(Received for publication, March 20, 1997, and in revised form, April 8, 1997)
From the Department of Medicine and the Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202
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.
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 -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.
[-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.
A canine cardiac 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
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).
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.
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 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 Assay45Ca2+ 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 ImmunoblottingPrior 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).
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.
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).
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).
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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.
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).
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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.
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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U94345[GenBank].
We gratefully acknowledge the technical assistance of Jeff Kelley.