica)

We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.     

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.     

Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains.     

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.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Characterization of a Listeria monocytogenes Ca(2+) pump: a SERCA-type ATPase with only one Ca(2+)-binding site

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) ～80 μm) and a high pH optimum (pH ～9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.     

Interference with phosphoenzyme isomerization and inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene

ATP hydrolysis and Ca(2+) transport by the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) are inhibited by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br(2)-TITU) in the micromolar range (Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566). In a study of the mechanism of inhibition, we found that Br(2)-TITU allows the enzyme to bind Ca(2+) and undergo phosphorylation by ATP. The level of ADP-sensitive phosphoenzyme (i.e. E1P-2Ca(2+)) observed in the transient state following addition of ATP is much higher in the presence than in the absence of the inhibitor. Br(2)-TITU does not interfere with enzyme phosphorylation by P(i) in the reverse direction of the cycle (i.e. E2P) and produces only a slight inhibition of its hydrolytic cleavage. The inhibitory effect of Br(2)-TITU on steady state ATPase velocity is attributed to interference with the E1P-2Ca(2+) to E2P-2Ca(2+) transition. In fact, experiments on conformation-dependent protection from proteolytic digestion suggest that, in the presence of Br(2)-TITU, the loops connecting the "A" domain to the ATPase transmembrane region undergo greater fluctuation than expected in the E2 and E2P states. Optimal stability of the gathered headpiece domains is thereby prevented. These effects are opposite to those of thapsigargin, in which the mechanism of inhibition is related to stabilization of a highly compact ATPase conformation and interference with Ca(2+) binding and phosphoenzyme formation. Our experiments with Br(2)-TITU provide the first demonstration of a kinetic limit posed by an inhibitor on the E1P-2Ca(2+) to E2P-2Ca(2+) transition in the wild-type enzyme.     

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca(2+)stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca(2+)entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca(2+)-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca(2+)content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca(2+)in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 microM TBHQ, individually or simultaneously, accelerated Ca(2+) store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca(2+) influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca(2+) extrusion by PMCA or the Na(+)/Ca(2+) exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.     

Modeling Ca2+ dynamics of mouse cardiac cells points to a critical role of SERCA's affinity for Ca2+

The SERCA2a isoform of the sarco/endoplasmic reticulum Ca(2+) pumps is specifically expressed in the heart, whereas SERCA2b is the ubiquitously expressed variant. It has been shown previously that replacement of SERCA2a by SERCA2b in mice (SERCA2(b/b) mice) results in only a moderate functional impairment, whereas SERCA activity is decreased by a 40% lower SERCA protein expression and by increased inhibition by phospholamban. To find out whether the documented kinetic differences in SERCA2b relative to SERCA2a (i.e., a twofold higher apparent Ca(2+) affinity, but twofold lower maximal turnover rate) can explain these compensatory changes, we simulated Ca(2+) dynamics in mouse ventricular myocytes. The model shows that the relative Ca(2+) transport capacity of SERCA2a and SERCA2b depends on the SERCA concentration. The simulations point to a dominant effect of SERCA2b's higher Ca(2+) affinity over its lower maximal turnover rate. The results suggest that increased systolic and decreased diastolic Ca(2+) levels in unstimulated conditions could contribute to the downregulation of SERCA in SERCA2(b/b) mice. In stress conditions, Ca(2+) handling is less efficient by SERCA2b than by SERCA2a, which might contribute to the observed hypertrophy in SERCA2(b/b) mice. Altogether, SERCA2a might be a better compromise between performance in basal conditions and performance during β-adrenergic stress.