From the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota 55455 and § Krannert Institute of Cardiology, Indiana University, Indianapolis, Indiana 46202
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ABSTRACT |
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Phospholamban (PLB), a 52-amino acid integral
membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac
sarcoplasmic reticulum through PLB phosphorylation mediated by
Phospholamban (PLB)1 is
a small membrane protein of 52 amino acid residues, which is composed
of an N-terminal cytoplasmic domain and a C-terminal membrane-spanning
domain (1, 2). In response to Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and low angle laser light scattering in SDS solutions (17, 18) showed
that PLB is predominantly a homopentamer in equilibrium with a small
fraction as monomer. Systematic replacement of single hydrophobic amino
acid residues, Leu or Ile, by Ala in the membrane-spanning region
transformed PLB from pentameric to monomeric form on SDS-PAGE (19).
Based on these results, a model for a tightly packed coiled-coil
pentamer (19-21) has been proposed: the However, there are several unanswered questions as follows.
(a) So far only pentamer/monomer (e.g. native
PLB) and purely monomeric (e.g. L37A-PLB) structural mutants
of PLB and their functional effects have been reported (22, 23), but
this does not exclude the possibility of other oligomeric forms of PLB
as inhibitory species. (b) In a coexpression system the
relative ratios of PLB and Ca-ATPase may vary, so it is possible that
the apparent enhanced inhibition of the pump by the monomeric mutants is due to variation in expression levels of PLB mutants and Ca-ATPase. In the present study, we have tested the inhibitory function of recombinant wild type-PLB (wt-PLB) and two different structural mutants, a phospholamban monomer and tetramer (19), using an in
vitro co-reconstitution system (15, 27). We have characterized the
inhibitory function of these structural mutants as a function of
increasing molar ratios of PLB/Ca-ATPase and as a function of
temperature. The correlation of these results with the oligomeric states of these mutants, as indicated by SDS-PAGE, provides new insight
into the structural basis of Ca-ATPase regulation by PLB.
Materials
The reagents used were octaethylene glycol monododecyl ether
(C12E8), Recombinant wt-PLB and the mutants were expressed in
Sf21/baculovirus insect cell system and purified by monoclonal
antibody (2D12) affinity column chromatography as described previously (15, 19). The concentration of PLB was determined by the Amido Black
assay (29). The purified protein was stored at METHODS
Ca-ATPase/PLB Co-reconstitution--
The method used for
functional reconstitution of Ca-ATPase with PLB has been described
previously (15, 27). In short, the required amount of PLB (5-25 µg)
in OG buffer was dried (lyophilized) and solubilized in 80 µl of
CHCl3 containing 20 µl of 2,2,2-trifluoroethanol and 0.8 mg of DOPC/DOPE (20% DOPE, by weight) for ATP hydrolysis measurements,
or EYPC/EYPA (20% EYPA, by weight) for Ca2+ uptake
measurements. The solvent was dried under nitrogen and the residual
solvent was removed by pumping the sample under vacuum. The dried film
of lipid and PLB was hydrated with 50 µl of 20 mM
imidazole, pH 7.0, by vortexing throughly followed by a brief sonication. The resulting vesicles, containing lipid and PLB, were made
to 20 mM imidazole, pH 7.0, 0.1 M KCl, 5 mM MgCl2, 10% glycerol; then 1.6 mg of Calcium Uptake Measurements--
45Ca2+
uptake measurements were carried using the microfiltration method (31)
as described previously (15, 27) at 25 °C. Each assay was done in
duplicate at pCa 5.4 and 6.8 in a volume of 0.25 ml which
contained 1-2 µg of Ca-ATPase (10-20 µl of vesicles after the
addition of PLB antibody), 50 mM imidazole, pH 7.0, 0.095 M K2SO4, 5 mM
MgCl2, 5 mM NaN3, 5 mM
K2 oxalate, 0.5 mM EGTA, and different
concentrations of CaCl2 to obtain pCa values of
5.4 and 6.8; free calcium concentrations were calculated by the method
of Fabiato and Fabiato (32). The final 45Ca2+
in the assay mix was 30 to 50 µCi. The assay mixture also contained 1 µM each of carbonyl cyanide
p-trifluoromethoxyphenylhydrazone and valinomycin to
dissipate gradients of pH and K+, respectively, and thus to
collapse any membrane potential that develops (33). Ca2+
uptake was initiated by the addition of 5 mM ATP. Aliquots
of 100 µl were filtered at 30 s and 2 min (pCa 5.4)
or 6 min (pCa 6.8) after the addition of ATP (we found that
these time points gave linear calcium uptake) using 0.22-µm filters
(GS type, Millipore) and were washed twice with 20 mM MOPS,
pH 7.0, 100 mM K2SO4, 10 mM MgCl2, and 3 mM
LaCl3. Quantitation of 45Ca2+ was
done by scintillation counting, and the initial calcium uptake rate was
calculated from the slope of the line joining the two time points.
ATPase Activity Measurements--
ATPase activity was assayed by
measuring the inorganic phosphate released from the hydrolysis of ATP
by Ca-ATPase at 25 and 37 °C, using the method of Lanzetta et
al. (34). Each assay was done in triplicate at pCa 5.4 and 6.8 in a volume of 0.12 ml that contained 0.5-1.0 µg of
Ca-ATPase (5-10 µl of vesicles), 50 mM imidazole, pH
7.0, 0.1 M KCl, 5 mM MgCl2, 0.5 mM EGTA, 1-2 µg/ml calcium ionophore (A23187), and
different concentrations of CaCl2 to obtain pCa
values of 5.4 and 6.8. ATP hydrolysis was initiated by the addition of
2.5 mM ATP. Aliquots of 50 µl were taken at zero time
(immediately after the addition of ATP) and at 2 (pCa 5.4)
or 6 min (pCa 6.8) and dispensed into 400 µl of malachite
green reagent followed by the addition of 50 µl of 34% sodium
citrate after 30 s. The absorbance was read at 650 nm, and the
rate of ATP hydrolysis was calculated using a standard curve from known
concentrations of inorganic phosphate.
Quantitation of PLB in the Vesicles--
Reconstituted vesicles
were subjected to sucrose gradient centrifugation to separate
unincorporated PLB from vesicles containing PLB and Ca-ATPase. After
functional assays, 20 µl of the remaining vesicles were loaded on top
of a stepwise gradient of 50 (50 µl), 20 (170 µl), 15 (170 µl),
10 (170 µl), and 5% (50 µl) sucrose in 0.8-ml centrifuge tubes.
The gradients were centrifuged for 14-16 h at 140,000 × g (Beckman, SW55 Ti rotor). Fractions of 100-120 µl were
collected from the bottom of the tube and subjected to immunoblot
analysis. A small volume of each fraction was diluted into transfer
buffer (25 mM Tris base, 193 mM glycine, 0.1%
SDS, and 10% methanol) and applied to PVDF (polyvinylidene difluoride) membranes (Bio-Rad) using a Slot-Blot apparatus (Bio-Rad). After blocking the membrane with 1% non-fat dry milk, it was incubated for
1 h with monoclonal anti-PLB antibody (2D12). The membrane was
washed and incubated for 1 h with goat anti-mouse antibody conjugated to horseradish peroxidase. The membrane was then washed and
the immunoreactive protein bands were developed with
3,3'-diaminobenzidine tetrahydrochloride (DAB) reagent (Sigma). The
immunoblots were scanned by a densitometer using the reflectance mode,
and the bands were quantitated using the volume (area × density)
analysis method. The densities of the bands were within the linear
range of intensities.
Quantitation of PLB Oligomers and Monomers--
PLB (wt-PLB) and
its mutants, C41L-PLB and L37A-PLB (50-100 ng), were
subjected to SDS-PAGE (16.5% Tris/Tricine gels, Bio-Rad) at various
temperatures, and proteins were transferred to PVDF membranes in 25 mM Tris (base), 193 mM glycine, pH 8.3, 10%
methanol, and 0.1% SDS for 1 h at 4 °C and 200 mA constant
current. After blocking the membrane with 1% non-fat dry milk, it was
incubated for 1 h with monoclonal anti-PLB antibody (2D12). The
membrane was washed and incubated for 1 h with goat anti-mouse
antibody conjugated to horseradish peroxidase. The membrane was then
washed, and the immunoreactive protein bands were developed with
3,3'-diaminobenzidine tetrahydrochloride (DAB) reagent (Sigma). The
immunoblots were scanned by a densitometer using the reflectance mode,
and the bands were quantitated using the volume (area × density)
analysis method.
On SDS-PAGE, wild type PLB (wt-PLB) migrates primarily as a
pentamer, with a small fraction as monomer (17). The mutants of PLB
used in this study, C41L-PLB (Cys 41 to Leu) and L37A-PLB (Leu 37 to Ala), migrated predominantly as tetramer and monomer, respectively, on SDS-PAGE at 25 °C (Fig.
1) (19).
-adrenergic stimulation. Based on site-directed mutagenesis and
coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in
HEK 293 cells, and on spin label detection of PLB oligomeric state in
lipid bilayers, it has been proposed that the monomeric form of PLB is
the inhibitory species, and depolymerization of PLB is essential for
its regulatory function. Here we have studied the relationship between
PLB oligomeric state and function by in vitro
co-reconstitution of PLB and its mutants with purified Ca-ATPase. We
compared wild type-PLB (wt-PLB), which is primarily a pentamer on
SDS-polyacrylamide gel electrophoresis (PAGE) at 25 °C, with two of
its mutants, C41L-PLB and L37A-PLB, that are primarily
tetramer and monomer, respectively. We found that the monomeric mutant
L37A-PLB is a more potent inhibitor than wt-PLB, supporting the
previous proposal that PLB monomer is the inhibitory species. On the
other hand, C41L-PLB, which has a monomeric fraction
comparable to that of wt-PLB on SDS-PAGE at 25 °C, has no inhibitory
activity when assayed at 25 °C. However, at 37 °C, a 3-fold
increase in the monomeric fraction of C41L-PLB on SDS-PAGE
resulted in inhibitory activity comparable to that of wt-PLB. Upon
increasing the temperature from 25 to 37 °C, no change in fraction
monomer or inhibitory activity for wt-PLB and L37A-PLB was observed.
Based on these results, the extent of inhibition of Ca-ATPase by PLB or
its mutants appears to depend not only on the propensity of PLB to
dissociate into monomers but also on the relative potency of the
particular PLB monomer when interacting with the Ca-ATPase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-adrenergic stimulation, PLB
phosphorylation regulates the rate of calcium pumping by the Ca-ATPase
of cardiac sarcoplasmic reticulum (3-6). Although the expression of
PLB is largely restricted to cardiac SR, to a lesser extent its
presence is reported in slow-twitch skeletal muscle SR (7, 8) and
smooth muscle SR (9). In its unphosphorylated form, PLB inhibits the
rate of Ca2+ pumping and ATP hydrolysis by the Ca-ATPase at
subsaturating [Ca2+]. Upon phosphorylation of PLB by
cAMP-dependent protein kinase A or
Ca2+/calmodulin-dependent protein kinase II,
this inhibition is relieved (4, 6, 10). Although PLB is not present in
fast-twitch muscle (8, 11, 12), it has been shown to be capable of regulating the fast-twitch Ca-ATPase isoform (SERCA1) in both reconstitution (13-15) and coexpression (16) experiments.
-helical transmembrane
domains of five monomers associate by intramembrane Leu/Ile
interactions forming a Leu/Ile zipper. Monomeric mutants of PLB were
shown to be more potent inhibitors of the cardiac isoform of the
Ca-ATPase (SERCA2a) when coexpressed in a heterologous system (22-24).
By using EPR spectroscopy, it was shown that PLB exists in an average
oligomeric size of 3.5 in DOPC vesicles, and upon phosphorylation the
average oligomeric size increased to 5.3 (26). This study suggested the
existence of a dynamic equilibrium between PLB subunits in the lipid
bilayer and that the regulation of the oligomeric state of PLB is
critical for its regulation of the Ca-ATPase. This led to the
hypothesis that the active inhibitory species of PLB is the monomer and
that increased oligomerization of PLB upon phosphorylation contributes to relief of the inhibition (22, 23, 25, 26).
EXPERIMENTAL PROCEDURES
-octyl glucoside (
-OG), and
Biobeads SM2 were purchased from Calbiochem (San Diego, CA). Dioleoyl
phosphatidylcholine (DOPC), dioleoyl phosphatidylethanolamine (DOPE),
egg yolk phosphatidylcholine (EYPC), and egg yolk phosphatidic acid
(EYPA) were obtained from Avanti Polar Lipids (Alabaster, AL). The
reagents for SDS-PAGE (16.5% Tris/Tricine ready gels and the
Tris/Tricine gel running buffer) and the PVDF membrane used for
immunoblots were from Bio-Rad. Anti-PLB monoclonal antibody (2D12) was
prepared as described previously (7, 15, 28). Horseradish
peroxidase-coupled goat anti-mouse antibody was supplied by Fisher
(Southern Biotechnology Laboratories, Inc.). The substrate for
horseradish peroxidase, 3,3'-diaminobenzidine tetrahydrochloride (DAB)
reagent, and Reactive Red affinity column were purchased from Sigma.
All the other reagents were of highest purity available and were
purchased from Sigma.
70 °C at a protein
concentration of 1.5-2.5 mg/ml, in a buffer ("OG buffer")
containing 88 mM MOPS, 18 mM glycine, 5 mM dithiothreitol, and 0.92%
-octyl glucoside (
-OG)
at pH 7.2. Rabbit skeletal sarcoplasmic reticulum (SR) Ca-ATPase was
purified from light SR using the reactive-red affinity chromatography
method (30).
-OG
was added followed by 20 µg of purified Ca-ATPase, in a final volume
of 100 µl with buffer. For Ca2+ uptake rate measurements,
the buffer contained 0.1 M K2 oxalate instead
of KCl. The detergent was then removed by incubation with 40 mg of wet
Biobeads (25 mg of Biobeads SM2/1 mg of detergent) for 3 h at room
temperature. The resultant Ca-ATPase/PLB lipid vesicles were separated
from Biobeads and immediately assayed for Ca2+ uptake or
ATP hydrolysis activity. Prior to the assay, the reconstituted vesicles
were incubated with or without PLB monoclonal antibody, 2D12 (22), at a
molar ratio of PLB/Ab = 1, for 15 min on ice.
RESULTS
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Fig. 1.
Oligomeric pattern of PLB (wt-PLB) and the
mutants C41L-PLB (C41L) and L37A-PLB (L37A) on
SDS-PAGE followed by immunoblotting. Samples were electrophoresed
on 16.5% Tris/Tricine gels and subjected to immunoblotting as
described under "Experimental Procedures."
The inhibitory function of recombinant PLB (wt-PLB) and its mutants, C41L-PLB and L37A-PLB, was tested in a reconstituted system. Co-reconstitution was done using purified skeletal SR Ca-ATPase and purified PLB in EYPC/EYPA lipid vesicles for Ca2+ uptake measurements, and in DOPC/DOPE lipid vesicles for ATP hydrolysis measurements. When EYPC/EYPA or pure DOPC was used for reconstitution, the ATP hydrolysis activity of the enzyme was too low at pCa 6.8 (low calcium) to obtain acceptable precision. Addition of 20% DOPE (by weight) to DOPC enhanced the activity by 40%, providing adequate precision at low calcium.
Inhibitory activity of PLB and the mutants, C41L-PLB and L37A-PLB, was assayed by measuring Ca2+ uptake and ATP hydrolysis rates in the presence and absence of anti-PLB monoclonal antibody (2D12). In coexpression studies, the extent of inhibition by PLB or its mutants has usually been reported as a shift (increase or decrease) in KCa (Ca2+ concentration at which Ca2+ stimulation of activity is half-maximal) compared with the control (no PLB), obtained from a curve of Ca-ATPase activity versus pCa (22, 23, 35). In the present study, only two calcium concentrations were used, pCa 5.4 ("high calcium"), where Ca2+ uptake and Ca-ATPase activities are maximal (Vmax), and pCa 6.8 ("low calcium"), where the shift in KCa caused by PLB results in a substantial inhibition. Thus at high calcium, only Vmax effects are observed, and at low calcium both effects are observed. We report the extent of inhibition as the percent decrease (percent inhibition) in the Ca-ATPase activity, compared with the activity in the presence of anti-PLB antibody (+PLB-Ab), which is functionally equivalent to PLB phosphorylation and therefore reports the disinhibited SERCA state (15).
At 25 °C and pCa 5.4, there is no significant difference
in calcium uptake rates in the absence and presence of anti-PLB
antibody (Fig. 2, left panel),
indicating that PLB or its mutants have no significant effect on the
maximal velocity (Vmax) of the Ca-ATPase. On the
other hand, at pCa 6.8 (Fig. 2, right panel),
wt-PLB, C41L-PLB, and L37A-PLB inhibit the pump by 48%
(0.41 ± 0.10 IU, PLB-Ab versus 0.80 ± 0.16 IU,
+PLB-Ab), 14% (1.06 ± 0.13 IU,
PLB-Ab versus
1.23 ± 0.15 IU, +PLB-Ab), and 64% (0.362 ± 0.09 IU,
PLB-Ab versus 1.020 ± 0.17 IU, +PLB-Ab),
respectively. Thus C41L-PLB has a much smaller inhibitory
effect than wt-PLB, and L37A-PLB has a much larger inhibitory effect
than wt-PLB. These inhibitory effects, for both wt-PLB and L37A-PLB,
are due to a shift in calcium sensitivity (an increase in
KCa) of the Ca-ATPase.
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As a more direct measure of PLB effects on SERCA activity, we also measured ATP hydrolysis by the reconstituted Ca-ATPase in the presence of calcium ionophore A23187 at 25 °C. With the use of the calcium ionophore, no transported Ca2+ is retained by the vesicles, allowing the ATPase to operate freely in the absence of generated Ca2+ gradients. With this method of assay, ATPase hydrolysis by the Ca2+ pump is not limited by the finite Ca2+ capacity of the reconstituted vesicles, and the permeability of the lipid vesicles themselves does not act as a confounding variable.
At saturating [Ca2+] (pCa 5.4), neither wt-PLB nor the two mutants (C41L-PLB and L37A-PLB) significantly affected the ATP hydrolysis rate (Fig. 3, left panel); ATP hydrolysis rates were identical in the presence and absence of the anti-PLB antibody. At pCa 6.8 (Fig. 3, right panel) wt-PLB and L37A-PLB inhibited the ATP hydrolysis rates significantly, whereas C41L-PLB was without appreciable effect. As observed with the coexpression system (22, 23), monomeric L37A-PLB inhibited the Ca-ATPase more effectively than pentameric wt-PLB. As observed with the Ca2+ transport assay, the inhibitory effect of co-reconstituted PLB on the ATP hydrolysis rates was prevented by the anti-PLB antibody. Thus, the co-reconstitution method with purified proteins gives results similar to those obtained with the cellular coexpression method for investigating PLB/SERCA regulatory interactions. Although the functional (inhibitory) effects of wt-PLB, C41L-PLB, and L37A-PLB obtained from ATP hydrolysis measurements are qualitatively similar to those obtained from Ca2+ uptake measurements, the overall rates of Ca2+ uptake at pCa 6.8 are significantly higher than the ATP hydrolysis rates. This could be due to the difference in lipid composition used for reconstitutions for these two different activity measurements. Also, the ATP hydrolysis rates are slightly lower in C41L-PLB/Ca-ATPase and L37A-PLB/Ca-ATPase reconstituted vesicles compared with wt-PLB/Ca-ATPase reconstituted vesicles and significantly lower compared with control (Ca-ATPase alone) vesicles. However, the Ca2+ uptake and ATP hydrolysis rates of Ca-ATPase at pCa 5.4 in the absence and presence of anti-PLB antibody are similar, indicating that the variations in the rates are not due to PLB or its mutants.
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The PLB-induced inhibitory effects discussed above (Figs. 2 and 3) were measured at added PLB/Ca-ATPase molar ratio of 10 in the reconstituted system. In cardiac SR, the molar ratio of PLB/Ca-ATPase is reported to be in the range of 5-8 (37). At an added PLB/Ca-ATPase molar ratio of 10, if the insertion of PLB in the lipid bilayers is symmetric and the Ca-ATPase is asymmetric (33), then the effective PLB/Ca-ATPase molar ratio is only 5, which is on the lower side of the reported molar ratio in cardiac SR. Also, if the inhibitory species of PLB is a monomer, then increasing the molar ratio of PLB/Ca-ATPase should also increase the amount of monomer, which should reflect in the inhibitory effects of wt-PLB and its mutant, specifically C41L-PLB. To test this, we have studied the inhibitory effects of PLB and its mutants, C41L-PLB and L37A-PLB, at different molar ratios of PLB/Ca-ATPase at pCa 5.4 and 6.8. In order to ensure that the effects were not due to differences in the efficiency of incorporation, we measured directly the fraction of PLB incorporated into vesicles.
PLB and its mutants, C41L-PLB and L37A-PLB, were reconstituted at added PLB/Ca-ATPase molar ratios of 5, 10, and 25 and measured the fraction of added PLB incorporated into lipid vesicles by separating free PLB from vesicle-bound PLB after sucrose gradient centrifugation of reconstituted vesicles (Fig. 4). Most of the lipid vesicles are present in 5-10% sucrose fractions (fractions 5 and 6). However, wt-PLB and C41L-PLB are present not only in the lipid-containing fractions but also in fractions containing 20% sucrose, representing unincorporated PLB. In contrast, L37A-PLB was distributed throughout the gradient with a marginally higher fraction of it associated with fractions 5 and 6, where most of the lipid is present. The fraction of unincorporated PLB increases with increasing amounts of total PLB added to the reconstitution system. The SDS-PAGE of sucrose gradient fractions indicate that the Ca-ATPase is present in fractions 5 and 6 only (Fig. 4C) indicating that almost all of the Ca-ATPase is incorporated into lipid vesicles. The molar ratio of PLB/Ca-ATPase associated with lipid vesicles (i.e. in fractions 5 and 6), versus the added PLB/Ca-ATPase molar ratio, is presented in Fig. 5. Although the fraction of unincorporated PLB increases with increasing amounts of total PLB added, it is apparent that the overall molar ratio of PLB/Ca-ATPase incorporated into lipid vesicles increases with increasing amounts of added PLB even at added molar ratios as high as 25 (Fig. 5).
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ATP hydrolysis rates of Ca-ATPase measured at various molar ratios of PLB incorporated into vesicles per Ca-ATPase are presented in Fig. 6. At pCa 6.8 (Fig. 6, bottom panels), the inhibitory effects of wt-PLB and L37A-PLB increase gradually with increasing molar ratios of PLB/Ca-ATPase. The inhibitory effects of wt-PLB appear to saturate at molar ratios above 7, at about 50% inhibition of the Ca-ATPase, whereas L37A-PLB appears to approach complete inhibition of the Ca-ATPase. This clearly shows that L37A-PLB is substantially more potent than wt-PLB and that this difference is not due to different efficiencies of incorporation. Substantial reversal of inhibition is produced by addition of the anti-PLB antibody. On the other hand, C41L-PLB has little or no inhibitory effects on the pump even at incorporated C41L-PLB/Ca-ATPase molar ratio above 10, clearly showing that C41L-PLB is much less potent than wt-PLB. At pCa 5.4, there is no effect of PLB or its mutants on Ca-ATPase function at any of the molar ratios tested. These results (Fig. 6, at pCa 6.8) also suggest that the inhibitory effects of wt-PLB, C41L-PLB, and L37A-PLB presented in Fig. 2 and Fig. 3 are not limited by the amount of PLB incorporated in the membrane and also indicate the approximate limit of inhibitory effects obtainable with these PLBs in our reconstitution system.
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Depolymerization of PLB has been suggested to be one of the prerequisites for its inhibitory effects on Ca-ATPase (22, 23, 25, 26, 35). If C41L-PLB is a more stable oligomer (tetramer) compared with the wt-PLB (pentamer), then its inability to depolymerize upon association with Ca-ATPase may cause its failure to inhibit the pump. We have tested the temperature-dependent oligomeric stability of C41L-PLB, compared with wt-PLB and L37A-PLB on SDS-PAGE. The results (Fig. 7) indicate that wt-PLB shows a pentamer/monomer pattern at all temperatures from 4 to 55 °C, with an increase in the monomeric fraction as the temperature is increased from 37 to 55 °C. C41L-PLB, while showing a tetramer/monomer pattern at 4 °C, completely dissociates into the monomeric form as the temperature is increased to 55 °C, indicating that C41L-PLB oligomer (tetramer) is less stable than that of wt-PLB (pentamer). L37A-PLB shows a pentamer/dimer/monomer pattern at 4 and 12 °C and a dimer/monomer pattern at higher temperatures. The dimeric form of L37A-PLB is persistent even at 55 °C.
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Whereas oligomer/monomer (ratio) pattern of wt-PLB and L37A-PLB on
SDS-PAGE was unchanged when the temperature was increased from 25 to
37 °C, a significant increase in the monomeric fraction of
C41L-PLB was observed at 37 °C (67% monomer) compared
with 25 °C (24% monomer). As the monomeric form of PLB was
suggested to be the inhibitory species (22, 23, 26), we tested the inhibitory activity of C41L-PLB along with wt-PLB and
L37A-PLB at 37 °C to see whether the increased amount of
C41L-PLB monomer has any gain in its inhibitory function.
ATP hydrolysis rates of Ca-ATPase co-reconstituted with wt-PLB,
C41L-PLB, and L37A-PLB measured at 37 °C (Fig.
8) show that while the inhibitory effects of wt-PLB and L37A-PLB are unchanged from 25 (Fig. 3) to 37 °C (Fig.
8), C41L-PLB showed a significant gain in its inhibitory activity, and this inhibition was comparable to wt-PLB. At 37 °C and
pCa 6.8 (Fig. 8, right panel), wt-PLB,
C41L-PLB, and L37A-PLB inhibit the pump by 42% (2.30 ± 0.27 IU, PLB-Ab versus 3.94 ± 0.10 IU, +PLB-Ab),
39% (2.22 ± 0.12 IU,
PLB-Ab versus 3.62 ± 0.36 IU, +PLB-Ab), and 70% (1.16 ± 0.251 IU,
PLB-Ab
versus 3.76 ± 0.35 IU, +PLB-Ab). These results
indicate that the gain in the inhibitory function of
C41L-PLB at 37 °C is in parallel with an increase in its
monomeric fraction observed on SDS-PAGE at 37 °C. On the same
reconstituted vesicles used for measurements at 37 °C, we measured
ATP hydrolysis rates at 25 °C, and the inhibitory effects were
similar to those described earlier (Fig. 3). Consistent with the
previous results observed at 25 °C, no inhibitory effects of wt-PLB
or the two PLB mutants on ATPase activity were noted when the assay was
conducted under Vmax conditions at 37 °C and pCa 5.4 (Fig. 8, left panel).
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DISCUSSION |
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We have characterized the functional effects of PLB and two of its structural mutants in an in vitro reconstitution system. Ca2+ uptake and ATP hydrolysis rate measurements (Figs. 2 and 3) show that the inhibitory effects exerted by wt-PLB are only at low Ca2+ (pCa 6.8) but not at high Ca2+ (pCa 5.4), and the extent of inhibition (~50% inhibition, or 2-fold stimulation by PLB antibody, see Table I) is consistent with our previously published results on wt-PLB (15). However, the inhibitory effects of L37A-PLB at low Ca2+ are much more pronounced (70-80%) than those of wt-PLB (Figs. 2 and 3, Table I), which is consistent with the report that L37A-PLB is a more potent inhibitor when coexpressed with SERCA2a in insect cell microsomes (22) or in HEK 293 cells (23). This establishes the validity of our reconstitution system for studying the functional effects of PLB and its mutants. There was a small decrease in Ca-ATPase activity in the presence of L37A-PLB at high Ca2+ (Fig. 6, L37A/pCa 5.4), but this was not reversed upon the addition of PLB antibody, suggesting that this change is not due to the effect of PLB on Vmax. Moreover, the effect was not observed when Ca2+ uptake rates were measured (Fig. 2). Some reports suggested that PLB inhibits the pump by altering both Vmax and KCa (39-42). Our results from both wt-PLB and L37A-PLB co-reconstituted with Ca-ATPase are consistent with the well established principle that PLB-induced inhibition of Ca-ATPase is due to a decrease in the Ca2+ sensitivity, (increase in KCa) but not due to a decrease in maximal velocity (Vmax) of the pump (reviewed in Ref. 6).
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If the monomeric form of PLB is the inhibitory species, as previously proposed (22, 23, 26), then increasing the molar ratios of PLB/Ca-ATPase should also increase the ratio of monomeric-PLB/Ca-ATPase and thereby the inhibitory effects of wt-PLB. Consistent with this prediction, we found that the inhibitory effects of wt-PLB or L37A-PLB increase with increasing molar ratios of PLB/Ca-ATPase, saturating at PLB/Ca-ATPase molar ratio of ~7-10 (Fig. 6). However, the tetrameric mutant C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB, has very little inhibitory effect on Ca-ATPase even at a C41L-PLB/Ca-ATPase molar ratio of ~13 (Fig. 6). Quantitation of PLB (Fig. 4) indicated that the fraction of PLB incorporated into lipid vesicles is more linear (Fig. 5) than the inhibitory effects (Fig. 6) with increasing molar ratio of PLB/Ca-ATPase. Therefore, the saturating inhibitory effects of wt-PLB and L37A-PLB and the absence of a significant inhibitory effect of C41L-PLB are not due to the lack of further incorporation of PLB into lipid vesicles with increasing molar ratios of PLB/Ca-ATPase.
On SDS-PAGE, wt-PLB was shown to have ~20% as monomer (Ref. 26 and see Table I), and our recent results from fluorescence energy transfer studies on wt-PLB co-reconstituted with Ca-ATPase in lipid have indicated the presence of ~30% as monomer (43). Assuming ~25% of wt-PLB as a monomer in equilibrium with pentamer, the molar ratio of monomeric-wt-PLB/Ca-ATPase in lipid vesicles would be ~1.8 and ~3.3 at wt-PLB/Ca-ATPase molar ratio of 7 and 13 (Fig. 5), respectively, and that of L37A-PLB is ~2.5 at L37A-PLB/Ca-ATPase molar ratio of 2.5 (Fig. 5). These comparable inhibitory effects exerted at comparable molar ratios of monomers to Ca-ATPase suggest that the monomeric form of wt-PLB is the most active inhibitory species. Similarly, the monomeric C41L-PLB/Ca-ATPase molar ratios, ~2.0 and ~3.3 (assuming 25% as monomer in C41L-PLB) in lipid vesicles at C41L-PLB/Ca-ATPase molar ratio of 8 and 13 (Fig. 5), respectively, are similar to those of wt-PLB. The similar monomer/Ca-ATPase ratios and much lower inhibitory function of C41L-PLB (Table I), compared with wt-PLB, suggest that the monomeric C41L-PLB is much less inhibitory compared with wt-PLB and L37A-PLB at 25 °C.
The effect of wt-PLB appears to saturate, achieving a level of about 50% inhibition, at molar ratios above ~7, whereas L37A-PLB inhibition appears to go nearly to completion (Fig. 6). Since the binding data (Figs. 4 and 5) indicate saturation of PLB-binding sites on the Ca-ATPase, these results indicate that L37A-PLB is a substantially more potent inhibitor when bound to the Ca2+ pump than is wt-PLB. If only monomers bind to the Ca-ATPase, as suggested by fluorescence measurements (43), this result indicates that the L37A-PLB monomer is a more potent inhibitor than the monomeric form of wt-PLB. Alternatively, wt-PLB may be less effective because both monomers and oligomers bind, but bound oligomers are less inhibitory than bound monomers. In any case, our data cannot be explained by the simple model that only monomers bind to the Ca2+ pump, and all bound monomers cause similar inhibition.
The tetrameric mutant C41L-PLB has little or no inhibitory effect on Ca-ATPase when studied at 25 °C (Figs. 2 and 3, Table I), but it has inhibitory activity comparable to that of wt-PLB when assayed at 37 °C (Fig. 8, Table I) in our reconstituted system. This increased inhibition correlates with an increase in the monomeric fraction (Fig. 7), suggesting that the monomeric form of C41L-PLB is the inhibitory species. However, the results also indicate that all monomers are not equally inhibitory, since the monomeric form of C41L-PLB appears to be less inhibitory than the monomeric form of wt-PLB, whereas the L37A-PLB monomer is more inhibitory than wt-PLB (discussed above).
A study in which PLB and its mutants were coexpressed with the SERCA2a isoform in insect cell microsomes also reported that C41L-PLB is less inhibitory than wt-PLB (24). However, that study reported results at 37 °C (very little inhibition and very little monomer) that are comparable to the results of the present study at 25 °C (24). The simplest explanation is that the temperature dependence of PLB inhibitory potency and pentamer stability is different in the two systems, due either to (a) different lipid composition in insect cell microsomes, (b) different ratio of PLB to Ca2+ pump, or (c) the different SERCA isoforms used in the two studies.
Based on site-directed mutagenesis and coexpression studies (22, 23), and on spin label measurements showing increased PLB oligomerization with phosphorylation in membranes (26), it has been suggested that the monomeric form of PLB is the inhibitory species (25). While the present results do show increased inhibitory effects with increased monomeric PLB fraction, the present results indicate that the inhibitory activity of PLB is not a function of monomeric fraction alone.
Some mutants of PLB have been found to have negligible inhibitory effects when coexpressed with the Ca-ATPase, despite running as monomers on SDS-PAGE (23), and some other mutants were shown to be more potent inhibitors than wt-PLB, while running as pentamers on SDS-PAGE (30). It was suggested that the inhibitory activity of PLB or its mutants depends not only on oligomeric state, but also on affinity for the Ca-ATPase (23, 35). This is consistent with our results for C41L-PLB at 37 °C, with three times as much monomer and a similar inhibitory activity as wt-PLB, and at 25 °C, with a monomeric fraction comparable to that of wt-PLB but much less inhibitory function (Table I). Thus the replacement of Cys-41 specifically with Leu may have resulted in very low affinity (weak binding) or decreased potency (binds but does not inhibit) for the monomeric species. It has been shown that the replacement of Cys-41 with Ala (23) or Phe (44) favors monomers on SDS-PAGE but does not affect the inhibitory function of PLB, indicating that Cys residue as such at position 41 is not essential for the inhibitory function. On the other hand, Cys-41 was shown to be important for the oligomeric structure of PLB (21), suggesting that the replacement of Cys-41 with Leu alters the structure of PLB, resulting in loss of functional interactions with Ca-ATPase.
Alternatively, the fraction monomer of C41L-PLB observed on SDS-PAGE at 25 °C may be misleading; it is possible that C41L-PLB is a more stable oligomer (tetramer) in lipid vesicles, preventing it from dissociating into monomers upon interaction with the pump. This indicates the importance of determining the oligomeric structure of PLB in lipid vesicles (26, 43).
From the results discussed above, the mechanism of regulation of
Ca-ATPase by PLB appears to depend on two factors as follows: Kd (dissociation constant for PLB oligomer) and
Ka (association constant for formation of an
inhibitory complex of PLB-monomer/Ca-ATPase) (Fig.
9). The maximum inhibition of Ca-ATPase is achieved when Kd and Ka are
large, and little or no inhibition is observed when
Kd and Ka are small. Assuming
that the wt-PLB has optimal Kd and Ka, then Kd of L37A-PLB
C41L-PLB (37 °C) > C41L-PLB (25 °C) = wt-PLB, On the other hand, Ka of L37A-PLB > wt-PLB
C41L-PLB (25 °C) = C41L-PLB
(37 °C).
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Conclusions--
The qualitatively similar functional effects of
wt-PLB and L37A-PLB in a reconstituted system compared with the results
obtained from the coexpression system validate the use of the in
vitro reconstitution system to study not only the functional
effects but to study the physical mechanism of interaction between PLB monomers and between PLB and the Ca-ATPase, using spectroscopy and
other biophysical methods. Also, the reconstitution system has the
advantage of quantitative control of the molar ratios of PLB to
Ca-ATPase. The inhibitory effects of PLB and its mutants, presented in
this study, are due to a shift in KCa (increase
in KCa) but not due to a decrease in
Vmax of the Ca2+ pump. The effects
of the C41L mutation on inhibitory function depend not only on the
monomeric fraction but also on structural perturbation. The inhibitory
effects of wt-PLB, C41L-PLB, and L37A-PLB at 25 and
37 °C suggest that two equilibrium constants, Kd,
the dissociation constant for oligomer to monomer, and
Ka, the association constant for formation of an inhibitory complex of monomeric PLB with Ca-ATPase, are the factors responsible for the strength of inhibitory effects.
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ACKNOWLEDGEMENT |
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We thank Howard Kutchai for critically reading and correcting the manuscript and suggesting good experiments.
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FOOTNOTES |
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* This work was supported in part by Grants GM27906 and AR32961 (to D. D. T.) from the National Institutes of Health and the Minnesota Supercomputer Institute.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.
Supported by a grant from the American Heart Association, National Center.
¶ Suported by Grants HL06308 and HL49428 from the National Institutes of Health.
To whom correspondence should be addressed.
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ABBREVIATIONS |
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The abbreviations used are:
PLB, phospholamban;
wt, wild type;
wt-PLB, native phospholamban expressed in Sf21
insect cell;
SR, sarcoplasmic reticulum;
PLB-Ab, a monoclonal antibody
to PLB (2D12);
PAGE, polyacrylamide gel electrophoresis;
-OG,
-octyl glucoside;
EYPC, egg yolk phosphatidylcholine;
EYPA, egg yolk
phosphatidic acid;
DOPC, dioleoyl phosphatidylcholine;
DOPE, dioleoyl
phosphatidylethanolamine;
DAB, 3,3'-diaminobenzidine
tetrahydrochloride;
PVDF, polyvinylidene difluoride;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MOPS, 4-morpholinepropanesulfonic acid;
SERCA, sarco (endo) plasmic reticulum
Ca-ATPase.
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REFERENCES |
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