Inhibition of plasma membrane Ca2+-ATPase by heparin is modulated by potassium

Heparin is related to several protein receptors that control Ca2+ homeostasis. Here, we studied the effects of heparin on the plasma membrane Ca2+-ATPase from erythrocytes. Both ATP hydrolysis and Ca2+ uptake were inhibited by heparin without modification of the steady-state level of phosphoenzyme formed by ATP. Calmodulin did neither modify the inhibition nor the binding of heparin. Inhibition by heparin was counteracted by K+ but not by Li+. This effect was extended to other sulfated polysaccharides with high number of sulfate residues. Hydrolysis of p-nitrophenylphosphate was equally inhibited by heparin. No evidence for enzyme uncoupling was observed: Ca2+ uptake and ATP hydrolysis remained tightly associated at any level of heparin, and heparin did not increase the passive Ca2+ efflux of inside-out vesicles. Vanadate blocked this efflux, indicating that the main point of Ca2+ escape from these vesicles was linked to the Ca2+ pump. It is discussed that sulfated polysaccharides may physiologically increase the steady-state level of Ca2+ in the cytosol by inhibiting the Ca2+ pumps in a K+ (and tissue) regulated way. It is suggested that heparin regulates the plasma membrane Ca2+-ATPase by binding to the E2 conformer.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Mechanism of action of the novel plasma membrane Ca(2+)-pump inhibitor caloxin

Caloxin 2A1 is a novel inhibitor of the plasma membrane (PM) Ca(2+)-pump [Am. J. Physiol. Cell Physiol. 280 (2001) C1027]. The PM Ca(2+)-pump is a Ca(2+)-Mg(2+)-ATPase that expels Ca(2+) from cells to help them maintain low concentrations of cytosolic Ca(2+). Caloxin 2A1 inhibits Ca(2+)-Mg(2+)-ATPase in human erythrocyte leaky ghosts. Here we report that this inhibition is non-competitive with respect to the substrates Ca(2+) and ATP and the activator calmodulin. This was anticipated since the high affinity binding site for Ca(2+) and sites for ATP and calmodulin are intracellular whereas caloxin 2A1 is a peptide selected for binding to the second extracellular domain of the pump. Caloxin 2A1 also inhibited the Ca(2+)-dependent formation of the acid stable 140 kDa acylphosphate intermediate from 32P-gamma-ATP. However, it did not inhibit the formation of the acylphosphate intermediate in the reverse direction-from 32P-orthophosphate. Consistent with results on mutagenesis of transmembrane residues in the pump protein, we suggest that caloxin 2A1 inhibits conformational changes required during the reaction cycle of the pump.     

Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy

Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.     

Inhibition of the intracellular Ca2+ transporter SERCA (Sarco-Endoplasmic Reticulum Ca2+-ATPase) by the natural polyphenol epigallocatechin-3-gallate

The use of a microsomal preparation from skeletal muscle revealed that both Ca(2+) transport and Ca(2+)-dependent ATP hydrolysis linked to Sarco-Endoplasmic Reticulum Ca(2+)-ATPase are inhibited by epigallocatechin-3-gallate (EGCG). A half-maximal effect was achieved at approx. 12?μM. The presence of the galloyl group was essential for the inhibitory effect of the catechin. The relative inhibition of the Ca(2+)-ATPase activity decreased when the Ca(2+) concentration was raised but not when the ATP concentration was elevated. Data on the catalytic cycle indicated inhibition of maximal Ca(2+) binding and a decrease in Ca(2+) binding affinity when measured in the absence of ATP. Moreover, the addition of ATP to samples in the presence of EGCG and Ca(2+) led to an early increase in phosphoenzyme followed by a time-dependent decay that was faster when the drug concentration was raised. However, phosphorylation following the addition of ATP plus Ca(2+) led to a slow rate of phosphoenzyme accumulation that was also dependent on EGCG concentration. The results are consistent with retention of the transporter conformation in the Ca(2+)-free state, thus impeding Ca(2+) binding and therefore the subsequent steps when ATP is added to trigger the Ca(2+) transport process. Furthermore, phosphorylation by inorganic phosphate in the absence of Ca(2+) was partially inhibited by EGCG, suggesting alteration of the native Ca(2+)-free conformation at the catalytic site.     

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Tricyclic antidepressants inhibit the Ca(2+)-dependent ATPase activity from plasma membrane.

Tricyclic antidepressants are moderately potent inhibitors of the plasma membrane Ca(2+)-ATPase activity measured in erythrocyte ghosts. For the calmodulin-activated activity, half-maximal inhibition was observed in the presence of 0.25 mM clomipramine. Desipramine, imipramine, and trimipramine show half-maximal inhibition in the range of 0.8 to 1 mM. The inhibition dependence on clomipramine concentration is the same whether the enzyme is activated by exogenous calmodulin or by tryptic digestion. A similar behavior was observed for desipramine. The inhibition mechanisms utilized by clomipramine and desipramine are different. The clomipramine effect is associated with the Ca(2+)-bound enzyme conformation and can be attributed to a decrease in the rate of phosphorylation by ATP. The desipramine effect appears more related to the Ca(2+)-free conformation, since the partial reaction involved in the release of inorganic phosphate is perturbed by this drug. There is also little or no effect of tricyclics on the enzyme's affinity for ligand (Ca(2+) or ATP) binding.     

Effect of eosin Y on Ca2+, Mg2+-ATPase of the smooth muscle sarcolemma

Eosin Y was studied with the aim to elucidate the mechanism of its inhibitory effect on the activity of Ca(2+)-transporting ATPase of myometrium cell plasma membrane. The inhibitor was studied for its effect on the maximal rate of the ATP-hydrolase reaction catalyzed by Ca2+, Mg(2+)-ATPase, on the enzyme affinity for the substrate and a possibility of enzyme activity protection under the inhibitor effect by the main reagents of ATP-hydrolase reaction. It was established that eosin Y decreased the turnover rate of this enzyme and his affinity for ATP. Preincubation of ATPase with ATP (or ATP plus MgCl2) had no effect on the extent of enzyme inhibition by eosin Y. This result proves that eosin Y and ATP do not compete for the site of binding on the enzyme.     

Association of vanadate-sensitive Mg(2+)-ATPase and shape change in intact red blood cells

Intact human erythrocytes, initially depleted of Mg2+ by EDTA incubation in the presence of A23187, exhibit Mg(2+)-dependent phosphate production of around 1.5 mmol per liter cells.h, half-maximally activated at around 0.4 mM added free Mg2+. This appears to correspond to Mg(2+)-stimulated adenosine triphosphatase (Mg(2+)-ATPase) activity found in isolated membranes, which is known to have a similar activity and affinity for Mg2+. Vanadate (up to 100 microM) inhibited Mg(2+)-dependent phosphate production and ATP breakdown in intact cells. Over a similar concentration range vanadate (3-100 microM) transformed intact cells from normal discocytes to echinocytes within 4-8 h at 37 degrees C, and more rapidly in Mg(2+)-depleted cells. The rate of Ca(2+)-induced echinocytosis was also enhanced in Mg(2+)-depleted cells. These results support previous studies in erythrocyte ghosts suggesting that vanadate-induced shape change is associated with inhibition of Mg(2+)-ATPase activity localized in the plasma membrane of the red blood cell.     

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.     

Calmodulin effect on purified rat cortical plasma membrane Ca(2+)-ATPase in different phosphorylation states

The plasma membrane Ca(2+)-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca(2+) concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca(2+)-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca(2+)-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca(2+)-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca(2+)-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca(2+)-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca(2+)-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca(2+)-ATPase.     

Specific inositol phosphates inhibit basal and calmodulin-stimulated Ca(2+)-ATPase activity in human erythrocyte membranes in vitro and inhibit binding of calmodulin to membranes.

D-Myo-inositol 1,4,5-trisphosphate (Ins[1,4-,5]P3) inhibits rat heart sarcolemmal Ca(2+)-ATPase activity (T. H. Kuo, Biochem. Biophys. Res. Commun. 152: 1111, 1988). We have studied the effect and mechanism of action of Ins(1,4,5)P3 and related inositol phosphates on human red cell membrane Ca(2+)-ATPase (EC 3.6.1.3) activity in vitro. At 10(-6) M, Ins(1,4,5)P3 and D-myo-inositol 4,5-bisphosphate (Ins[4,5]P2) inhibited human erythrocyte membrane Ca(2+)-ATPase activity in vitro by 42 and 31%, respectively. D-Myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 1-phosphate were not inhibitory. Enzyme inhibition by Ins(1,4,5)P3 was blocked by heparin. Exogenous purified calmodulin also stimulated red cell membrane Ca(2+)-ATPase activity; this stimulation was inhibited by Ins(1,4,5)P3. Ins(4,5)P2 and Ins(1,4,5)P3, but not Ins(1,4)P2, inhibited the binding of [125I]calmodulin to red cell membranes. Thus, specific inositol phosphates reduce plasma membrane Ca(2+)-ATPase activity and enhancement of the latter in vitro by purified calmodulin. The mechanism of these effects may in part relate to inhibition by inositol phosphates of binding of calmodulin to erythrocyte membranes.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.     

Effect of eosin Y on Ca2+-dependent activity of Ca2+-Mg2+-ATPase in smooth muscle cell membrane

With the aim to elucidate mechanism of eosin Y inhibitory effect on the Ca(2+)-transporting ATPase activity of myometrial cell plasma membrane effect of this inhibitor on the maximal initial rate of ATP hydrolysis reaction, catalyzed by Ca2+, Mg(2+)-ATPase, and on the enzyme affinity for Ca2+ was studied. It was established that eosin Y decreased the rate of Ca2+, Mg(2+)-ATPase catalitic turnover determined by Ca2+ and had no effect on enzyme affinity for this cation.     

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.