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Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function
Biochemical and Biophysical Research Communications 401 (2010) 370–375
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Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function Eleri Hughes, Rachel Edwards, David A. Middleton ⇑ School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom
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Article history: Received 3 September 2010 Available online 17 September 2010 Keywords: Isothermal titration calorimetry Heart failure Oligosaccharides Heparin Solid-state NMR
a b s t r a c t The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) regulates the active transport of Ca2+ into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca2+ uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Phospholamban (PLB) is a pentameric transmembrane protein located in the sarcoplasmic reticulum (SR) of cardiac ventricular myocytes [1]. PLB regulates the active transport of Ca2+ into the SR lumen via a reversible inhibitory association with the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) [2–4]. Inhibition is relieved following phosphorylation of PLB at residues Ser16 and Thr17 in response to b-adrenergic stimulation [2]. Disruption of Ca2+ homeostasis in cardiac cells affects the regulation of muscle contraction and relaxation, and may contribute to cardiovascular disorders, such as heart failure (HF) [5–8]. There is substantial evidence that HF and cardiac hypertrophy are linked to defects in Ca2+ uptake by the cardiac SR [9–12]. Molecules that stimulate cardiac calcium transport by targeting the inhibitory SERCA-PLB interaction are thus interesting from a therapeutic perspective [13]. Several studies have identified agents that interfere with the PLB-SERCA interaction and stimulate calcium transport [14–18]. It was shown over 20 years ago that large polyanions such as heparin and dextran sulphate stimulate cardiac SERCA activity, and their action was attributed to the relief of inhibition by PLB [19]. Abbreviations: PLB, phospholamban; SSNMR, solid-state nuclear magnetic resonance; MAS, magic-angle spinning; ITC, isothermal titration calorimetry; SERCA, sarco(endo)plasmic reticulum calcium ATPase; DOPC, dioleoylphosphatidylcholine; HDO, heparin-derived oligosaccharide; HDec, heparin-derived decasaccharide; HHex, heparin-derived hexasaccharide; HDi, heparin-derived disaccharide; HF, heart failure. ⇑ Corresponding author. E-mail address: [email protected] (D.A. Middleton). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.09.056
In the search for such compounds it would be useful to identify a suitable target site or region within PLB. It is known that PLB regulates SERCA at least in part through interactions between the transmembrane regions of the two proteins, which lowers the affinity of SERCA for calcium [20,21]. The cytoplasmic domain is more attractive as an accessible target site for drugs in terms of its accessibility, but the functional importance of the PLB cytoplasmic domain has not been fully resolved. Mutations of polar residues in the cytoplasmic domain of PLB reveal that several residues in this region are essential for the regulatory interaction with SERCA [22–24], but it is unclear whether the cytoplasmic domain is autonomously regulatory. Functional studies with peptides representing the PLB cytoplasmic domain in isolation have been contradictory. Some investigators showed that peptides comprising PLB cytoplasmic residues are capable of inhibiting SERCA by lowering maximal activity (Vmax) [25–27]. Others have reported that peptides encompassing all or some of the first 31 residues of PLB have no effect at all on SERCA activity [28–30]. This lack of consensus is compounded by the uncertainty about the effect of full-length PLB on Vmax for SERCA; some studies have reported no significant effect on Vmax [31], some show a clear reduction [32,33] and other report an increase in maximal activity [34]. Biochemical and biophysical methods are used here to demonstrate that a series of synthetic, heparin-derived oligosaccharides (HDOs) perturb the SERCA-PLB interaction in membrane preparations. Our findings add further support to the argument that the PLB cytoplasmic domain plays a regulatory role by lowering the maximal rate of ATP hydrolysis by SERCA. Using HDOs as a research tool we suggest that the PLB cytoplasmic domain is a realistic target for small molecules with more drug-like properties.
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2. Materials and methods 2.1. Materials Synthetic NAc-PLB1–23 (>95%), a peptide comprising the first 23 cytoplasmic residues of PLB was supplied by Peptide Protein Research (PPR Ltd) (Fareham). Synthetic full-length PLB and PLB29–52, comprising only the transmembrane residues, were purchased from Activotec (Cambridge). HDOs were supplied by Carbosynth Ltd (Berkshire), and all other chemicals supplied by Sigma Chemicals Ltd (UK). The HDOs used were: heparin derived decasaccharide (HDec; C60H75N5O95S15K20); heparin derived hexasaccharide (HHex; C36H47N3O58S9K12); heparin derived disaccharide (HDi; C12H15NO19S3Na4). SERCA1a Ca2+-ATPase was prepared from fast-twitch rabbit skeletal muscle according to a method adapted from East and Lee [35].
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ature (RT) for 15 min, and then at 4 °C for 45 min. Detergent was removed with the addition of Amberlite XAD2 resin (approximately 25 mg per hour), for 3 h with stirring at RT. Samples were removed from resin beads and frozen at 20 °C until required for assay. Assays were carried out in a final volume of 200 ll, using 5 lg purified SERCA1a membranes or 10 lg reconstituted SERCA1a per sample and CaCl2 added to the required free calcium concentration. Samples were initially prepared in a total volume of 120 ll of reaction buffer (30 mM Tris, pH 8). Following addition of 80 ll of assay medium (4 mM ATP, 4 mM MgCl2, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), 30 mM imidazole, pH 7.3), generating a final pH of 7.5, the samples were incubated for 10 min at 37 °C. Specific Ca2+-ATPase activity was quantified as the amount of inorganic phosphate (Pi) liberated upon hydrolysis of ATP as measured by formation of a phospho-molybdate complex under acidic conditions [36,37].
2.2. Co-reconstitution of SERCA and full length PLB 2.3. Expression of PLB Co-reconstitutions with full length PLB, at defined ratios of 160:5:1 or 160:10:1 DOPC/PLB/SERCA were carried out with DOPC and PLB solubilised in 50:50 chloroform/methanol, dried to a film under nitrogen then left on the vacuum pump overnight. For reconstitution, samples were dissolved in 500 ll OG buffer (6 mg/ml octyl glucoside in 10 mM Tris/0.25 M sucrose, pH 7.5), then SERCA1a, treated with C12E8 to 1 mg/ml was added to the required concentration. Samples were left to stir at room temper-
Uniformly 13C and 15N labelled PLB was expressed in Escherichia coli BL21(DE3) as a maltose binding fusion protein (MBP), following transformation with the pMAL-c2X plasmid (New England Biolabs Inc.), containing the human wild-type PLB gene sequence preceded by an engineered TEV cleavage site [38]. Cells were harvested by centrifugation, resuspended in lysis buffer (20 mM KH2PO4, pH 7.3, 120 mM NaCl, 1 mM ethylenediaminetetraacetic
Fig. 1. ITC data for 50 lM PLB1–23 titrated with HDOs at 25 °C. Raw data of power versus time (top) and titration curves of integrated enthalpy versus mole ratio of reactants (bottom) are shown for HDec (A), HHex (B) and HDi (C). Solid lines in the bottom panels are the best fitting curves corresponding to the values of Ka and DH and stoichiometry N given.
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acid (EDTA), 0.1 mM dithiothreitol and 0.5% glycerol) and frozen overnight at 20 °C prior to lysis by 2 cycles of French Press. Following 30 min incubation at 4 °C with shaking, cells were centrifuged (20 000g, 4 °C, 15 min) and the supernatant bound to amylose resin, washed (20 mM KH2PO4, pH 7.3, 120 mM NaCl, 1 mM EDTA and 0.02% NaN3), and eluted with wash buffer, containing 46 mM maltose. The fusion protein was cleaved with AcTEV protease (Invitrogen), resulting in precipitation of PLB, which was collected by ultracentrifugation (100 000g, 4 °C, 1 h; Sorvall T-865.1). The pellet was resuspended in 10 ml 50 mM KH2PO4, pH 7.2; 1 M guanidine hydrochloride (GuHCl) and centrifugation repeated at 50 000g for 30 min. The final pellet was then solubilised in up to 5 ml 50 mM KH2PO4, pH 7.2; 7 M GuHCl followed by HPLC purification on a C5 analytical column (Phenomenex) using a 5–95% gradient of AcN (0.1% TFA). Successful expression and purification of PLB was determined by Western blot using anti-PLB antibody A1 (Badrilla). 2.4. Isothermal titration calorimetry Heat flow resulting from binding of HDOs to PLB1–23 was measured using the ultrasensitive iTC200 MicroCalorimeter (MicroCal LLC, Northampton, MA). Reaction cell volume and total injection volume were 200 ll and 40 ll respectively. Experiments were performed at 25 °C, at a power reference setting of 6 l cal/s, with stirring at 1000 rpm. Data analysis was carried out using the Origin v.7 software (MicroCal) and fitted using the ‘one set of sites’ model. The reaction cell contained a 25 lM or 50 lM solution of PLB1–23 in
10 mM Tris, 1 mM EDTA, pH 7.4. Solutions of oligosaccharides were prepared in the same buffer at concentrations of 250 lM, and injected via the syringe. Titrations were carried out at intervals up to 2 min in 1 ll aliquots following an initial discard aliquot of 0.3 ll. The heat of dilution was determined in control experiments titrating oligosaccharides into a buffer solution minus the peptide. Subtraction of the heat of dilution values from experimental values allows the determination of heat flow resulting from peptide binding. 2.5. Solid-state NMR Magic angle-spinning (MAS) 13C solid-state (SS) NMR experiments were performed using a Bruker Avance 400 spectrometer operating at a magnetic field of 9.3 Tesla. Experiments were carried out at 4 °C. Membrane pellets obtained by centrifugation were packed into a 4 mm zirconium rotor and rotated at the magic angle at 8 kHz, maintaining the spinning rate automatically to within ± 1 Hz. All experiments utilized an initial 4.0-ls 13C 90° excitation pulse length and TPPM proton decoupling [39] at a field of 85 kHz during signal acquisition and a 2-s recycle delay. 3. Results and discussion 3.1. Screening HDO-PLB interactions Earlier work showed that the water-soluble peptide PLB1-23, representing the cytoplasmic residues of PLB, interacts with the cytoplasmic face of SERCA in SR vesicles and reduces Vmax for
Fig. 2. Effect of HDOs on the inhibition of SERCA by the PLB cytoplasmic domain peptide PLB1–23. Rates of ATP hydrolysis by SERCA (at 1.6 lM Ca2+) in rabbit skeletal muscle membranes was measured at different concentrations of HDec (A), HHex (B) or HDi (C) in the presence of 75 lM PLB1–23 (hatched white bars) or in the absence of peptide (black bars). The three saccharides have a minimal effect on ATPase activity in the absence of the peptide. Activities are expressed as a percentage of the rate of ATP hydrolysis in the absence of HDO and peptide (3.58 lmol Pi/mg per min).
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calcium transport [25]. ITC measurements were conducted on PLB1–23 in aqueous solution titrated with the heparin-derived decasaccharide HDec, hexasaccharide HHex or disaccharide HDi. The ITC isotherms show that HDec binds to the peptide with an association constant Ka of 5.31 105 M1 and a binding enthalpy DH of 10.8 kcal/mol (Fig. 1A). The stoichiometry N of 0.27 indicates that each HDec molecule binds approximately three peptide molecules, presumably because of the large size and high formal charge of the saccharide (20 compared to +4 for the peptide). HHex also interacts with the peptide, but with a lower affinity Ka (2.72 105 M1) and total binding enthalphy (DH = 5.5 kcal/mol) (Fig. 1B). In this case the stoichiometry N is 0.40, indicating a lower binding capacity of the saccharide. Virtually no interaction between HDi and PLB1–23 is detected and reliable thermodynamic data could not be calculated from the data (Fig. 1C).
concentration of HHex is required to abolish the inhibition of SERCA by PLB1-23. The reason for this discrepancy is not clear, but may be related to the higher degrees of freedom of the water soluble peptide compared to full-length PLB, which is confined within a two-dimensional lipid matrix. Reconstitution of SERCA with a 10fold excess of the peptide PLB29-52 representing the PLB transmembrane domain results in a 20% reduction in the rate of ATP hydrolysis compared to SERCA alone under the same conditions used for full-length PLB (Fig. 3B, triangles). Addition of HHex up to 25 lM has no significant effect on ATPase activity in the presence of PLB29-52. This observation, taken together with the studies on PLB1-23, suggests that the dis-inhibitory effect of HHex on fulllength PLB is mediated through the PLB cytoplasmic domain.
3.2. Functional effects of HDOs
The interaction between HHex and uniformly 13C and 15N labelled PLB ([U-13C,15N]PLB) in DOPC membranes was examined by 13C MAS SSNMR. Fig. 4A shows one-dimensional MAS SSNMR spectra (at 4 °C) obtained with direct polarisation (DP) of the 13C nuclei (top) and with cross-polarisation (CP) from 1H to 13C (bottom). The CP-MAS spectrum is rather broad and contains little fine detail, but much sharper peaks are seen in the DP spectrum as
The HDOs were next screened for their effects on the inhibitory interaction between PLB1–23 and SERCA. HDOs were added at various concentrations to SERCA in a native membrane preparation and the rate of ATPase activity (at 1.6 lM Ca2+) was determined with and without 75 lV PLB1–23 (Fig. 2A–C). In the absence of HDO, 75 lV PLB1–23 reduced Vmax by 35% compared to the control in the absence of peptide as observed earlier [25]. After the addition of HDec activity recovered with increasing decasaccharide concentration and at 10 lV HDec the rate of hydrolysis was close to the level measured in the absence of peptide (Fig. 2A). Inhibition by PLB1–23 is also reversed by HHex but higher concentrations of the oligosaccharide (200 lV) are required to restore ATPase activity to uninhibited levels (Fig. 2B). Addition of heparin disaccharide (HDi) to concentrations of up to 400 lV has no effect on SERCA inhibition (Fig. 2C). NaCl or KCl at 4 mM (the approximate sodium and potassium content of the HDOs) had no effect on ATPase activity in the presence of 75 lV PLB1–23 or in the absence of peptide (data not presented). The effectiveness of the HDOs in dis-inhibiting SERCA thus parallels the affinity of HDOs for PLB1–23. HHex was investigated for its functional effect on the interaction between full-length PLB and SERCA reconstituted into DOPC membranes. Calcium-dependent activity curves for SERCA, normalised to Vmax, are shifted to the right in the presence of PLB signifying a reduction in the calcium affinity of the enzyme [34]. A normalised plot of the ATPase activity of SERCA reconstituted into DOPC in the absence of PLB shows calcium-dependent hydrolysis of ATP with half maximal rate (KCa) at 0.34 lM Ca2+ and Vmax at approximately 1.6 lM Ca2+ (Fig. 3A). With PLB in fivefold molar excess, KCa increases to 0.92 lM signifying a reduction in the calcium affinity of the enzyme, following the trend reported previously. Addition of 25 lM HHex to the PLB/SERCA membranes did not have a significant effect on the calcium affinity of the enzyme either in the presence of PLB (Fig. 3A) or in the absence of PLB (not presented). We next investigated whether HHex affects Vmax for SERCA when reconstituted alone or with PLB. Using the standard reconstitution procedure [35] the recovery of active SERCA in the presence of PLB can be highly variable and comparisons between the absolute values of Vmax from preparation to preparation must be treated with caution. It is valid to draw conclusions about the trend in Vmax in response to the titration of an external agent, however. With a 10-fold molar excess of PLB addition of 25 lM HHex partially relieves the inhibition of SERCA and increases Vmax to approximately 70% of the rate for SERCA alone (Fig. 3B, filled circles). At a lower (5:1) PLB:SERCA ratio, Vmax in the absence of saccharide is reduced to 80% of SERCA alone, but increases to almost 100% of SERCA when 25 lM HHex is added to the membranes (Fig. 3B, open circles). Recalling Fig. 2B, it is noted that a much higher (200 lM)
3.3. Detection of HDO-PLB interactions using solid-state NMR
Fig. 3. The ATPase activity of SERCA reconstituted into DOPC membranes at a lipid:SERCA molar ratio of 160:1. (A) Calcium dependence of ATP hydrolysis by SERCA reconstituted into DOPC membranes at a lipid:protein molar ratio of 160:1. Activation of ATP hydrolysis by Ca2+ is shown for SERCA alone in the absence of peptide or HDO (triangles), for 5:1 PLB:SERCA (squares) and for 5:1 PLB:SERCA in the presence of 25 lM HHex (circles). (B) The effect of HHex on maximal hydrolytic activity of SERCA reconstituted alone (squares), with full length PLB at PLB:SERCA ratios of 5:1 (open circles) or 10:1 (filled circles) or with PLB29–52 (open triangles). Activities are presented as percentage Vmax for reconstituted SERCA in the absence of HDO and PLB peptide (2.28 lmol Pi/mg per min). For each set of data, mean values and standard errors are given for measurements on three individual reconstituted samples.
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Fig. 4. 13C MAS SSNMR spectra of uniformly 13C and 15N labelled PLB incorporated into DOPC membranes (lipid:peptide molar ratio of 10:1). (A) Full spectra obtained with direct polarisation (DP) and cross-polarisation (CP) in the absence of oligosaccharide. (B) Regions of DP spectra obtained for PLB alone (top) and in the presence of HHex at oligosaccharide:PLB molar ratios of 0.5:1, 1:1 and 2:1. The peak assigned to the guanidinium carbon of arginine residues is labelled Rf. Each spectrum is the result of accumulating between 80 000 and 120 000 transients.
reported previously [40]. DP detects the signals for all regions including the dynamic cytoplasmic region whereas CP favours detection of signals for the motionally- and conformationally- constrained transmembrane region. The DP spectrum is therefore more suitable for observing interactions between HHex and the cytoplasmic region of PLB. Fig. 4B shows a series of DP-MAS spectra of [U-13C,15N]PLB titrated with HHex in a saccharide:protein molar ratio of 0:5:1, 1:1 and 2:1. The high-field region of the spectrum is very crowded with signals from the lipid and labelled protein (Fig. 4B, right). It is not possible to attribute any changes in this region to specific amino acids or residues at this stage because of the complexity and overlap in the spectrum. Multidimensional spectroscopy will be required for a more detailed analysis of this region. The low-field region is less crowded and dominated by peaks for the backbone amide carbonyl groups around 170– 180 ppm (Fig. 4C, left). These also become slightly broader with increasing HHex concentration. A small peak for the arginine guanidinium carbon(s) (Rf) is well separated from the other peaks and shows a movement up-field from 160.0 ppm in the absence of HHex to 158.4 ppm at a HHex:PLB ratio of 2:1. These changes imply that HHex interacts with the PLB cytoplasmic domain around one or more of the four arginine residues in the PLB cytoplasmic
domain. Although an interaction between polyanionic HHex and cationic arginine side groups is not surprising, these results strengthen the argument that HDOs associate with the cytoplasmic domain of full-length PLB in a membrane environment. These experiments also demonstrate that SSNMR is sensitive to ligand binding to the PLB cytoplasmic domain. 4. Conclusions HDOs serve as a useful research tool for the dual purpose of investigating the nature of the PLB-SERCA interaction and for assessing the potential of disrupting this interaction to stimulate calcium uptake by the SR. It is generally agreed that the PLB transmembrane domain lowers the affinity of SERCA for calcium [20,21], but whether the PLB cytoplasmic domain enhances this effect or plays a secondary role in regulating the maximal activity of SERCA has been more difficult to establish. Progress has been hampered by the problems in obtaining functionally reproducible preparations of SERCA with PLB, with which to make reliable comparisons. Here we show that heparin-derived oligosaccharides, and in particular HHex, can interact with the cytoplasmic domain of PLB and enhance maximal levels of SERCA activity in a
E. Hughes et al. / Biochemical and Biophysical Research Communications 401 (2010) 370–375
well-defined membrane environment. HHex has no significant effect on the calcium affinity of SERCA. Taken together, these results imply that the PLB cytoplasmic domain regulates maximal SERCA activity at high calcium concentrations, but not KCa, which is regulated by the PLB transmembrane domain. Although HDOs are not suitable as drug candidates for several reasons, this work suggests that it is feasible to target the cytoplasmic region of PLB with other, more drug-like molecules in order to disrupt the PLB-SERCA interaction and enhance SERCA activity. Acknowledgments The British Heart Foundation is acknowledged for financial support of this work (Project Grant PG/07/069/23357). The authors thank Prof. Mikael Esmann (University of Aarhus) for his constructive comments on this manuscript. References [1] J. Fujii, A. Ueno, K. Kitano, et al., Clin. Invest. 79 (1987) 301–304. [2] M. Tada, M.J. Inui, J. Mol. Cell. Cardiol. 15 (1983) 565–575. [3] J.E. Mahaney, J.M. Autry, L.R. Jones, Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: new insights on Ca-ATPase regulation by phospholamban, Biophys. J. 78 (2000) 1306–1323. [4] J.M. Autry, L.R. Jones, Functional co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation, J. Biol. Chem. 272 (1997) 15872–15880. [5] M. Meyer, W. Schillinger, B. Pieske, et al., Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy, Circulation 92 (1995) 778– 784. [6] K.L. Koss, E.G. Kranias, Phospholamban: a prominent regulator of myocardial contractility, Circ. Res. 79 (1996) 1059–1063. [7] R.H.G. Schwinger, G. Munch, B. Bolck, et al., Reduced Ca2+-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation, J. Mol. Cell. Cardiol. 31 (1999) 479–491. [8] A.G. Brittsan, A.N. Carr, A.G. Schmidt, E.G. Kranias, Maximal inhibition of SERCA2 Ca2+ affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban, J. Biol. Chem. 275 (2000) 12129–12135. [9] G. Hasenfuss, H. Reinecke, R. Studer, et al., Relation between myocardialfunction and expression of sarcoplasmic-reticulum Ca2+-Atpase in failing and nonfailing human myocardium, Circ. Res. 75 (1994) 434–442. [10] I.A. Hobaim, B. O’Rourke, Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure, Circulation 103 (2001) 1577–1584. [11] H. Kubo, K.B. Margulies, V. Piacentino, et al., Patients with end-stage congestive heart failure treated with beta-adrenergic receptor antagonists have improved ventricular myocyte calcium regulatory protein abundance, Circulation 104 (2001) 1012–1018. [12] M. Lindner, E. Erdmann, D.J. Beuckelmann, Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure, J. Mol. Cell. Cardiol. 30 (1998) 743–749. [13] A. Schmidt, I. Edes, E.G. Kranias, Phospholamban: a promising therapeutic target in heart failure? Cardiovasc. Drugs Ther. 15 (2001) 387–396. [14] I. BerrebiBertrand, P. Lahouratate, V. Lahouratate, et al., Mechanism of action of sarcoplasmic reticulum calcium-uptake activators – discrimination between sarco(endo)plasmic reticulum Ca2+ ATPase and phospholamban interaction, Eur. J. Biochem. 247 (1997) 801–809. [15] E. McKenna, J.S. Smith, K.E. Coll, et al., Dissociation of phospholamban regulation of cardiac sarcoplasmic reticulum Ca2+ ATPase by quercetin, J. Biol. Chem. 271 (1996) 24517–24525. [16] M. Chiesi, R. Schwaller, Reversal of phospholamban induced-inhibition of cardiac sarcoplasmic-reticulum Ca2+-ATPase by tannin, Biochem. Biophys. Res. Comm. 202 (1994) 1668–1673. [17] Y. Kimura, M. Inui, M. Kadoma, et al., Effects of monoclonal-antibody against phospholamban on calcium-pump ATPase of cardiac sarcoplasmic-reticulum, J. Mol. Cell. Cardiol. 23 (1991) 1223–1230.
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Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function Eleri Hughes, Rachel Edwards, David A. Middleton ⇑ School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom
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Article history: Received 3 September 2010 Available online 17 September 2010 Keywords: Isothermal titration calorimetry Heart failure Oligosaccharides Heparin Solid-state NMR
a b s t r a c t The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) regulates the active transport of Ca2+ into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca2+ uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Phospholamban (PLB) is a pentameric transmembrane protein located in the sarcoplasmic reticulum (SR) of cardiac ventricular myocytes [1]. PLB regulates the active transport of Ca2+ into the SR lumen via a reversible inhibitory association with the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) [2–4]. Inhibition is relieved following phosphorylation of PLB at residues Ser16 and Thr17 in response to b-adrenergic stimulation [2]. Disruption of Ca2+ homeostasis in cardiac cells affects the regulation of muscle contraction and relaxation, and may contribute to cardiovascular disorders, such as heart failure (HF) [5–8]. There is substantial evidence that HF and cardiac hypertrophy are linked to defects in Ca2+ uptake by the cardiac SR [9–12]. Molecules that stimulate cardiac calcium transport by targeting the inhibitory SERCA-PLB interaction are thus interesting from a therapeutic perspective [13]. Several studies have identified agents that interfere with the PLB-SERCA interaction and stimulate calcium transport [14–18]. It was shown over 20 years ago that large polyanions such as heparin and dextran sulphate stimulate cardiac SERCA activity, and their action was attributed to the relief of inhibition by PLB [19]. Abbreviations: PLB, phospholamban; SSNMR, solid-state nuclear magnetic resonance; MAS, magic-angle spinning; ITC, isothermal titration calorimetry; SERCA, sarco(endo)plasmic reticulum calcium ATPase; DOPC, dioleoylphosphatidylcholine; HDO, heparin-derived oligosaccharide; HDec, heparin-derived decasaccharide; HHex, heparin-derived hexasaccharide; HDi, heparin-derived disaccharide; HF, heart failure. ⇑ Corresponding author. E-mail address: [email protected] (D.A. Middleton). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.09.056
In the search for such compounds it would be useful to identify a suitable target site or region within PLB. It is known that PLB regulates SERCA at least in part through interactions between the transmembrane regions of the two proteins, which lowers the affinity of SERCA for calcium [20,21]. The cytoplasmic domain is more attractive as an accessible target site for drugs in terms of its accessibility, but the functional importance of the PLB cytoplasmic domain has not been fully resolved. Mutations of polar residues in the cytoplasmic domain of PLB reveal that several residues in this region are essential for the regulatory interaction with SERCA [22–24], but it is unclear whether the cytoplasmic domain is autonomously regulatory. Functional studies with peptides representing the PLB cytoplasmic domain in isolation have been contradictory. Some investigators showed that peptides comprising PLB cytoplasmic residues are capable of inhibiting SERCA by lowering maximal activity (Vmax) [25–27]. Others have reported that peptides encompassing all or some of the first 31 residues of PLB have no effect at all on SERCA activity [28–30]. This lack of consensus is compounded by the uncertainty about the effect of full-length PLB on Vmax for SERCA; some studies have reported no significant effect on Vmax [31], some show a clear reduction [32,33] and other report an increase in maximal activity [34]. Biochemical and biophysical methods are used here to demonstrate that a series of synthetic, heparin-derived oligosaccharides (HDOs) perturb the SERCA-PLB interaction in membrane preparations. Our findings add further support to the argument that the PLB cytoplasmic domain plays a regulatory role by lowering the maximal rate of ATP hydrolysis by SERCA. Using HDOs as a research tool we suggest that the PLB cytoplasmic domain is a realistic target for small molecules with more drug-like properties.
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2. Materials and methods 2.1. Materials Synthetic NAc-PLB1–23 (>95%), a peptide comprising the first 23 cytoplasmic residues of PLB was supplied by Peptide Protein Research (PPR Ltd) (Fareham). Synthetic full-length PLB and PLB29–52, comprising only the transmembrane residues, were purchased from Activotec (Cambridge). HDOs were supplied by Carbosynth Ltd (Berkshire), and all other chemicals supplied by Sigma Chemicals Ltd (UK). The HDOs used were: heparin derived decasaccharide (HDec; C60H75N5O95S15K20); heparin derived hexasaccharide (HHex; C36H47N3O58S9K12); heparin derived disaccharide (HDi; C12H15NO19S3Na4). SERCA1a Ca2+-ATPase was prepared from fast-twitch rabbit skeletal muscle according to a method adapted from East and Lee [35].
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ature (RT) for 15 min, and then at 4 °C for 45 min. Detergent was removed with the addition of Amberlite XAD2 resin (approximately 25 mg per hour), for 3 h with stirring at RT. Samples were removed from resin beads and frozen at 20 °C until required for assay. Assays were carried out in a final volume of 200 ll, using 5 lg purified SERCA1a membranes or 10 lg reconstituted SERCA1a per sample and CaCl2 added to the required free calcium concentration. Samples were initially prepared in a total volume of 120 ll of reaction buffer (30 mM Tris, pH 8). Following addition of 80 ll of assay medium (4 mM ATP, 4 mM MgCl2, 0.5 mM ethylene glycol bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), 30 mM imidazole, pH 7.3), generating a final pH of 7.5, the samples were incubated for 10 min at 37 °C. Specific Ca2+-ATPase activity was quantified as the amount of inorganic phosphate (Pi) liberated upon hydrolysis of ATP as measured by formation of a phospho-molybdate complex under acidic conditions [36,37].
2.2. Co-reconstitution of SERCA and full length PLB 2.3. Expression of PLB Co-reconstitutions with full length PLB, at defined ratios of 160:5:1 or 160:10:1 DOPC/PLB/SERCA were carried out with DOPC and PLB solubilised in 50:50 chloroform/methanol, dried to a film under nitrogen then left on the vacuum pump overnight. For reconstitution, samples were dissolved in 500 ll OG buffer (6 mg/ml octyl glucoside in 10 mM Tris/0.25 M sucrose, pH 7.5), then SERCA1a, treated with C12E8 to 1 mg/ml was added to the required concentration. Samples were left to stir at room temper-
Uniformly 13C and 15N labelled PLB was expressed in Escherichia coli BL21(DE3) as a maltose binding fusion protein (MBP), following transformation with the pMAL-c2X plasmid (New England Biolabs Inc.), containing the human wild-type PLB gene sequence preceded by an engineered TEV cleavage site [38]. Cells were harvested by centrifugation, resuspended in lysis buffer (20 mM KH2PO4, pH 7.3, 120 mM NaCl, 1 mM ethylenediaminetetraacetic
Fig. 1. ITC data for 50 lM PLB1–23 titrated with HDOs at 25 °C. Raw data of power versus time (top) and titration curves of integrated enthalpy versus mole ratio of reactants (bottom) are shown for HDec (A), HHex (B) and HDi (C). Solid lines in the bottom panels are the best fitting curves corresponding to the values of Ka and DH and stoichiometry N given.
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acid (EDTA), 0.1 mM dithiothreitol and 0.5% glycerol) and frozen overnight at 20 °C prior to lysis by 2 cycles of French Press. Following 30 min incubation at 4 °C with shaking, cells were centrifuged (20 000g, 4 °C, 15 min) and the supernatant bound to amylose resin, washed (20 mM KH2PO4, pH 7.3, 120 mM NaCl, 1 mM EDTA and 0.02% NaN3), and eluted with wash buffer, containing 46 mM maltose. The fusion protein was cleaved with AcTEV protease (Invitrogen), resulting in precipitation of PLB, which was collected by ultracentrifugation (100 000g, 4 °C, 1 h; Sorvall T-865.1). The pellet was resuspended in 10 ml 50 mM KH2PO4, pH 7.2; 1 M guanidine hydrochloride (GuHCl) and centrifugation repeated at 50 000g for 30 min. The final pellet was then solubilised in up to 5 ml 50 mM KH2PO4, pH 7.2; 7 M GuHCl followed by HPLC purification on a C5 analytical column (Phenomenex) using a 5–95% gradient of AcN (0.1% TFA). Successful expression and purification of PLB was determined by Western blot using anti-PLB antibody A1 (Badrilla). 2.4. Isothermal titration calorimetry Heat flow resulting from binding of HDOs to PLB1–23 was measured using the ultrasensitive iTC200 MicroCalorimeter (MicroCal LLC, Northampton, MA). Reaction cell volume and total injection volume were 200 ll and 40 ll respectively. Experiments were performed at 25 °C, at a power reference setting of 6 l cal/s, with stirring at 1000 rpm. Data analysis was carried out using the Origin v.7 software (MicroCal) and fitted using the ‘one set of sites’ model. The reaction cell contained a 25 lM or 50 lM solution of PLB1–23 in
10 mM Tris, 1 mM EDTA, pH 7.4. Solutions of oligosaccharides were prepared in the same buffer at concentrations of 250 lM, and injected via the syringe. Titrations were carried out at intervals up to 2 min in 1 ll aliquots following an initial discard aliquot of 0.3 ll. The heat of dilution was determined in control experiments titrating oligosaccharides into a buffer solution minus the peptide. Subtraction of the heat of dilution values from experimental values allows the determination of heat flow resulting from peptide binding. 2.5. Solid-state NMR Magic angle-spinning (MAS) 13C solid-state (SS) NMR experiments were performed using a Bruker Avance 400 spectrometer operating at a magnetic field of 9.3 Tesla. Experiments were carried out at 4 °C. Membrane pellets obtained by centrifugation were packed into a 4 mm zirconium rotor and rotated at the magic angle at 8 kHz, maintaining the spinning rate automatically to within ± 1 Hz. All experiments utilized an initial 4.0-ls 13C 90° excitation pulse length and TPPM proton decoupling [39] at a field of 85 kHz during signal acquisition and a 2-s recycle delay. 3. Results and discussion 3.1. Screening HDO-PLB interactions Earlier work showed that the water-soluble peptide PLB1-23, representing the cytoplasmic residues of PLB, interacts with the cytoplasmic face of SERCA in SR vesicles and reduces Vmax for
Fig. 2. Effect of HDOs on the inhibition of SERCA by the PLB cytoplasmic domain peptide PLB1–23. Rates of ATP hydrolysis by SERCA (at 1.6 lM Ca2+) in rabbit skeletal muscle membranes was measured at different concentrations of HDec (A), HHex (B) or HDi (C) in the presence of 75 lM PLB1–23 (hatched white bars) or in the absence of peptide (black bars). The three saccharides have a minimal effect on ATPase activity in the absence of the peptide. Activities are expressed as a percentage of the rate of ATP hydrolysis in the absence of HDO and peptide (3.58 lmol Pi/mg per min).
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calcium transport [25]. ITC measurements were conducted on PLB1–23 in aqueous solution titrated with the heparin-derived decasaccharide HDec, hexasaccharide HHex or disaccharide HDi. The ITC isotherms show that HDec binds to the peptide with an association constant Ka of 5.31 105 M1 and a binding enthalpy DH of 10.8 kcal/mol (Fig. 1A). The stoichiometry N of 0.27 indicates that each HDec molecule binds approximately three peptide molecules, presumably because of the large size and high formal charge of the saccharide (20 compared to +4 for the peptide). HHex also interacts with the peptide, but with a lower affinity Ka (2.72 105 M1) and total binding enthalphy (DH = 5.5 kcal/mol) (Fig. 1B). In this case the stoichiometry N is 0.40, indicating a lower binding capacity of the saccharide. Virtually no interaction between HDi and PLB1–23 is detected and reliable thermodynamic data could not be calculated from the data (Fig. 1C).
concentration of HHex is required to abolish the inhibition of SERCA by PLB1-23. The reason for this discrepancy is not clear, but may be related to the higher degrees of freedom of the water soluble peptide compared to full-length PLB, which is confined within a two-dimensional lipid matrix. Reconstitution of SERCA with a 10fold excess of the peptide PLB29-52 representing the PLB transmembrane domain results in a 20% reduction in the rate of ATP hydrolysis compared to SERCA alone under the same conditions used for full-length PLB (Fig. 3B, triangles). Addition of HHex up to 25 lM has no significant effect on ATPase activity in the presence of PLB29-52. This observation, taken together with the studies on PLB1-23, suggests that the dis-inhibitory effect of HHex on fulllength PLB is mediated through the PLB cytoplasmic domain.
3.2. Functional effects of HDOs
The interaction between HHex and uniformly 13C and 15N labelled PLB ([U-13C,15N]PLB) in DOPC membranes was examined by 13C MAS SSNMR. Fig. 4A shows one-dimensional MAS SSNMR spectra (at 4 °C) obtained with direct polarisation (DP) of the 13C nuclei (top) and with cross-polarisation (CP) from 1H to 13C (bottom). The CP-MAS spectrum is rather broad and contains little fine detail, but much sharper peaks are seen in the DP spectrum as
The HDOs were next screened for their effects on the inhibitory interaction between PLB1–23 and SERCA. HDOs were added at various concentrations to SERCA in a native membrane preparation and the rate of ATPase activity (at 1.6 lM Ca2+) was determined with and without 75 lV PLB1–23 (Fig. 2A–C). In the absence of HDO, 75 lV PLB1–23 reduced Vmax by 35% compared to the control in the absence of peptide as observed earlier [25]. After the addition of HDec activity recovered with increasing decasaccharide concentration and at 10 lV HDec the rate of hydrolysis was close to the level measured in the absence of peptide (Fig. 2A). Inhibition by PLB1–23 is also reversed by HHex but higher concentrations of the oligosaccharide (200 lV) are required to restore ATPase activity to uninhibited levels (Fig. 2B). Addition of heparin disaccharide (HDi) to concentrations of up to 400 lV has no effect on SERCA inhibition (Fig. 2C). NaCl or KCl at 4 mM (the approximate sodium and potassium content of the HDOs) had no effect on ATPase activity in the presence of 75 lV PLB1–23 or in the absence of peptide (data not presented). The effectiveness of the HDOs in dis-inhibiting SERCA thus parallels the affinity of HDOs for PLB1–23. HHex was investigated for its functional effect on the interaction between full-length PLB and SERCA reconstituted into DOPC membranes. Calcium-dependent activity curves for SERCA, normalised to Vmax, are shifted to the right in the presence of PLB signifying a reduction in the calcium affinity of the enzyme [34]. A normalised plot of the ATPase activity of SERCA reconstituted into DOPC in the absence of PLB shows calcium-dependent hydrolysis of ATP with half maximal rate (KCa) at 0.34 lM Ca2+ and Vmax at approximately 1.6 lM Ca2+ (Fig. 3A). With PLB in fivefold molar excess, KCa increases to 0.92 lM signifying a reduction in the calcium affinity of the enzyme, following the trend reported previously. Addition of 25 lM HHex to the PLB/SERCA membranes did not have a significant effect on the calcium affinity of the enzyme either in the presence of PLB (Fig. 3A) or in the absence of PLB (not presented). We next investigated whether HHex affects Vmax for SERCA when reconstituted alone or with PLB. Using the standard reconstitution procedure [35] the recovery of active SERCA in the presence of PLB can be highly variable and comparisons between the absolute values of Vmax from preparation to preparation must be treated with caution. It is valid to draw conclusions about the trend in Vmax in response to the titration of an external agent, however. With a 10-fold molar excess of PLB addition of 25 lM HHex partially relieves the inhibition of SERCA and increases Vmax to approximately 70% of the rate for SERCA alone (Fig. 3B, filled circles). At a lower (5:1) PLB:SERCA ratio, Vmax in the absence of saccharide is reduced to 80% of SERCA alone, but increases to almost 100% of SERCA when 25 lM HHex is added to the membranes (Fig. 3B, open circles). Recalling Fig. 2B, it is noted that a much higher (200 lM)
3.3. Detection of HDO-PLB interactions using solid-state NMR
Fig. 3. The ATPase activity of SERCA reconstituted into DOPC membranes at a lipid:SERCA molar ratio of 160:1. (A) Calcium dependence of ATP hydrolysis by SERCA reconstituted into DOPC membranes at a lipid:protein molar ratio of 160:1. Activation of ATP hydrolysis by Ca2+ is shown for SERCA alone in the absence of peptide or HDO (triangles), for 5:1 PLB:SERCA (squares) and for 5:1 PLB:SERCA in the presence of 25 lM HHex (circles). (B) The effect of HHex on maximal hydrolytic activity of SERCA reconstituted alone (squares), with full length PLB at PLB:SERCA ratios of 5:1 (open circles) or 10:1 (filled circles) or with PLB29–52 (open triangles). Activities are presented as percentage Vmax for reconstituted SERCA in the absence of HDO and PLB peptide (2.28 lmol Pi/mg per min). For each set of data, mean values and standard errors are given for measurements on three individual reconstituted samples.
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Fig. 4. 13C MAS SSNMR spectra of uniformly 13C and 15N labelled PLB incorporated into DOPC membranes (lipid:peptide molar ratio of 10:1). (A) Full spectra obtained with direct polarisation (DP) and cross-polarisation (CP) in the absence of oligosaccharide. (B) Regions of DP spectra obtained for PLB alone (top) and in the presence of HHex at oligosaccharide:PLB molar ratios of 0.5:1, 1:1 and 2:1. The peak assigned to the guanidinium carbon of arginine residues is labelled Rf. Each spectrum is the result of accumulating between 80 000 and 120 000 transients.
reported previously [40]. DP detects the signals for all regions including the dynamic cytoplasmic region whereas CP favours detection of signals for the motionally- and conformationally- constrained transmembrane region. The DP spectrum is therefore more suitable for observing interactions between HHex and the cytoplasmic region of PLB. Fig. 4B shows a series of DP-MAS spectra of [U-13C,15N]PLB titrated with HHex in a saccharide:protein molar ratio of 0:5:1, 1:1 and 2:1. The high-field region of the spectrum is very crowded with signals from the lipid and labelled protein (Fig. 4B, right). It is not possible to attribute any changes in this region to specific amino acids or residues at this stage because of the complexity and overlap in the spectrum. Multidimensional spectroscopy will be required for a more detailed analysis of this region. The low-field region is less crowded and dominated by peaks for the backbone amide carbonyl groups around 170– 180 ppm (Fig. 4C, left). These also become slightly broader with increasing HHex concentration. A small peak for the arginine guanidinium carbon(s) (Rf) is well separated from the other peaks and shows a movement up-field from 160.0 ppm in the absence of HHex to 158.4 ppm at a HHex:PLB ratio of 2:1. These changes imply that HHex interacts with the PLB cytoplasmic domain around one or more of the four arginine residues in the PLB cytoplasmic
domain. Although an interaction between polyanionic HHex and cationic arginine side groups is not surprising, these results strengthen the argument that HDOs associate with the cytoplasmic domain of full-length PLB in a membrane environment. These experiments also demonstrate that SSNMR is sensitive to ligand binding to the PLB cytoplasmic domain. 4. Conclusions HDOs serve as a useful research tool for the dual purpose of investigating the nature of the PLB-SERCA interaction and for assessing the potential of disrupting this interaction to stimulate calcium uptake by the SR. It is generally agreed that the PLB transmembrane domain lowers the affinity of SERCA for calcium [20,21], but whether the PLB cytoplasmic domain enhances this effect or plays a secondary role in regulating the maximal activity of SERCA has been more difficult to establish. Progress has been hampered by the problems in obtaining functionally reproducible preparations of SERCA with PLB, with which to make reliable comparisons. Here we show that heparin-derived oligosaccharides, and in particular HHex, can interact with the cytoplasmic domain of PLB and enhance maximal levels of SERCA activity in a
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well-defined membrane environment. HHex has no significant effect on the calcium affinity of SERCA. Taken together, these results imply that the PLB cytoplasmic domain regulates maximal SERCA activity at high calcium concentrations, but not KCa, which is regulated by the PLB transmembrane domain. Although HDOs are not suitable as drug candidates for several reasons, this work suggests that it is feasible to target the cytoplasmic region of PLB with other, more drug-like molecules in order to disrupt the PLB-SERCA interaction and enhance SERCA activity. Acknowledgments The British Heart Foundation is acknowledged for financial support of this work (Project Grant PG/07/069/23357). The authors thank Prof. Mikael Esmann (University of Aarhus) for his constructive comments on this manuscript. References [1] J. Fujii, A. Ueno, K. Kitano, et al., Clin. Invest. 79 (1987) 301–304. [2] M. Tada, M.J. Inui, J. Mol. Cell. Cardiol. 15 (1983) 565–575. [3] J.E. Mahaney, J.M. Autry, L.R. Jones, Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: new insights on Ca-ATPase regulation by phospholamban, Biophys. J. 78 (2000) 1306–1323. [4] J.M. Autry, L.R. Jones, Functional co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation, J. Biol. Chem. 272 (1997) 15872–15880. [5] M. Meyer, W. Schillinger, B. Pieske, et al., Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy, Circulation 92 (1995) 778– 784. [6] K.L. Koss, E.G. Kranias, Phospholamban: a prominent regulator of myocardial contractility, Circ. Res. 79 (1996) 1059–1063. [7] R.H.G. Schwinger, G. Munch, B. Bolck, et al., Reduced Ca2+-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation, J. Mol. Cell. Cardiol. 31 (1999) 479–491. [8] A.G. Brittsan, A.N. Carr, A.G. Schmidt, E.G. Kranias, Maximal inhibition of SERCA2 Ca2+ affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban, J. Biol. Chem. 275 (2000) 12129–12135. [9] G. Hasenfuss, H. Reinecke, R. Studer, et al., Relation between myocardialfunction and expression of sarcoplasmic-reticulum Ca2+-Atpase in failing and nonfailing human myocardium, Circ. Res. 75 (1994) 434–442. [10] I.A. Hobaim, B. O’Rourke, Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure, Circulation 103 (2001) 1577–1584. [11] H. Kubo, K.B. Margulies, V. Piacentino, et al., Patients with end-stage congestive heart failure treated with beta-adrenergic receptor antagonists have improved ventricular myocyte calcium regulatory protein abundance, Circulation 104 (2001) 1012–1018. [12] M. Lindner, E. Erdmann, D.J. Beuckelmann, Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure, J. Mol. Cell. Cardiol. 30 (1998) 743–749. [13] A. Schmidt, I. Edes, E.G. Kranias, Phospholamban: a promising therapeutic target in heart failure? Cardiovasc. Drugs Ther. 15 (2001) 387–396. [14] I. BerrebiBertrand, P. Lahouratate, V. Lahouratate, et al., Mechanism of action of sarcoplasmic reticulum calcium-uptake activators – discrimination between sarco(endo)plasmic reticulum Ca2+ ATPase and phospholamban interaction, Eur. J. Biochem. 247 (1997) 801–809. [15] E. McKenna, J.S. Smith, K.E. Coll, et al., Dissociation of phospholamban regulation of cardiac sarcoplasmic reticulum Ca2+ ATPase by quercetin, J. Biol. Chem. 271 (1996) 24517–24525. [16] M. Chiesi, R. Schwaller, Reversal of phospholamban induced-inhibition of cardiac sarcoplasmic-reticulum Ca2+-ATPase by tannin, Biochem. Biophys. Res. Comm. 202 (1994) 1668–1673. [17] Y. Kimura, M. Inui, M. Kadoma, et al., Effects of monoclonal-antibody against phospholamban on calcium-pump ATPase of cardiac sarcoplasmic-reticulum, J. Mol. Cell. Cardiol. 23 (1991) 1223–1230.
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