 |
INTRODUCTION |
The SR1 Ca pump is an
important cellular protein that transduces the energy stored in
cytosolic ATP (
GATP) into energy that is stored as a [Ca] gradient across the SR membrane
(
GSRCa). The pump establishes this
gradient at steady-state through a balance of the following 3 fluxes:
an SR Ca pump-dependent influx balanced by a
pump-dependent efflux (or "backflux") and a passive
leak flux (mediated by the ryanodine receptor or other pathways).
Backflux is a unidirectional flux that results from reversal of the SR
Ca pump in the forward mode. High Ca within the SR is transported
through the pump back into the cytosol, and ATP can be made from ADP
and inorganic phosphate as has been demonstrated directly in SR
membrane vesicles (1-4). Backflux has also been described in both
digitonin-permeabilized isolated cardiac myocytes (5) and in isolated
myocytes under whole-cell voltage clamp (6).
Phospholamban (PLB) is an important regulator of the SR Ca pump.
In the unphosphorylated state it associates with the SR Ca pump to
inhibit its activity. This inhibition can be relieved through
phosphorylation by protein kinase A and/or
Ca-calmodulin-dependent protein kinase (7, 8). Although it
is well established that PLB inhibits the forward mode of SR Ca
transport by increasing the forward
KCa
(KCa-f) to higher [Ca], the effects
of PLB on reverse SR Ca-ATPase and
KCa
(KCa-r) are unknown. This becomes an
important issue, especially in the case where the Ca pump approaches
thermodynamic equilibrium, as may be the case in intact myocytes under
some conditions (9). That is, the forward and reverse Ca flux can reach
the point where they are equal and opposite, and the [Ca] gradient is
maximal at about 7000:1 (10). If PLB shifts
KCa-f without shifting KCa-r by a comparable amount, the
maximal [Ca] gradient that the SR Ca pump can generate may change,
and this could alter the energetic efficiency of the pump.
PLB coexists in the cardiac myocyte in both a monomeric and pentameric
form. The pentameric form has been reported to form a channel that may
mediate Ca leak from the SR (11, 12).
In this report we measure forward and reverse Ca pump flux and also
pump-independent leak in SR vesicles from wild-type (WT) mice,
phospholamban knockout mice (PLB-KO), and mice that express equal
amounts of either wild-type PLB (PLB-70) or nonpentamer-forming mutant
PLB (PLB-C41F) upon a knockout background. Nonpump-mediated Ca leak was
not different among these groups, which does not support any
significant role for a PLB-mediated leak under our conditions.
We hypothesize that PLB inhibits SR Ca pump influx at the same
time that it stimulates SR Ca pump backflux. Such an effect would
result in a decreased
GSRCa and
decreased SR Ca pump energetic efficiency. The data in WT, PLB-KO, and
PLB-70 membranes all support this hypothesis.
 |
MATERIALS AND METHODS |
All chemicals were from Sigma, except as indicated.
Mathematical data manipulation was performed using Lotus 1-2-3 (Lotus Development Corp., Cambridge, MA) and Excel (Microsoft Corp., Seattle,
WA). Nonlinear regression fits and statistics were done with GraphPad
(iSi Software, Philadelphia, PA).
SR Ca Influx and Backflux Assay--
For the purposes of this
paper the efficiency of the SR Ca pump is defined as
follows:
|
(Eq. 1)
|
where
GATP is the free
energy stored in the form of ATP in the cytosol or 59 kJ/mol (13) and
GSRCa is the free energy required
by the pump to establish the SR [Ca] gradient. This is defined as
follows:
|
(Eq. 2)
|
where [Ca]SR is free SR Ca
and
[Ca]SR/[Ca]c is the free SR Ca gradient. Therefore when the pump generates a higher
SR [Ca] gradient, it is operating at a higher energetic efficiency.
In this paper we measure [Ca]SRT
(and relate it to [Ca]SR) in SR
membrane vesicles at 100 nM
[Ca]c as a measure of the SR [Ca] gradient.
Experimental Procedures--
PLB-KO, PLB-70, and WT mice
were anesthetized with 0.25 mg of pentobarbital/g of body weight.
Thoracotomy was performed, and hearts were removed, cannulated via the
aorta, and perfused with 5 ml of 10 mM caffeine, 10 mM EGTA, normal tyrode. Normal tyrode consisted of (in
mM) 140 NaCl, 4 KCl, 10 glucose, 5 HEPES, and 1 MgCl2, pH 7.4, with NaOH. Hearts were then perfused with 15 ml of 0.5 mM BAPTA in (in mM) 140 sucrose, 70 KCl, 40 HEPES, pH 7.2 (membrane buffer). Subsequently, the ventricular
tissue was separated from the rest of the heart and put into 0.5 BAPTA membrane buffer, minced, and homogenized with two 15-s pulses of
a polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY).
This procedure depletes the membranes of endogenous Ca. The membranes that resulted were centrifuged at 100,000 × g,
resuspended in 1 ml 0.5 BAPTA, 100 µg/ml aprotinin, 100 µg/ml leupeptin, 0.5 ryanodine, and 20 µM
digitonin in membrane buffer, and glass-Teflon homogenized.
The membranes were split into two portions. One was treated with
25 nmol/mg thapsigargin (Thg) and the other with
Me2SO vehicle. The membranes were incubated for 20 min at room temperature to allow ryanodine and thapsigargin binding and
were then put on ice.
Triplicate incubates were set up for plus and minus Thg groups.
Each incubate contained 86 µl of uptake buffer. MgATP (4 µl, 100 mM stock) and 10 µl of membranes (~5 mg/ml) were beaded
on the side of the test tube, and the assay for each time point was begun by vortexing, thus washing the ATP and the membranes into the
incubate. The incubates contained (final concentration in mM during incubation) 0.69 MgCl2, 0.5 BAPTA,
0.151 CaCl2 (100 nM
[Ca]c, 100-200 µCi of
45Ca), 4 MgATP, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
14 sucrose, 127 KCl, 40 HEPES, pH 7.2. Phosphocreatine (12.5) and 5 units/ml creatine phosphokinase were present to regenerate ATP.
Mitochondrial Ca uptake was inhibited by 2 µM ruthenium
red and 4 µM oligomycin. Digitonin (20 µM)
inhibited sarcolemmal Ca uptake, and ryanodine receptors were blocked
by 0.5 mM ryanodine. Uptake was stopped with an ice-cold
solution (in mM) of 1 EGTA, 200 KCl, 20 Mops, Tris to pH
7.4 (stop solution). The incubates were vacuum-filtered through Whatman
GF/C glass fiber filters (Fisher Scientific, Pittsburgh, PA). The
tubes were washed 3×, and the filter was washed an additional 2×
with stop solution.
Membranes were allowed to take up Ca for 0, 10, 20, 30, 60, and 90 s, thus forming the uptake part of the curve (see Fig. 2). The backflux
part of the curve was constructed from incubates that were allowed to
take up Ca for 90 s, at which point an efflux buffer was added.
Efflux buffer consisted of (final concentration in mM
during incubation) 50 EGTA, 17.1 CaCl2 (100 nM
[Ca]c), 40 HEPES, pH 7.2. This
solution caused 45Ca efflux while holding
[Ca]c at 100 nM and
reducing extracellular 45Ca specific activity 100-fold. The
indicated time points were taken after the addition of efflux buffer.
Membrane protein concentration was determined using the Bio-Rad
total protein assay reagent kit (Bio-Rad Laboratories, Hercules, CA).
Data Analysis--
The influx part of the experiment was fit
with a rising exponential function,
|
(Eq. 3)
|
where kinf is the rate
constant and t is time. Steady-state
[Ca]SRT
([Ca]SRT-SS) was therefore the
plateau of the relationship. Note that the described data are
"efflux-sensitive" uptake. We found that a varying percentage of
the total Ca taken up did not come out of the membranes upon addition
of efflux buffer. We therefore characterized the kinetics of the SR Ca
pump only in terms of Ca available for transport into and out of the membranes.
The initial rate of uptake was determined as the derivative of this
function at t = 0.
|
(Eq. 4)
|
Similarly, the efflux part of the time course was described with
an exponential decay,
|
(Eq. 5)
|
where [Ca]SRT+efl is efflux
buffer-sensitive [Ca]SRT, and
[Ca]SRT-efl is efflux
buffer-insensitive [Ca]SRT
(i.e. see Fig. 2, bottom plateau of the curve). kinf is the rate constant, and
t is the time from dilution. The rate of efflux at identical
[Ca]SRT+efl (0.8 nmol/mg) in all
groups was determined from the following derivative:
|
(Eq. 6)
|
where t is the time from dilution to
[Ca]SRT-efl = 0.8 nmol/mg.
Calculation of
GSRCa from Ca Uptake
Kinetics--
The initial rate of 45Ca uptake when
[Ca]SR = 0 is described by the
classic Hill equation,
|
(Eq. 7)
|
where Jpumpf is the forward
pump rate and Vmax,
KCa-f, and n are the
maximal velocity, [Ca]c at
half-maximal velocity, and the Hill coefficient in the forward direction, respectively. We set n to 2, KCa-f to 0.25, and 0.14 for WT and
PLB-KO, respectively (as measured by Frank, et al. (14)).
Given these parameters and Jpumpf,
Vmax was determined.
The rate of backflux at steady-state was also determined by using
Equation 6, where t = 0 (i.e. the
rate when excess 40Ca is added; see Fig. 2). This rate is
described by the following:
|
(Eq. 8)
|
where Jpumpr is the
unidirectional reverse pump rate and
KCa-r has its usual meaning for the
reverse pump rate. This is a special case of the generic reversible
equation,
|
(Eq. 9)
|
where Vmaxf = Vmaxr and nf = nr. For 45Ca efflux, the left term in the
numerator is zero, giving Equation 8. Given
Jpumpr and the
Vmax above, the
KCa-r can be inferred from Equation 8.
At steady-state Jpump is zero, so the
numerator of Equation 9 is zero, and this reduces to the Haldane
relationship.
|
(Eq. 10)
|
Combining with Equation 2, we arrive at Equation 11.
|
(Eq. 11)
|
Thus, if we know KCa-f and
derive KCa-r from Equation 8 the
GSRCa can be inferred.
SR Ca Leak--
Membranes from PLB-KO, PLB-70, and PLB-C41F mice
were prepared as above except 30 µM EGTA, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin, 140 KCl, 40 HEPES, pH 7.4 was used
for perfusion, homogenization, and resuspension instead of BAPTA.
The protocol for measuring passive SR Ca leak from the membranes is
illustrated in Fig. 1A. Mouse membranes were added to a
cuvette with stirring at a final concentration of ~1-2 mg/ml. Also
present were (final concentrations) EGTA (30 µM),
MgCl2 (1 mM free), the protease inhibitors
aprotinin and leupeptin (10 µg/ml), oligomycin (2 µM)
and ruthenium red (2 µM) to inhibit mitochondrial uptake,
and 0.5 mM ryanodine to block ryanodine receptors.
[Ca]c was measured with 2 µM indo-1 (Molecular Probes, Eugene, OR). An 8100 series
spectrofluorometer (Spectronic Instruments, Rochester, NY) was used to
excite the indo-1 at 355 nm. Fluorescence emission at 400 and 470 nm
was measured. The 400:470 ratio was converted to
[Ca]c using the Grynkiewicz equation (9, 15).
Uptake was started by 4 mM ATP addition. ATP was
regenerated with 5 units/ml creatine phosphokinase and 12.5 mM phosphocreatine. EGTA or Ca was added such that a
plateau was reached at ~100 nM [Ca]c.
[Ca]c gradually rose after 10 nmol/mg Thg was added until the SR Ca pump was completely
blocked. Passive leak continued until the SR was empty of Ca.
The leak rate constant (see below) was used to characterize the leak in
the different groups of membranes.
[Ca]c was converted to total Ca
([Ca]T) using known Ca binding constants for all buffers within the cuvette (see Table I). These binding constants were collected from the literature, and nearly all of
the endogenous affinity constants come from in vitro
measurements where physiological intracellular conditions were
simulated (normal ionic strength and pH value), usually at room temperature.
Exogenous EGTA and indo-1 are overwhelmingly the dominant Ca buffering
species accounting for >98% of the Ca bound (
32 µM versus ~0.3 µM). Fortunately these are the
buffers that we know the most about and of which we can be most sure.
Constants for EGTA in particular were fully corrected for ionic
strength and pH value using the Maxchelator program (see Ref. 16; free
for download on the World Wide Web).
Referenced values were converted from nmol/mg to µmol/liter cytosol
using the conversion factors 0.4 liter of cell volume/kg of wet weight,
120 mg of homogenate protein/g of wet weight (17) and a measured value
of 0.312 mg of membranes/mg of homogenate.
Therefore for each time point in Fig. 1A, we know
[Ca]c and
[Ca]T. The difference between the
[Ca]T before Thg and
[Ca]T after all of the Ca has
leaked out of the SR is the [Ca]SRT
just prior to Thg addition. Because we know how much Ca leaks out of
the SR during the experiment, we can now calculate
[Ca]SRT at each time point as the amount of Ca that hasn't leaked out yet.
Given [Ca]SRT and the SR Ca
buffering parameters, [Ca]SR can be
calculated. SR volume was assumed to be 3% of cellular volume (18,
19). [Ca]SRT was converted to
[Ca]SR using the following
relationships.
|
(Eq. 12)
|
|
(Eq. 13)
|
Bmax-SR and
Kd-SR have been previously determined
to be 14 mmol/liter of SR and 638 µM, respectively (10). Equation 13 was substituted into Equation 12, and
[Ca]SR as a function of
[Ca]SRT was calculated from the
quadratic solution of the result.
The leak rate (Jleak) is the change
in [Ca]T over time and is assumed
to be proportional to the concentration difference
([Ca]SR
[Ca]c),
|
(Eq. 14)
|
where kleak is a rate constant
of leak flux, determined by linear regression of leak flux data (see
Fig. 1B; gray line).
 |
RESULTS |
PLB Dependence of SR Ca Leak--
There are two primary routes of
Ca efflux from the SR when the ryanodine receptors are blocked with
high ryanodine (as is the case with all of the experiments here). These
are backflux (1-6) and passive SR Ca leak across the membrane. To
measure Epump with and without PLB we
must first determine the extent to which passive leak rate contributes
to SR Ca efflux in our system.
Fig. 1A shows the protocol
that we used to measure this leak rate. When
[Ca]c was ~100 nM,
Thg (25 nmol/mg) was added. Thg forms a dead-end complex with the SR Ca
pump (20) thus inhibiting both SR Ca influx and backflux through the
pump. [Ca]c gradually rose until
the SR Ca pump was completely blocked. Passive leak continued until the
SR was empty of Ca. [Ca]T was computed at each time point using known Ca binding constants
(Table I).
[Ca]SRT was computed as
[Ca]T at the end of the leak minus
[Ca]T before Thg, and both
[Ca]SRT and
d[Ca]SRT/dt
could be calculated for each time point. [Ca]SR was calculated using Equations 12 and 13 (10). The slope of the relationship between the
leak flux (Jleak) and the free Ca
difference across the SR membrane is
kleak (Fig. 1B).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Protocol for measuring passive SR Ca leak
from the membranes. A, 1-2-mg/ml membranes were added
to a cuvette with stirring. The cuvette contained (final
concentrations) 30 µM EGTA, 1 mM free
MgCl2, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µM oligomycin, 2 µM ruthenium red, and 0.5 mM ryanodine. Fluorescence outside the vesicles was
measured with 2 µM indo-1 and converted to
[Ca]c. ATP (4 mM) was
added to start uptake. Creatine phosphokinase (PCr; 5 units/ml) and 12.5 mM phosphocreatine regenerated ATP (note
the increased fluorescence upon phosphocreatine addition because of Ca
contamination). EGTA or Ca was added such that a plateau was reached at
approximately 100 nM
[Ca]c. Thg (25 nmol/mg protein) was
added, and [Ca]c rose until the SR
Ca pump was completely blocked. Passive leak continued until the SR was
empty of Ca. [Ca]c was converted to
total Ca using known Ca binding constants for all buffers within the
cuvette (Table I). Endogenous affinity constants come from in
vitro measurements where physiological intracellular conditions
were simulated as nearly as possible (normal ionic strength and pH
value) usually at room temperature. Exogenous EGTA and indo-1 are far
and away the dominant Ca buffering species accounting for >98% of the
Ca bound. [Ca]SRT was computed as
Ca at the end of the leak minus Ca before Thg allowing calculation of
[Ca]SRT and
[Ca]SR for all times. Thg,
B, kleak was determined as
the slope of the relationship between leak rate and
([Ca]SR [Ca]c) (6). The gray
line is the regression through the data (black
points).
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Cuvette Ca Buffering Parameters
Although all are accounted for, note that EGTA and indo-1 account for
the majority of the buffering capacity.
|
|
The following three groups of mice were compared: 1) transgenic mice
that expressed only mutant nonpentamer-forming PLB (Ref. 21; PLB-C41F),
2) PLB-KO, and 3) PLB-70 (mice that express WT PLB at the same level as
the mutant PLB is expressed in PLB-C41F). There was no significant
difference in SR Ca leak between the PLB-C41F group, the PLB-70 group,
and the PLB-KO group (0.058 ± 0.011 versus 0.061 ± 0.010 versus 0.045 ± 0.006/min, Student's t test, p > 0.05, n = 6).
We conclude that passive SR Ca leak is unchanged in mice containing
mutant or wild-type PLB or in PLB-KO. Furthermore, there was no
evidence of SR Ca leak through a pentameric PLB channel under these conditions.
SR Ca Influx and Backflux Assay--
Fig.
2A shows the protocol for
measuring unidirectional SR Ca fluxes along with a typical experiment.
45Ca uptake is started with the addition of ATP and
ventricular membranes to the uptake medium as described under
"Materials and Methods." Incubates were filtered at the indicated
times to measure [Ca]SRT. At
90 s excess nonradioactive 40Ca buffered with 50 mM EGTA was added. The high Ca/EGTA concentration diluted
the 45Ca to negligible levels while maintaining a
[Ca]c of 100 nM. Under
this condition, unidirectional SR 45Ca efflux took place.
Note that the actual [Ca]SRT and the fluxes at steady-state have not changed here. The backflux has only
been "uncovered" by the dilution of 45Ca outside the
vesicles. This is demonstrated in Fig. 2B, where Ca flux
rates were calculated from SR Ca content data as in Fig. 2A.
In this case mean kinetic parameters were taken from the mean PLB-KO
data (see below). Fig. 2A (dashed line) shows
that although the [45Ca] has changed,
[Ca]c and
[Ca]SRT (and
[Ca]SR) have not. The
45Ca efflux gives a measure of the unidirectional backflux
at steady-state, but the net total Ca flux is still zero. Also note the
rapid loss of 45Ca compared with the leak in Fig.
1A (translated into the gray dashed line in Fig.
2A). Therefore nearly all of this 45Ca efflux is
backflux through the SR Ca pump (Fig. 2A).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
A, the protocol for measuring
unidirectional SR Ca efflux is shown. 45Ca uptake is
started with the addition of ATP. Incubates were filtered at the
indicated times to measure intravesicular Ca content. At 90 s,
excess nonradioactive 40Ca buffered with 50 mM
EGTA was added, diluting the 45Ca on the extravesicular
(cytosolic) side of the membranes. This resulted in 45Ca
efflux with [Ca]c maintained at 100 nM. The points are the individual time points
(i.e. the raw data from a single experiment). The
black dashed line indicates the theoretical steady-state
[Ca]SRT
(45Ca + 40Ca).
The gray dashed line is calculated from the leak data of
Fig. 1. It is the estimated Ca that is lost because of leak (as opposed
to pump reversal). Note the rapid loss of Ca compared with Fig. 1
indicating that the majority of this 45Ca efflux is
pump-mediated backflux. B, balance of unidirectional Ca
influx and efflux rates during net SR Ca uptake (for example as in
A). Flux rates are calculated from the mean PLB-KO data
(Fig. 3). Note that the efflux rate increases and the influx rate
decreases as SR Ca content rises until these nearly balance and the net
uptake rate becomes 0.
|
|
As can be seen in Fig. 3A,
45Ca began to accumulate in the SR immediately upon ATP and
membrane addition in both WT and PLB-KO. [Ca]SRT came nearly to steady-state
where Ca influx and efflux are equal by 90 s.
[Ca]SRT-SS as determined by Equation 3 in WT was ~25% of that in PLB-KO (0.9 versus
4.1 nmol/mg of protein). This important result may reflect a lower SR
[Ca] gradient and therefore a lower
GSRCa (Equation 2) at steady-state.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
The figure shows the full time course of
uptake (A) and efflux (B) experiments
in WT and PLB-KO mice. The assay is performed as in Fig. 2. WT
mice took up Ca with a lower initial rate (0.13 versus 0.31 nmol/mg/s) to a lower steady-state
[Ca]SRT (0.9 versus 4.1 nmol/mg protein). The efflux rate is higher in WT (0.065 versus 0.037 nmol/mg/s) at a
[Ca]SRT of 0.8 nmol/mg. Data are
mean ± S.E.; n = 5-6. The data are significantly
different (two-way ANOVA, p < 0.05).
|
|
Note that in some experiments, we found a varying percentage of the
total Ca taken up did not come out of the membranes upon addition of
efflux buffer (the nonzero plateau in Fig. 3B).
This Ca appears to be pump-insensitive. Characterization of the
kinetics of the SR Ca pump is therefore only in terms of Ca that was
available for both uptake and efflux from the membranes.
[Ca]SRT-SS is therefore the plateau
in Fig. 3A minus the efflux-insensitive Ca (i.e.
the plateau in Fig. 3B). Note that this treatment
is conservative in that it tends to minimize differences between WT and
PLB-KO.
From the higher [Ca]SRT in PLB-KO
we conclude that the efficiency of SR Ca uptake is lower in the
presence of PLB (Equation 1). The only alternatives would be (1) a
difference in leak flux (not the case, see above) or (2) different
intra-SR Ca buffering. The latter is highly unlikely, because the
calsequestrin concentration is the same in PLB-KO and WT mice (22), and
the concentration would have to be five times higher to explain the results. These results are also consistent with findings of much higher
SR Ca load in PLB-KO intact myocytes (23).
Having examined the raw data, we can now further characterize these
experiments in a quantitative manner. Two different methods can be used
to determine the SR Ca pump efficiency from the data. First previously
determined SR Ca buffering characteristics (10) were used to convert
vesicular [Ca]SRT to
[Ca]SR (using Equations 12 and 13;
see also Table II). Note that these values are similar to the 7000:1
[Ca]SR:[Ca]c ratio (~700 µM
[Ca]SR) expected from the results
of Shannon and Bers (10). From this ratio, the SR Ca pump efficiency
was determined using Equations 1 and 2. We found a basal
GSRCa in WT mice of 33.5 kJ/mol (56.8% efficiency). PLB-KO had a much higher
GSRCa (43.9 kJ/mol; 74.5%). These
results are summarized in the top half of Table II and
graphically in Fig. 5.
View this table:
[in this window]
[in a new window]
|
Table II
SR Ca pump efficiency as determined from the SR [Ca] gradient and
from the uptake kinetics, respectively
The analysis assumes SR Ca buffering parameters from Shannon and Bers
(10), GATP = 59 kJ/mol (13), and a Hill
coefficient of 2. The KCa-f values in the bottom
part of the table are from Ref. 14. Vmax is
calculated from the initial forward uptake rate. This same
Vmax value is used to calculate
KCa-r from the rate of pump-mediated backflux.
GSRCa is calculated using Equation 11.
|
|
Secondly, we used the measured uptake kinetics to calculate
GSRCa in a different manner. We
characterized the SR Ca uptake in terms of
Vmax,
KCa-f, and n in the
forward direction from Jpumpf
(i.e. the initial uptake rate) as described under
"Materials and Methods" (Table II). Similarly, backflux was also
described assuming Vmax = Vmaxr and nf = nr. Given Jpumpr and the
Vmax above, the
KCa-r was determined from Equation 8.
Table II shows that the KCa-r in
PLB-KO is dramatically increased (1.401 versus 0.298 mM SR) consistent with the decreased rate of efflux observed in this phenotype.
If the initial uptake rate is lower in WT for the same
Vmax, n, and
[Ca]c, then
KCa-f must be higher. This will tend
to reduce
GSRCa according to
Equation 11. Indeed initial uptake rates are lower in WT (0.13 versus 0.31 nmol/mg/s; see Fig. 3A), and this
higher KCa-f is consistent with
extensive data on the effect of PLB on forward SR Ca pumping (21,
22).
Similarly for a given Vmax,
[Ca]SRT, and
[Ca]SR a higher rate of efflux in
WT versus PLB-KO may be because of a lower
KCa-r. A decreased
KCa-r in WT would also tend to reduce
GSRCa (Equation 11). The rate of
backflux at [Ca]SRT = 0.8 nmol/mg was therefore evaluated using Equation 6 (Fig. 3B). Note
that the rate of efflux is measured at 0.8 nmol/mg efflux-sensitive Ca
(i.e. above the plateau in Fig.
3B). The rate of efflux in WT (0.065 nmol/mg/s) was higher
than the rate of efflux in PLB-KO (0.037 nmol/mg/s). These results
together with Equations 8 and 11 allows calculation of
GSRCa. The results are summarized in Table II. The values compare favorably with those determined by the
first method above. Taken together the values are consistent with a
PLB-dependent decrease in SR Ca pump efficiency (see
Equation 1 and Fig. 5).
In addition, if the apparent decrease in SR Ca pump efficiency is
because of the presence of PLB, we would predict that the response to
phospholamban will be graded (i.e. the less PLB,
the less the decrease in Epump). The
PLB-70 mice express PLB at 70% of the level seen in WT. This group
shows SR Ca influx and backflux characteristics that fall between the
PLB-KO and WT groups (Fig. 4). The
initial rate of influx for PLB-70 was 0.17 with a backflux rate at 0.8 nmol/mg of 0.051 nmol/mg/s resulting in a
[Ca]SRT-SS of 2.2 nmol/mg protein.
All of these values fell between the values for PLB-KO and WT in Fig.
3.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
The time course of uptake and efflux
experiments in PLB-70 (70% of WT [PLB]) are demonstrated. Data
are mean ± S.E.; n = 3. The assay is performed as
in Fig. 2. PLB-70 mice took up Ca with an initial influx rate of 0.17 nmol/mg/s to a steady-state [Ca]SRT
of 2.2 nmol/mg protein. The efflux rate was 0.051 nmol/mg/s. This value
is between the levels seen in WT and PLB-KO groups, indicating that the
difference in [Ca]SRT is graded
with PLB. C, D, and E summarize the
results from A and B and from Fig. 3.
[Ca]SRT is higher in PLB-KO
(C) with a higher initial influx rate (D) and a
lower efflux rate at 0.8 nmol/mg (E). PLB-70 is intermediate
reflecting an intermediate [PLB]. These results indicate that PLB may
decrease the efficiency of the SR Ca pump. Standard errors in
C are for the plateau values in A and in Fig.
3A and are generated by the fitting program. All values in
C are statistically different (one-way ANOVA).
|
|
Further analysis, as above for WT and PLB-KO, indicate a basal
GSRCa in PLB-70 of 38.9 kJ/mol
with 66.0% efficiency. These values are also intermediate between
those of the WT and PLB-KO mice (33.5 kJ/mol, 56.8% and 43.9 kJ/mol,
74.5%, respectively). These results are summarized in the top
half of Table II and graphically in Fig. 4, C-E and in
Fig. 5.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
The figure demonstrates the efficiency of the
SR Ca pump ± PLB.
[Ca]SR/[Ca]c
values are taken from Table II, and the thick line
corresponding to GSRCa is based
upon Equation 2. The right ordinant shows apparent energetic
efficiency
( GSRCa/ GATP;
Equation 1) as a percent value.
|
|
 |
DISCUSSION |
In the present study, we have provided the following new
information: 1) passive leak from the SR with RyR
(ryanodine receptors) blocked is small relative to
backflux through the Ca pump, 2) passive leak from SR membranes is not
PLB-dependent, 3) the efficiency of the Ca pump is
decreased in the presence of PLB, and (4) PLB regulates this pump
efficiency by decelerating influx while accelerating backflux through
the SR Ca pump.
Passive Ca Leak from SR Vesicles--
In all of the experiments
presented, the SR RyR have been blocked with high (0.5 mM)
concentrations of ryanodine. Under these conditions, only two efflux
pathways are present, Ca efflux through the reverse mode of the SR Ca
pump (Refs. 1-6, backflux) and passive Ca leak from the SR
(which could in principal include flux via residual unblocked ryanodine
receptors). To evaluate the backflux in our experiments, we first
needed to make sure that leak would not interfere with our measurements
to any significant extent.
We accomplished this by measuring the passive leak from the SR with
both SR Ca pump influx and backflux blocked with thapsigargin (Fig. 1).
SR Ca leak from the vesicles was less than 1% of our backflux
measurements (Fig. 2) indicating that leak was a minimal amount of the
total efflux and would not be expected to alter SR Ca content (9).
PLB coexists in the cardiac myocyte in both a monomeric and pentameric
form (11, 24). The pentameric form might form a channel within the
membrane and thus mediate SR Ca leak (12). We therefore measured leak
in 3 different types of mice (PLB-70, PLB-C41F, and PLB-KO). The
PLB-C41F group contained only mutant PLB, which does not form
pentamers. The data showed no difference between the leak rates in
PLB-70 and PLB-C41F (0.061 ± 0.010 versus 0.058 ± 0.011/min rate constants) indicating that the putative pentameric
PLB channel did not conduct Ca under the conditions of our assay.
Indeed, the leak rate from PLB-KO mice was not different from these two
groups indicating that the presence or absence of SR PLB made no
difference in our results for Ca leak. Thus our data do not provide
support for a functionally relevant leak pathway created by PLB under
our experimental conditions.
SR Ca Pump Fluxes--
Forward and reverse fluxes are by
definition equal and opposite at steady-state
[Ca]SRT when leak is negligible. As
uptake takes place and SR Ca accumulates, reverse pump-flux rate rises
to meet the forward pump-flux rate (Fig. 2B). When leak is
small (as it is here) the pump is expected to reach this point where
the
[Ca]SR/[Ca]c gradient is at its thermodynamic limit. We found that
[Ca]SRT rose exponentially to a
steady-state value of [Ca]SRT-SS in
WT, which was 22% of that in PLB-KO (Fig. 3). From this result, we
suggest that PLB may lower the energetic efficiency of the SR Ca pump.
Consistent with this interpretation, kinetic analysis of the forward
and reverse Ca pump flux indicated that the initial rate of uptake was
higher in PLB-KO, whereas the rate of backflux at the same
[Ca]SRT was lower. This implies a
substantially lower
KCa-r:KCa-f
ratio in WT, which would again imply lower
GSRCa and energetic efficiency of
the SR Ca pump in WT versus PLB-KO. In addition, the
response to PLB appears to be graded with less of an effect in
membranes from mice that have only 70% of the normal PLB levels (Fig.
4).
Physiological and Pathophysiological Significance--
The
decrease in SR Ca pump efficiency suggested here may have importance to
both the understanding of normal physiology and of cardiac disease
states. Both in PLB-KO mice and when PLB is phosphorylated
[Ca]SRT goes up (23). The increase
in [Ca]STR leads to higher peak
twitch [Ca]c both in part because
there is more Ca available for SR Ca release and because the fraction
of that total SR Ca that is released rises (25-32).
The question of why [Ca]SRT goes up
is a thorny one, however. It is well established that PLB
phosphorylation increases the rate of SR Ca uptake and
[Ca]c decline (7, 8, 21, 23). This
has led to the hypothesis that diastolic
[Ca]SRT increases simply because
the SR Ca pump takes up more Ca in the given diastolic period. However,
such an explanation presupposes that the Ca within the SR does not have
time to approach a steady-state under normal physiological conditions
and/or that the SR Ca pump balances a large SR Ca leak at rest. Though
the pump is unlikely to reach a true thermodynamically limited gradient
during the normal diastolic interval, data from our laboratory indicate
that a large diastolic leak from the SR is unlikely (5, 6, 9, 33). If
the pump efficiency was not changed, the PLB-dependent decrease in Ca uptake rate would cause
[Ca]SRT to reach steady-state more
slowly but would result in the same maximal [Ca]SRT.
Our data here support the following alternative (though not mutually
exclusive) hypothesis: that the decreased diastolic
[Ca]SRT in WT is because of a
decrease in the SR Ca pump efficiency as defined by the steady-state SR
[Ca] gradient. A reduced ability to generate such a gradient would
naturally lead to a decrease in the steady-state SR Ca content. Our
data indicate that such a mechanism is plausible and may contribute at
least in part to the higher SR Ca accumulation typically observed in
PLB-KO mice. In apparent contrast to this, isoproterenol treatment does
not necessarily result in an appreciable increase in maximal
[Ca]SRT (9). However, this result
may be complicated by cAMP-dependent protein kinase
effects on ryanodine receptors, causing an increased SR Ca leak via Ca
release channels (34). Such a leak would tend to offset the ability of
the pump to build a larger [Ca] gradient. This would also explain why
blockage of the ryanodine receptors in isolated myocytes can sometimes
lead to a much higher [Ca]STR, especially in situations where higher SR Ca loads are favored (35). It
would also promote increased SR Ca release as Ca sparks (36, 37).
In a concurrent study, Frank et al. (14) showed that PLB
reduces the measured coupling ratio of the SR Ca pump (ratio of Ca
taken up to ATP consumed) for [Ca]c
at or below 300 nM (relevant to conditions here and
physiological). How does the apparently lower stoichiometry from rate
measurements relate to the present data and conclusions concerning
thermodynamic efficiency? Their rate measurements were done in the
virtual absence of Ca pump backflux, because they used oxalate to keep
[Ca]SR from rising. Their
complementary set of results may be another manifestation of the same
depressant effect of PLB on SR Ca pump efficiency. A decline in
coupling ratio means that one ATP cannot support transport of two Ca
ions. This could mean that PLB allows the pump to slip backward more
easily when [Ca]c is very low
(without making ATP). In the present study this would show up as a
lower limiting
[Ca]SR/[Ca]c gradient and lower energetic efficiency. Of course, this is
speculative, and elucidation of the molecular mechanism for this
apparent reduction in SR Ca pump efficiency by PLB will require further study.
By dissecting the effect into the component kinetic parameters, we can
observe that the influx and efflux of Ca through the pump may both
contribute their respective effects to the whole. This may become an
important distinction when considering both pathological and
pharmacological pump-associated phenomena. For instance,
GATP is decreased during ischemia,
which may lead to a decrease in
[Ca]SRT. Increasing SR Ca pump efficiency in this situation may allow
[Ca]SR and
[Ca]c to remain relatively normal.
Otherwise [Ca]SR would decline and
[Ca]c would rise. This would reduce both systolic and diastolic function. Indeed the elevated
[Ca]c may stimulate
Ca-dependent proteases and lead to mitochondrial Ca
loading, both of which can lead to cell death.
The data may also give us a better understanding of cardiac disease
states where the PLB:SR Ca pump ratio may be increased (as reviewed by
Koss et al. (38)). Such an increase would result in
decreased SR Ca not only because of reduced pump rate (reducing the
chances of reaching diastolic SR steady-state) but also because the
ability of the pump to build the normal
[Ca]SR/[Ca]c gradient would be reduced. Given this, increasing not only the rate but
the efficiency of the pump may be especially important in both
physiologic responses, as well as therapeutics.
In summary, we have measured the parameters for SR Ca efflux both
through the SR Ca pump and through passive SR Ca leak. Though we found
no PLB-dependent increase in the leak rate, a
PLB-dependent increase in SR Ca backflux through the SR Ca
pump is apparent. This increased backflux contributes to an apparent
decrease in SR Ca pump efficiency, which may be important in both
pathophysiological and physiological states.