Functional Co-expression of the Canine Cardiac Ca2+ Pump and Phospholamban in Spodoptera frugiperda (Sf21) Cells Reveals New Insights on ATPase Regulation*

(Received for publication, March 20, 1997, and in revised form, April 8, 1997)

Joseph M. Autry and Larry R. Jones Dagger

From the Department of Medicine and the Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

The utility of the baculovirus cell expression system for investigating Ca2+-ATPase and phospholamban regulatory interactions was examined. cDNA encoding the canine cardiac sarco(endo)plasmic Ca2+-ATPase pump (SERCA2a) was cloned for the first time and expressed in the presence and absence of phospholamban in Spodoptera frugiperda (Sf21) insect cells. The recombinant Ca2+ pump was produced in high yield, contributing 20% of the total membrane protein in Sf21 microsomes. At least 70% of the expressed pumps were active. Co-expression of wild-type, pentameric phospholamban with the Ca2+-ATPase decreased the apparent affinity of the ATPase for Ca2+, but had no effect on the maximum velocity of the enzyme, similar to phospholamban's action in cardiac sarcoplasmic reticulum vesicles. To investigate the importance of the oligomeric structure of phospholamban in ATPase regulation, SERCA2a was co-expressed with a monomeric mutant of phospholamban, in which leucine residue 37 was changed to alanine. Surprisingly, monomeric phospholamban suppressed SERCA2a Ca2+ affinity more strongly than did wild-type phospholamban, demonstrating that the pentamer is not essential for Ca2+ pump inhibition and that the monomer is the more active species. To test if phospholamban functions as a Ca2+ channel, Sf21 microsomes expressing either SERCA2a or SERCA2a plus phospholamban were actively loaded with Ca2+ and then assayed for unidirectional 45Ca2+ efflux. No evidence for a Ca2+ channel activity of phospholamban was obtained. We conclude that the phospholamban monomer is an important regulatory component inhibiting SERCA2a in cardiac sarcoplasmic reticulum membranes, and that the channel activity of phospholamban previously observed in planar bilayers is not involved in the mechanism of ATPase regulation.


INTRODUCTION

Phospholamban is a pentameric transmembrane phosphoprotein regulator of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum (1, 2). In the dephosphorylated state, phospholamban inhibits the Ca2+ pump by decreasing the apparent affinity of the ATPase for Ca2+ (3, 4). Inhibition of the Ca2+ pump is relieved by phosphorylation of phospholamban at serine 16 or threonine 17 or by the binding of a phospholamban monoclonal antibody to this cytoplasmic phosphorylation domain, resulting in a substantial increase in Ca2+ transport into cardiac sarcoplasmic reticulum vesicles at low ionized Ca2+ concentration (5-7). Purified phospholamban also forms Ca2+ channels in lipid bilayers (8), but the functional role of this channel activity is ill defined (9). The physiological importance of phospholamban is demonstrated by recent work with cardiomyocytes (7) and phospholamban knockout mice (10), where it was shown that ablation of phospholamban regulatory function greatly augments the intracellular Ca2+ transient and myocardial contractility, and at the same time attenuates the cardiac response to beta -adrenergic agents such as isoproterenol.

To understand the molecular mechanism of phospholamban regulation, several mammalian cell expression systems have recently been developed in which phospholamban and the Ca2+-ATPase are co-expressed after transient transfection of cells with plasmid expression vectors (11-13). These studies have provided useful insights into the mechanism of phospholamban inhibition, including identification of some of the amino acid residues of phospholamban required for Ca2+ pump regulation (14). However, the cell expression systems used to date have several drawbacks, including low transfection efficiencies, low expression levels, and low membrane yields, making detailed biochemical and kinetic characterizations with use of these systems difficult (11-13).

In the work described here, we have examined the utility of the baculovirus cell expression system for investigating phospholamban and Ca2+-ATPase regulatory interactions. An important strength of this system is that virtually all of the insect cells are infected using viral expression vectors, ensuring that very high levels of foreign protein expression are achieved (15). We recently reported on the use of this system for the expression and mass purification of canine cardiac phospholamban and several of its protein mutants from Sf211 cells (16, 17). The purified, recombinant protein was successfully reconstituted into proteoliposomes to study its secondary structure (18) and oligomeric organization in the lipid bilayer (19). Successful co-reconstitution with Ca2+ pumps purified from rabbit skeletal muscle (16) and canine myocardium (20) was also achieved. Here, we report on the further development of this system for functional co-expression of phospholamban with the canine cardiac Ca2+ pump (SERCA2a). Microsomes isolated from infected Sf21 cells exhibit high levels of ATP hydrolysis and active Ca2+ transport, and, furthermore, cardiac-like coupling between phospholamban and SERCA2a is retained. With the baculovirus system, we also demonstrate that a monomer-forming mutant of phospholamban (17, 19), unexpectedly, is a stronger inhibitor of SERCA2a activity than is the pentamer, suggesting that the monomer may be the key molecular species regulating the Ca2+ pump in sarcoplasmic reticulum membranes. No evidence for a Ca2+ channel activity of phospholamban was obtained.


EXPERIMENTAL PROCEDURES

Materials

[gamma -32P]ATP, 45CaCl2, and 125I-labeled protein A were purchased from DuPont NEN. Nucleic acid-synthesizing and -modifying enzymes were obtained from Promega. Sf21 cells were purchased from Invitrogen, and the BaculoGoldTM system was obtained from Pharmingen.

Cloning Canine SERCA2a cDNA

A canine cardiac lambda gt10 cDNA library (21) was screened in duplicate with 5' end-labeled oligonucleotide probes corresponding to base pairs 1-33 and 2935-2970 of rabbit SERCA2a cDNA (22). SERCA2a cDNA encoding the full-length canine cardiac Ca2+ pump was excised from lambda  phage genomic DNA using EcoRI and subcloned into the EcoRI polylinker site of pBluescript. The SERCA2a cDNA clone contained approximately 250 base pairs of 5'-untranslated sequence, a 2991-base pair open reading frame, and approximately 850 base pairs of 3'-untranslated sequence, which included a 39-base pair poly(A) tail. The entire protein coding region of the canine SERCA2a cDNA was sequenced in both directions by the dideoxy method (21).

Expression of SERCA2a and Phospholamban in Sf21 Cells

Wild-type phospholamban and L37A-PLB were expressed in Sf21 insect cells as recently described (16, 17). To express canine SERCA2a in Sf21 cells, the XmaI insert encoding the Ca2+ pump was excised from pBluescript and inserted into the XmaI site of the baculovirus transfer vector pVL1393 (15). This XmaI insert contained the entire protein coding region of the Ca2+ pump, 90 base pairs of 5'-untranslated sequence, and the entire 3'-untranslated region including 11 base pairs excised from the pBluescript polylinker. Recombinant baculovirus containing the canine SERCA2a cDNA was obtained after co-transfection of Sf21 cells with pVL1393 and linearized baculovirus DNA using the BaculoGoldTM system (17). The Ca2+ pump and phospholamban were expressed in Sf21 insect cells grown in suspension (1.5 × 106 cells/ml) at 27 °C in Grace's insect cell medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Atlanta Biologicals), 0.1% Pluronic F-68 (Life Technologies, Inc.), 50 µg/ml gentamicin, and 2.5 µg/ml amphotericin B. Microsomes were isolated from insect cells harvested 48 h after infection with baculoviruses. For expression of the Ca2+ pump alone, a multiplicity of infection of 10 (viruses per cell) was used. For co-expression of the Ca2+ pump and phospholamban, a multiplicity of infection of 15 was used for SERCA2a, and of 5 for wild-type phospholamban and L37A-PLB.

Isolation of Microsomes from Sf21 Cells

Virus-infected Sf21 cells in 600 ml of suspension (9 × 108 cells) were sedimented, washed twice with phosphate-buffered saline, and resuspended in 50 ml of medium containing 10 mM NaHCO3, 0.2 mM CaCl2, plus the following protease inhibitors: aprotinin (10 µg/ml), leupeptin (2 µg/ml), pepstatin A (1 µg/ml), and Pefabloc (0.1 mM), which were included throughout the entire preparative procedure. Cells were disrupted by N2 cavitation using a Parr Cell Disruption Bomb 4635 (Parr Instruments, Moline, IL), and cellular homogenates were diluted into an equal volume of ice-cold medium containing 500 mM sucrose, 300 mM KCl, 6 mM MgCl2, and 60 mM histidine (pH 7.4). Homogenates were centrifuged at 1000 × g for 20 min. Supernatants were recovered, diluted with 0.25 volume of 3 M KCl, and centrifuged at 10,000 × g for 20 min. Supernatants were again recovered and centrifuged at 100,000 × g for 30 min. Pellets were washed in 250 mM sucrose, 600 mM KCl, 3 mM MgCl2, 30 mM histidine (pH 7.4) and sedimented as before. Final pellets (Sf21 microsomes) were resuspended at approximately 5 mg/ml in 250 mM sucrose, 30 mM histidine (pH 7.4) and stored in small aliquots at -40 °C. The average yield per 600 ml of infection was 40 mg of microsomal protein.

Ca2+ Uptake and Ca2+-ATPase Assays

Ca2+ uptake was measured radiometrically using the Millipore filtration technique, and ATPase activity was determined by measuring release of Pi from ATP colorimetrically (23). Assays were conducted at 37 °C with 100 µg of Sf21 microsomal protein in 1 ml of reaction medium consisting of 50 mM MOPS (pH 7.0), 100 mM KCl, 3 mM MgCl2, 2.7 mM ATP, 10 mM oxalate, 5 mM NaN3, with 2 mM EGTA and 0.20 to 1.8 mM CaCl2 (containing tracer amounts of radioactive 45Ca) to give the desired ionized Ca2+ concentrations, determined as described previously (23). Ca2+ uptake was terminated at selected times by vacuum filtering 10 µg of membrane protein through glass-fiber filters, which were washed twice with 5 ml of 150 mM NaCl. 45Ca2+ accumulated inside membrane vesicles was monitored by liquid scintillation counting. ATPase activity was terminated at selected times by adding 50 µl of reaction medium to 300 µl of ice-cold 25 mM EDTA (pH 8.0). Pi liberated was monitored by adding 2 ml of malachite green reagent and reading the absorbance at 660 nm (23). Basal ATPase activity, measured in the absence of added Ca2+ with 2 mM EGTA, was subtracted from total ATPase activity to yield the Ca2+-ATPase activities reported. Prior to initiating assays, microsomes were preincubated for 20 min on ice with or without affinity-purified anti-phospholamban monoclonal antibody 2D12, at a membrane protein to antibody ratio of 1:1 (4, 23).

45Ca2+ Efflux Assay

45Ca2+ efflux from Sf21 microsomes was measured after active Ca2+ loading in the presence of 25 mM potassium phosphate, a Ca2+-precipitating agent that allows ready exchange of accumulated 45Ca2+ (24). Ca2+ loading was performed at 37 °C with 100 µg of microsomal protein with or without the phospholamban monoclonal antibody in 10 ml of reaction medium containing 50 mM histidine (pH 7.0), 100 mM KCl, 3 mM MgCl2, 2.7 mM ATP, 5 mM NaN3, plus 25 mM potassium phosphate and 50 µM added 45Ca2+. After membrane vesicles had accumulated approximately 700 nmol of Ca2+/mg of protein, efflux of radioactive Ca2+ from the vesicles was initiated by two methods: under conditions in which Ca2+ uptake was stopped completely by addition of 2 mM EGTA, and under conditions in which Ca2+-ATPase remained active, by addition of 9 mM nonradioactive Ca2+ and 10 mM EGTA (2 µM ionized Ca2+ concentration) to give a 180-fold dilution of the radioactive Ca2+ in the medium. Ca2+ load remaining within the microsomes was monitored at selected times by vacuum filtering 10 µg of protein through glass-fiber filters, which were washed once with 5 ml of 150 mM NaCl. As a control, calcium efflux was also initiated by adding 3 µg/ml of the Ca2+ ionophore A23187.

SDS-PAGE and Immunoblotting

Prior to electrophoresis, microsomal proteins were solubilized at 37 °C for 5 min in dissociation medium that contained 62.5 mM Tris (pH 6.8), 5% glycerol, 5% SDS, 5 mM dithiothreitol, and 0.0025% bromphenol blue. SDS-PAGE was conducted by the method of Laemmli (25) using 7.5-15% polyacrylamide (see Fig. 2) or by the method of Porzio and Pearson (26) using 8% polyacrylamide (see Fig. 6) (27). Gels were stained with Coomassie Blue, or proteins were transferred to nitrocellulose for immunoblotting. Nitrocellulose sheets were probed with anti-SERCA2a monoclonal antibody 2A7-A1 for detection of cardiac Ca2+ pumps or with monoclonal antibody 2D12 for detection of phospholamban (28). Antibody binding proteins were visualized using 125I-protein A followed by autoradiography. Labeling intensities were quantified with use of a GS-250 molecular imager (Bio-Rad).


Fig. 2. SDS-PAGE and immunoblotting of canine SERCA2a and phospholamban expressed in Sf21 microsomes. Sf21 microsomes from wild-type (WTV), SERCA2a (SERCA), and SERCA2a plus phospholamban (SERCA/PLB) baculovirus-infected cells, as well as canine cardiac sarcoplasmic reticulum vesicles (CSR), were subjected to SDS-PAGE and immunoblotting. Panel A shows a Coomassie Blue-stained gel (50 µg of protein/lane), and panel B shows a corresponding immunoblot (5 µg of protein/lane) developed with SERCA2a (upper) and phospholamban (lower) monoclonal antibodies. ± Boil indicates whether microsomes were boiled in SDS prior to PAGE. ATPase, SERCA2a; PLBP, phospholamban pentamer; PLBM, phospholamban monomer.
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Fig. 6. Immunoblot showing co-expression of monomeric phospholamban and Ca2+ pump. 10 µg of microsomes from Sf21 cells expressing monomeric phospholamban and the Ca2+ pump (SERCA/L37A) were subjected to SDS-PAGE and immunoblotting, along with 10 µg of microsomes expressing wild-type phospholamban and the Ca2+ pump (SERCA/PLB). Blots were probed with antibodies as described in Fig. 2. Note that boiling in SDS (± Boil) was required to dissociate the wild-type phospholamban pentamer into monomers, whereas L37A-PLB was entirely monomeric without boiling in SDS.
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Miscellaneous Methods

Procedure I canine cardiac microsomes enriched in sarcoplasmic reticulum were isolated as described previously (27). Purification of recombinant canine phospholamban from Sf21 cells was conducted as described elsewhere (16, 17). The Ca2+-ATPase was purified from canine cardiac sarcoplasmic reticulum vesicles by 2A7-A1 monoclonal antibody affinity chromatography, using the methodology described by Reddy et al. (16). Protein concentrations were determined by the Lowry method using bovine serum albumin as the standard.


RESULTS

Cloning of Canine Cardiac SERCA2a cDNA

Most of the detailed biochemical work characterizing phospholamban and Ca2+-ATPase regulatory interactions has been conducted with sarcoplasmic reticulum preparations isolated from dog heart (1, 3). These well characterized preparations are enriched in the two proteins and are prepared in relatively high yield (27), which has greatly facilitated the biochemical studies. Ironically, however, the canine cardiac Ca2+ pump has not been cloned or expressed to date. Therefore, to express canine SERCA2a in Sf21 cells, we first had to obtain the cDNA clone for this isoform. The SERCA2a cDNA clone was isolated and found to contain a 2991 base pair open reading frame encoding a polypeptide of 997 amino acid residues with a calculated molecular weight of 109,619. The deduced amino acid sequence of dog SERCA2a displays 98-99% identity with mammalian SERCA2a Ca2+-ATPases cloned from human (29), rabbit (22), cat (30), rat (31), or pig (32) species, and 95% identity with avian SERCA2a Ca2+-ATPase cloned from chicken (33) (Fig. 1).



Fig. 1. Deduced amino acid sequence of canine cardiac Ca2+ pump. The canine SERCA2a amino acid sequence deduced from the cDNA sequence is reported in single-letter amino acid code. SERCA2a sequences from human (29), rabbit (22), cat (30), rat (31), pig (32), and chicken (33) species are shown for comparison. Identical residues are indicated by hyphens and the asterisk denotes the stop codon. Residue numbers are in the right margin. The DNA sequence has been submitted to GenBankTM (accession no. [GenBank]).
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Expression and Functional Assay of SERCA2a in Sf21 Cells

Microsomes were isolated from Sf21 insect cells infected with the SERCA2a-encoding baculovirus. Separation of the microsomal membrane proteins by SDS-PAGE followed by Coomassie Blue staining revealed the abundant expression of an 110-kDa protein, which migrated with identical mobility as the Ca2+ pump in canine cardiac sarcoplasmic reticulum vesicles (Fig. 2A). The expressed protein was not visible in microsomes obtained from control (i.e. wild-type virus-infected) Sf21 cells. The identity of the expressed protein as the Ca2+ pump was confirmed by immunoblotting with a monoclonal antibody recognizing SERCA2a (28) (Fig. 2B), demonstrating that the recombinant Ca2+ pump is expressed in Sf21 cell microsomes at levels approaching those of the native Ca2+-ATPase in cardiac sarcoplasmic reticulum vesicles. Quantitative immunoblotting using the purified canine cardiac Ca2+-ATPase as a standard revealed that the recombinant Ca2+ pump accounted for 20-25% of the total protein of Sf21 microsomes (data not shown), similar to Ca2+ pump content reported in cardiac sarcoplasmic reticulum vesicles (34). Thus Sf21 cells infected with the SERCA2a-encoding baculovirus readily express the full-length Ca2+ pump.

To confirm the functional integrity of recombinant SERCA2a, Ca2+ transport assays were conducted with microsomal membranes isolated after N2 cavitation of Sf21 cells. When assayed at the saturating Ca2+ concentration of 1 µM, the Ca2+ transport rate was increased 10-fold in Sf21 microsomes expressing SERCA2a compared with control microsomes, confirming that the recombinant Ca2+ pump was indeed functional (Fig. 3A). The Ca2+ transport rate and the maximal level of Ca2+ accumulation achieved was similar to that previously observed with use of canine cardiac microsomes (46). Ca2+ activation of Ca2+ transport was found to be half-maximal at approximately 0.1 µM ionized Ca2+ (Fig. 3B), demonstrating that SERCA2a displays a high Ca2+ affinity when expressed in the absence of phospholamban (Fig. 3B; see Table II). This apparent Ca2+ affinity of SERCA2a is similar to that exhibited by the rabbit skeletal muscle Ca2+ pump when expressed in Sf9 cells and assayed under similar conditions (35). As expected, a phospholamban monoclonal antibody (4, 36) had no effect on Ca2+ transport when SERCA2a was expressed by itself (Fig. 3, A and B). Note that at the lower Ca2+ concentrations tested (<= 0.1 µM), microsomes containing SERCA2a accumulated at least 20 times more Ca2+ than did control microsomes, due to the negligible activity of the endogenous Ca2+ pump in Sf21 cells at low Ca2+ concentration (Fig. 3B). Failure of the endogenous Ca2+ pump in insect cells to react with SERCA1 (35)- or SERCA2-specific antibodies coupled with its low apparent affinity for Ca2+ suggests that it may be the product of the SERCA3 gene (37), but this remains to be tested. In control experiments we observed that the low background level of Ca2+ uptake, as well as the Ca2+ uptake attributable to SERCA2a expression, was completely inhibited by thapsigargin (data not shown).


Fig. 3. Ca2+ uptake by Sf21 microsomes expressing canine SERCA2a. Panel A shows a time course of Ca2+ uptake measured at 1 µM ionized Ca2+, and panel B shows the ionized Ca2+ dependence of Ca2+ transport, determined at 8 min of Ca2+ uptake. Sf21 microsomes used were from cells infected with SERCA2a-encoding baculovirus (SERCA) or wild-type baculovirus (WTV). +Ab (filled symbols) denotes microsomes pretreated with phospholamban monoclonal antibody 2D12.
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Table II. Phospholamban regulation of SERCA2a Ca2+ affinity in Sf21 microsomes

The ionized Ca2+ concentrations giving half-maximal stimulation of Ca2+-ATPase and Ca2+ uptake activities (KCa values) in Sf21 microsomes are reported. Microsomes expressed SERCA2a alone, SERCA2a plus wild-type phospholamban (WT-PLB), or SERCA2a plus L37A-PLB. Assays were conducted in the presence and absence of phospholamban monoclonal antibody 2D12 (±Ab) as described under "Experimental Procedures." Results were obtained from three separate microsomal preparations for each condition and are the means ± S.E.

Protein expressed KCa values
Ca2+-ATPase
Ca2+ uptake
(-)Ab (+)Ab (-)Ab (+)Ab

nM
SERCA2a 105  ± 10 106  ± 8 95  ± 11 101  ± 10
SERCA2a + WT-PLB 224  ± 44 125  ± 19 168  ± 13 123  ± 6
SERCA2a + L37A-PLB 537  ± 77 185  ± 24 400  ± 30 154  ± 13

Functional Coupling between Recombinant SERCA2a and Phospholamban Assayed by Ca2+ Uptake

Previously we demonstrated that baculovirus-infected Sf21 cells express recombinant canine cardiac phospholamban with high efficiency (16, 17). To test for functional coupling between canine cardiac SERCA2a and phospholamban we therefore co-infected Sf21 cells with viruses carrying the cDNAs encoding both proteins and isolated membranes for Ca2+ transport assays. Fig. 2 shows that co-expression of phospholamban with SERCA2a in Sf21 cells produced similar levels of the two proteins in insect cell microsomes compared with the protein levels detected in canine cardiac sarcoplasmic reticulum vesicles. Phospholamban was visible in insect cell microsomes either by Coomassie Blue staining (Fig. 2A) or by immunoblot analysis (Fig. 2B) and exhibited the characteristic conversion from the pentameric to monomeric forms (2) by boiling in SDS. Control Sf21 microsomes (WTV) contained no detectable phospholamban.

To test for functional coupling between SERCA2a and phospholamban in Sf21 microsomes, Ca2+ transport assays were conducted at low (30 nM) and high (1 µM) Ca2+ concentrations in the presence and absence of anti-phospholamban monoclonal antibody 2D12, which blocks the inhibitory interaction between phospholamban and the cardiac Ca2+-ATPase (4, 7, 23, 36). At low Ca2+ concentration, Ca2+ transport by Sf21 microsomes containing both proteins was stimulated 8-fold by addition of the phospholamban monoclonal antibody (Fig. 4A). However, at high Ca2+ concentration, Ca2+ transport by the same microsomes was unaffected by the antibody (Fig. 4B). Since the same monoclonal antibody increased Ca2+ uptake by canine cardiac sarcoplasmic reticulum vesicles approximately 10-fold at a low Ca2+ concentration, but had no effect at saturating Ca2+ concentration (4, 23), we conclude that the recombinant SERCA2a Ca2+ pump is tightly coupled to phospholamban in Sf21 microsomes in a similar fashion to that in cardiac sarcoplasmic reticulum vesicles.


Fig. 4. Ca2+ uptake by Sf21 microsomes co-expressing canine SERCA2a and phospholamban. Panel A shows results of Ca2+ uptake measured at 30 nM ionized Ca2+ concentration, and panel B shows results of Ca2+ uptake measured at 1 µM ionized Ca2+ concentration. Sf21 microsomes were obtained from cells co-infected with SERCA2a and phospholamban-encoding baculoviruses (SERCA/PLB) or wild-type baculovirus (WTV). Filled symbols denotes microsomes pretreated with phospholamban monoclonal antibody (+Ab).
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Ca2+ Affinities Monitored by ATP Hydrolysis

It has proven problematical to measure ATP hydrolysis by recombinantly expressed Ca2+ pumps in previous studies, due to the low protein expression levels obtained and the interference by endogenous ATPase activities present in the microsomal preparations (38, 39). The abundant expression of canine SERCA2a in Sf21 cells suggested that it would be possible to measure ATP hydrolysis by this recombinant Ca2+ pump, which is a more direct method than assay of Ca2+ transport for estimating apparent Ca2+ affinities. Table I demonstrates that Ca2+-independent (basal) ATPase activity was very low in Sf21 microsomes (~200 nmol of Pi/mg of protein/8 min). In membranes containing SERCA2a, the Ca2+-dependent ATPase activity was at least 20 times greater than the basal ATPase activity when measured at a saturating Ca2+ concentration. Even at the lowest ionized Ca2+ concentration tested (30 nM), ATP hydrolysis by SERCA2a was still four times greater than basal ATPase activity (Table I). ATP hydrolysis by the endogenous Ca2+ pump was barely detectable (WTV) compared with that exhibited by SERCA2a. The maximal Ca2+-ATPase activity reported in Table I for SERCA2a expressed in Sf21 microsomes (33 µmol of Pi/mg of protein/h) is 55 times greater than the ATPase activity recently reported for SERCA2a expressed in HEK-293 membranes (0.6 µmol of Pi/mg of protein/h) (40).

Table I. Ca2+-ATPase activity of SERCA2a expressed in Sf21 microsomes

Ca2+-ATPase activity was measured in microsomes isolated from Sf21 cells infected with wild-type baculovirus or baculovirus encoding canine SERCA2a. Pi release was monitored colorimetrically as described under "Experimental Procedures" and was found to be linear with time for at least 20 min. ATP hydrolysis at 8 min is reported.

[Ca2+] ATPase activity
SERCA2a WTV

nM nmol Pi/mg protein/8 min
0 207 204
30 1025 222
47 1455 257
67 1992 300
89 2422 335
115 2760 361
179 3636 430
328 4471 544
625 5008 661
2412 4851 727

Plots depicting Ca2+ activation of ATP hydrolysis by SERCA2a, expressed in the presence and absence of phospholamban, are shown in Fig. 5. SERCA2a expressed alone had a high apparent Ca2+ affinity (KCa value = 105 ± 10 nM), which was unaffected by the phospholamban antibody (Fig. 5A and Table II). Co-expression of phospholamban with SERCA2a decreased the Ca2+ affinity by a factor of two, but this decrease in Ca2+ affinity was removed by the phospholamban monoclonal antibody, shifting the Ca2+ activation curve to the left (Fig. 5B and Table II). At the saturating Ca2+ concentration of 2.4 µM, the antibody had no effect on ATP hydrolysis (Fig. 5B). Thus phospholamban primarily decreases the Ca2+ affinity of the pump, whether measured by assay of Ca2+ transport or by ATP hydrolysis, but has no effect on the Vmax of the enzyme. It should be pointed out that the Ca2+-ATPase activities determined in these studies were about 2-3 times greater than the Ca2+ transport rates measured, giving apparent coupling coefficients (Ca2+ ions transported per ATP molecule hydrolyzed) of approximately 0.3-0.5. Similar low coupling coefficients are obtained with the use of canine cardiac sarcoplasmic reticulum vesicles, and are believed to be due to a significant proportion of leaky vesicles that hydrolyze ATP but are unable to retain accumulated Ca2+ (4, 23). We detected no differences in coupling coefficients between microsomes expressing SERCA2a alone, or microsomes expressing SERCA2a plus phospholamban.


Fig. 5. Phospholamban regulation of Ca2+-ATPase activity in Sf21 microsomes. Ca2+ activation of ATPase activity was measured in Sf21 microsomes from SERCA2a (SERCA)-infected cells (panel A) and SERCA2a plus phospholamban (SERCA/PLB) co-infected cells (panel B). Microsomes from wild-type virus (WTV) infected cells were also assayed. Microsomes pretreated with the phospholamban monoclonal antibody (+Ab) are denoted by the filled symbols. ATPase activities were determined at 8 min of incubation, and basal (Ca2+-independent) ATPase activities have been subtracted from the values reported.
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Co-expression of a Monomeric Mutant of Phospholamban with SERCA2a

L37A-PLB is monomeric in SDS solution (17) or when reconstituted in phospholipid membranes (19). Since L37A-PLB is essentially depolymerized in lipid membranes, we co-expressed it with SERCA2a in Sf21 cells to test if the phospholamban monomer is sufficient for inhibition of the ATPase. Fig. 6 is an immunoblot showing that L37A-PLB was entirely monomeric on SDS-PAGE when co-expressed with SERCA2a in Sf21 cell microsomes. The expression levels of L37A-PLB and the Ca2+ pump were similar to that achieved with co-expression of wild-type phospholamban and the pump (Fig. 6). Ca2+-transport assays conducted at 30 nM Ca2+ revealed that L37A-PLB is a potent inhibitor of SERCA2a, and that this inhibition is reversed by addition of the phospholamban monoclonal antibody (Fig. 7A). ATPase activity was measured to assess Ca2+ activation of SERCA2a when co-expressed with L37A-PLB (Fig. 7B). Like wild-type phospholamban, L37A-PLB shifted the Ca2+ activation curve to the right, but, remarkably, the extent of the shift in the KCa value was significantly greater with monomeric phospholamban (approximately 5-fold) than with wild-type phospholamban (approximately 2-fold) (Table II). The reduction in Ca2+ affinity by L37A-PLB was largely reversed by addition of the monoclonal antibody, and at the highest Ca2+ concentration tested, only a marginal effect of the antibody was noted (Fig. 7B). (At ionized Ca2+ concentrations greater than 10 µM, no effect of the antibody was observed.) Similar Ca2+ affinity shifts were observed when Ca2+ uptake assays were conducted instead of measuring ATP hydrolysis (Table II). These results demonstrate that monomeric phospholamban is actually a stronger inhibitor of the Ca2+ pump than is wild-type phospholamban. However, the basic mechanism of inhibition of SERCA2a by the two phospholambans is the same, in that the apparent KCa value is increased with little effect on the Vmax of the enzyme measured at saturating Ca2+ concentration.


Fig. 7. Phospholamban monomer effect on Ca2+ uptake and Ca2+-ATPase activities. Microsomes were isolated from Sf21 cells infected with SERCA2a and L37A-PLB encoding baculoviruses (SERCA/L37A) or wild-type baculovirus (WTV). Panel A shows a time course of Ca2+ uptake measured at 30 nM ionized Ca2+ concentration, and panel B shows the Ca2+ dependence of Ca2+-ATPase activity determined at 8 min of incubation. Phospholamban antibody was used as described in the legend to Fig. 3.
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45Ca2+ Efflux from Sf21 Microsomes Expressing Phospholamban

Purified phospholamban incorporated into planar lipid bilayers forms Ca2+ channels (8), and it has been proposed that Ca2+ efflux through phospholamban is involved in its mechanism of ATPase regulation (9). This hypothesis has been difficult to test with cardiac sarcoplasmic reticulum vesicles, due to the presence of other channels in these membranes and the lack of adequate control membranes that contain SERCA2a but no phospholamban. Here we tested for a Ca2+ efflux role for phospholamban by using Sf21 microsomes expressing SERCA2a alone, or microsomes expressing SERCA2a plus wild-type phospholamban. Microsomes were actively preloaded with 45Ca2+ in the presence of 25 mM phosphate, a Ca2+-precipitating agent that readily exchanges Ca2+ (24). 45Ca2+ efflux was then initiated under active transport conditions by diluting extravesicular 45Ca2+ with excess unlabeled Ca2+-EGTA buffer, or under conditions in which the Ca2+ pump was inactivated by chelating extravesicular Ca2+ completely with EGTA. When Ca2+ efflux was initiated by the unlabeled 40Ca2+ chase (Fig. 8A), no difference in the rate of efflux was detected between SERCA2a-containing microsomes and microsomes containing SERCA2a plus phospholamban. Measurement of 45Ca2+ efflux when the Ca2+ pump was not cycling, by chelation of all of the extravesicular Ca2+ with EGTA, also showed that co-expression of phospholamban with SERCA2a did not increase the Ca2+ efflux rate from microsomes (Fig. 8B). Addition of 3 µg/ml of the Ca2+ ionophore A23187 resulted in the rapid release of all of the accumulated Ca2+ under both conditions (Fig. 8, A and B), demonstrating that the Ca2+ inside the microsomes was readily releasable. Furthermore, the phospholamban monoclonal antibody had no significant effect on Ca2+ efflux under either efflux condition (Fig. 8, A and B). Therefore, phospholamban does not exhibit a significant Ca2+ channel activity in Sf21 microsomes when co-expressed with a functional SERCA2a Ca2+ pump.


Fig. 8. Ca2+ efflux from Sf21 microsomes. Microsomes were isolated from Sf21 cells expressing SERCA2a (SERCA) or SERCA2a plus phospholamban (SERCA/PLB). Microsomes were actively loaded with 45Ca2+ in the presence of 25 mM phosphate, and Ca2+ efflux was then initiated by cold Ca2+ chase (panel A) or by chelation of all of the extravesicular Ca2+ with EGTA (panel B), as described under "Experimental Procedures." Rapid Ca2+ efflux was also induced by addition of a Ca2+ ionophore (+A23187). +Ab denotes incubations conducted in the presence of the phospholamban monoclonal antibody.
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DISCUSSION

To understand the molecular mechanism of phospholamban regulation of the Ca2+ pump, an efficient expression system for these two proteins is desirable, which yields reasonable quantities of the recombinant proteins with functional interactions preserved. Here we have achieved this goal with the use of the baculovirus cell expression system. With this system, the SERCA2a ATPase contributes 20% of the total microsomal protein, a protein content comparable to that found in cardiac sarcoplasmic reticulum vesicles, and an expression level far greater than that previously reported (see below). Moreover, phospholamban co-expressed with SERCA2a is functional, producing regulatory effects identical to those observed with use of cardiac sarcoplasmic reticulum vesicles. Since preparation of microsomes from 600 ml of infected insect cells yields 40 mg of membrane protein (almost one-half the yield of sarcoplasmic reticulum vesicles from one dog heart), it is now possible to quickly and easily synthesize milligram quantities of the recombinant Ca2+ pump using the baculovirus system.

Previous studies investigating recombinant SERCA Ca2+ pumps resulted in levels of cellular protein expression much lower than those presently reported. For example, expression of SERCA1a in COS cells yielded 150 µg of microsomal protein containing 2 µg of Ca2+-ATPase (38). SERCA2a and SERCA3 Ca2+ pumps have been expressed at comparable levels (37), where microsomal membranes contained approximately 50-100 pmol of Ca2+ pump per mg of total protein, or about 1-2% Ca2+-ATPase. SERCA1a has also been expressed in yeast cells (yielding 0.6% of total membrane protein in secretory vesicles) (39) and in baculovirus-infected Sf9 cells (yielding 1-3% of total membrane protein in microsomes) (35). A drawback to some of these systems, however, is that Ca2+-ATPase activities could not be measured, due to the low expression levels achieved and interference from basal ATPase activities present in the microsomal preparations (38, 39). Additionally, in some of these studies a significant fraction of the recombinant Ca2+ pumps were apparently inactive (12, 41). As reported here, SERCA2a accounted for 20% or more of the total microsomal protein from infected Sf21 cells. Moreover, we estimate that 70-80% of the expressed Ca2+ pumps were active, relative to activity in cardiac sarcoplasmic reticulum membranes, by comparison of the ATPase hydrolysis rate to the amount of protein expressed determined by immunoblotting. Thus we have achieved expression levels of a highly active SERCA Ca2+ pump 10-20 times greater than those previously reported. In the study of Skerjanc et al. (35) the same baculovirus system was used to express the rabbit skeletal muscle Ca2+ pump in Sf9 cells, but the level of the recombinant protein obtained was about 10-fold lower than that presently reported. The higher expression level observed presently could be due to the use of Sf21 cells instead of Sf9 cells or simply due to the fact that canine SERCA2a expresses more efficiently in insect cells than does rabbit SERCA1a; however, we have not investigated either of these possibilities. Even with the lower expression level in insect cells achieved by Skerjanc et al. (35), it was still possible to measure direct binding of 45Ca2+ to a recombinant Ca2+ pump for the first time. Thus, we anticipate that Sf21 microsomes expressing canine SERCA2a will be a very useful system for future studies investigating detailed biochemical aspects of enzyme function.

Previously we demonstrated that Sf21 cells express canine cardiac phospholamban very effectively, allowing purification of milligram quantities of the protein (16, 17), and here we have shown that, when canine SERCA2a is co-expressed with phospholamban, the two proteins are functionally coupled. Both Ca2+-ATPase and Ca2+-uptake activities of Sf21 microsomes were stimulated manyfold at low ionized calcium concentration by the phospholamban monoclonal antibody, which produces the same effect as phosphorylation of phospholamban by protein kinases (7), but no effect of the antibody was noted at saturating Ca2+ concentration. Thus, use of the baculovirus cell expression system provides additional strong evidence that the main regulatory effect of phospholamban is on the apparent Ca2+ affinity of the Ca2+ pump, but not on the Vmax of the enzyme. A similar regulatory effect of phospholamban on the Ca2+ pump has been reported with use of canine cardiac sarcoplasmic reticulum vesicles (4, 6, 23, 42), with use of the purified and reconstituted proteins (16, 20), with use of the recombinant proteins expressed in HEK-293 membranes (43), and with use of phospholamban knockout mice (10). Although Kirchberger and co-workers (44) have recently challenged the idea that the main regulatory effect of phospholamban is on the KCa value for Ca2+ transport, most evidence concurs that dephosphorylated phospholamban strongly suppresses the Ca2+ affinity of the enzyme, with an insignificant effect on Vmax. The argument by Kirchberger and co-workers (44) that the 45Ca2+ uptake assay used by others (4, 6, 16, 43) artifactually reports a Ca2+ affinity change is negated by our results with ATPase activity measurements, where a similar KCa shift by phospholamban is observed by a completely independent and more direct method.

Use of the Sf21 system allowed us to test if the phospholamban pentamer is essential for SERCA2a regulation. To examine this issue, SERCA2a was co-expressed with L37A-PLB, which is monomeric on SDS-PAGE (17) and, more importantly, also when reconstituted in lipid membranes (19). Surprisingly, we observed that L37A-PLB was a more effective suppressor of SERCA2a Ca2+ affinity than wild-type phospholamban. Using an electron paramagnetic resonance technique, Cornea et al. (19) recently demonstrated that wild-type phospholamban exists in two physical states in the lipid bilayer; one state is composed of pentamers (80% of total phospholamban) and the other monomers (20% of total phospholamban), giving about an equimolar ratio of monomers to pentamers. These two states are in dynamic equilibrium, and phosphorylation of phospholamban by cAMP-dependent protein kinase shifts the equilibrium completely toward pentamers (19). Since we show here that the phospholamban monomer is a more effective inhibitor of ATPase activity than is the pentamer, the results suggest that phosphorylation of phospholamban in the sarcoplasmic reticulum membrane could relieve inhibition of the Ca2+ pump by at least two mechanisms: one, by disrupting the physical interaction between phospholamban and the Ca2+ pump (45) and, two, by promoting the more complete association of phospholamban into pentamers (19), which are relatively ineffective inhibitors compared with monomers. More sophisticated techniques will be required to test if the phospholamban pentamer by itself is capable of inhibiting the Ca2+ pump, but the results presented presently in combination with those of Cornea et al. (19) clearly indicate that at least one monomeric mutant of phospholamban is a much stronger inhibitor of SERCA2a activity than is wild-type phospholamban. Our results are consistent with a recent preliminary report of Kimura et al. (47), who identified several additional monomer-forming mutations in the leucine zipper region of phospholamban (17), which produced similar potent inhibition of the Ca2+ pump like L37A-PLB reported presently. Thus the increased effectiveness of L37A-PLB detected here is probably a direct consequence of its monomer status and not related to the change in the amino acid per se.

Although we reported earlier that purified phospholamban forms Ca2+ channels in lipid bilayers (8), we found no evidence here for phospholamban acting as a Ca2+ efflux channel when co-expressed with SERCA2a under conditions in which the two proteins are tightly coupled. A similar inability of phospholamban to form Ca2+ channels was noted by Reddy et al. (16) in a study in which phospholamban was successfully co-reconstituted with the skeletal muscle Ca2+ pump in phospholipid vesicles. Thus we believe that a Ca2+ efflux role for phospholamban in its mechanism of regulation of SERCA2a is very unlikely and that recent models proposing such a role (9) should be viewed with some skepticism.

In conclusion, we have demonstrated that the baculovirus cell expression system is ideally suited for investigating phospholamban and SERCA2a regulatory interactions. High levels of protein expression are achieved, with preservation of functional coupling. With use of this system, it is now possible to carry out detailed biochemical and kinetic analyses while investigating both normal and mutated proteins.


FOOTNOTES

*   This research was supported by National Institutes of Health Grants HL06308 and HL49428.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U94345[GenBank].


Dagger    Charles Fisch Professor of Cardiology. To whom correspondence should be addressed: Krannert Institute of Cardiology, 1111 W. 10th St., Indianapolis, IN 46202. Tel.: 317-630-6695; Fax: 317-630-8595. E-mail: jones{at}kimail.dmed.iupui.edu.
1   The abbreviations used are: Sf21 cells, Spodoptera frugiperda insect cells; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SERCA2a, cardiac SERCA isoform; SERCA1a, adult fast twitch skeletal muscle isoform; SERCA3, ubiquitous SERCA isoform; L37A-PLB, canine phospholamban with leucine residue 37 in the transmembrane domain replaced by alanine; KCa value, ionized Ca2+ concentration giving half-maximal activation of the Ca2+ pump; WTV, wild-type virus.

ACKNOWLEDGEMENT

We gratefully acknowledge the technical assistance of Jeff Kelley.


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