(Received for publication, March 21, 1995)
From the
Once two radioactive Ca
Sarcoplasmic reticulum Ca
Figure S1:
Scheme 1
Ca
Figure 2:
Ca
Such a
description of a channel-like structure for the transport sites was
first proposed by Inesi(5) . Inesi (5) took advantage
of the possibility to selectively place one The
question whether the dissociation of the Ca The discrepancy between the results of Inesi (5) on the one hand and those of Hanel and Jencks (9) and Orlowski and Champeil (10) on the other hand is
difficult to understand, because they all used similar experimental
conditions, typically pH 6.8-7.5, room temperature, 80-100
mM KCl. Here we show that with 300 mM KCl, at pH 8
and 5 °C, the lumenal dissociation of the two Ca SR vesicles were prepared and tested as described in (1) from rabbits subjected to a 48-h starvation diet to lower
the contamination by phosphorylase(11) . All experiments were
carried out at 5 °C in a cold room, and the buffer was always 100
mM Tes-Tris, pH 8, 300 mM KCl. It was prepared with
water filtered through a Milli-Q Water Purification System (Millipore).
All salts were added as chlorides. Vesicles were made leaky by an
incubation of at least 1 h at 2 mg/ml in 50 mM Tris, 10 mM KCl, 2 mM EDTA, at room temperature.
All solutions containing
[ [
If it is assumed that the dissociation of the two
Ca
Fig. 1shows Ca
Figure 1:
Ca
Steady-state ATPase
activity was measured as described above, using tight vesicles
permeabilized by 4% (w/w) A23187 or leaky vesicles. The steady-state
activity measured in the presence of 3 mM Mg
The
two sites are sketched in Fig. 2, which also reports the results
of cytoplasmic Ca
Figure 3:
Ca
This so-called occluded
Ca Almost all the occluded Ca
Figure 4:
Ca
Figure 5:
Ca
The
experiments reported in Fig. 3and Fig. 4were identical,
except that the vesicles in Fig. 4were leaky. Thus, although in
the phosphorylated ATPase the Ca
Figure 6:
Bound
[
Phosphorylated ATPase was formed as described in the legend to Fig. 5and perfused with 1 mM
Figure 7:
Effect of Mg
Understanding how Ca
Under these conditions, the
ATPase activity was effectively low, and the vesicles were able to
accumulate Ca The
luminal Ca
where the departure of one Such a sequence would provide a stable phosphoenzyme in the presence
of cold Ca Our data show a close similarity between the transport sites of the
nonphosphorylated ATPase that are accessible from the cytoplasm and
those of the phosphorylated ATPase that are accessible from the vesicle
lumen. In both states, the transport sites interact with each other as
bulk Ca Our results agree with the conclusion of
Inesi (5) whose model includes luminal sequential dissociation,
but they differ from those of Hanel and Jencks (9) and Orlowski
and Champeil (10) who found that the two Ca Finally, as Hanel and
Jencks (9) found the same rate of internalization of
Ca Our finding that Ca We
have shown here that the lumenal dissociation of the Ca
coming from the
cytoplasm are bound to the transport sites of the nonphosphorylated
ATPase, excess EGTA induces rapid dissociation of both ions, whereas
excess nonradioactive Ca
only reaches one of the two
bound Ca
. This difference has been explained assuming
that the two Ca
sites are in a single file channel in
which the superficial Ca
is freely exchangeable from
the cytoplasm, whereas the deeper Ca
is exchangeable
only when the superficial site is vacant. The same experiment was done
using phosphorylated ATPase to determine whether Ca
dissociation toward the lumen is sequential as well. Under
conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky
vesicles, 5 °C, pH 8, 300 mM KCl), we found the same two
pools of Ca
. Excess EGTA induced dissociation of both
ions together with dephosphorylation. Excess nonradioactive
Ca
induced the exchange of half the radioactive
Ca
without any effect on the phosphoenzyme level. Our
results show a close similarity between the transport sites of the
nonphosphorylated and the phosphorylated enzymes, although the
orientation, affinities, and dissociation rate constants are
different.
-ATPase is a
membranous enzyme that pumps Ca
from the cytoplasm of
muscle cells into the reticulum lumen, requiring ATP hydrolysis. The
ATPase cycle transports 2Ca
per molecule of ATP and
per monomer of ATPase, as described in Fig. S1. During the
cycle, the transport sites change their orientation and affinity,
depending on whether the ATPase is phosphorylated. The high-affinity
transport sites of the nonphosphorylated ATPase are accessible from the
cytoplasm, whereas once the ATPase has been phosphorylated the
transport sites have lower affinity and are accessible from the lumen.
This allows Ca
release into the SR (
)lumen and is followed by dephosphorylation of the
phosphoenzyme.
binding to E, the
Ca
-deprived nonphosphorylated ATPase, has been well
characterized. Two ions bind sequentially with a high affinity and a
positive cooperativity which both depend on the experimental
conditions, Ca = 0.1-10 µM and n
= 1.3-2, for pH 8-6, and
0-3 mM Mg
at 20 °C (1) .
The two Ca
ions are known to be kinetically
distinguishable, since Dupont (2) showed that the dissociation
of half the
Ca
bound to E was
impaired by the presence of excess
Ca
in
the medium. This has been confirmed by several authors under various
experimental conditions, different temperatures, pH, Mg
concentrations . .
.(3, 4, 5, 6, 7, 8) .
A simple example of two sites being sequentially accessible from the
cytoplasm by the two Ca
ions is a channel with a deep
site and a superficial site (see the sketch in Fig. 2). The
first ion must reach the deep site to leave the superficial site vacant
for the second ion. The Ca
bound to the superficial
site is freely exchangeable with the outer medium, whereas the
Ca
bound to the deep site is not.
dissociation from the
nonphosphorylated ATPase (leaky vesicles). Cytoplasmic sites were
saturated with Ca
by manual perfusion with 10
µM [
Ca]Ca
as
described and then perfused with either 1 mM EGTA (
) or 1
mM
Ca
(
) for various
times. Cartoons illustrating the experiment: A, initial state; B, final state for EGTA; C, final state for
Ca
.
Ca
on top of one
Ca
, to determine
whether their dissociation toward the lumen is sequential. By
monitoring the internalization of the Ca
ions after
phosphorylation, Inesi (5) concluded that their dissociation
toward the lumen is sequential and that the first Ca
bound to E is the first to be internalized.
ions
toward the lumen is sequential or not has been reinvestigated more
recently by Hanel and Jencks (9) and Orlowski and
Champeil(10) . In observing the dissociation of Ca
from the phosphorylated ATPase, Orlowski and Champeil (10) found no difference between the dissociation kinetics
induced by EGTA or cold Ca
and no difference in the
dissociation rates of each individual ion. Hanel and Jencks (9) found no difference between the Ca
internalization rates observed using empty vesicles, or vesicles
loaded with 20 mM Ca
, and also no difference
between the internalization rates of each individual ion. In both
papers, the authors concluded that the two ions cannot be kinetically
distinguished. The explanation given by Hanel and Jencks (9) is that a slow conformational change corresponding to
deocclusion of Ca
precedes fast dissociation of
Ca
from the phosphoenzyme, therefore making the
measurement of the individual Ca
dissociation steps
impossible.
ions from the phosphorylated ATPase is sequential, as is the
cytoplasmic dissociation of the two Ca
ions from the
nonphosphorylated ATPase.
[
Kinetic measurements involving
[Ca]Ca
and
[
-
P]ATP
Measurements
Ca]Ca
or
[
-
P]ATP all started with the same
incubation and rinsing steps. Vesicles (0.2 mg/ml) were first incubated
in the pH 8 buffer, plus Mg
, as specified. 1 ml of
this suspension was deposited on a filter (Millipore HA 0.45), and the
adsorbed vesicles were rinsed with 1 ml of 100 µM EGTA to
deprive the enzyme of contaminating Ca
. For
cytoplasmic Ca
dissociation experiments, ATPase was
converted to the Ca
E state by manually perfusing
the filters for 5 s with 1 ml of 10 µM [
Ca]Ca
. For lumenal
Ca
dissociation experiments, ATPase was converted to
the Ca
E-P state by manually perfusing the filters
for 5 s with 2 ml of 100 µM
[
Ca]Ca
or
Ca
, 100 µM [
-
P]ATP or ATP, and Mg
as specified. The kinetic measurements were started immediately
after this step using a rapid filtration apparatus (Biologic, Claix,
France). They were done by perfusing 1 mM EGTA, or 1
mM
Ca
, plus Mg
as specified, for various times.
Ca]Ca
or
[
-
P]ATP also contained 1 mM
[
H]glucose, which allows evaluation of the filter
wet volume, usually about 30 µl.
H and
Ca
or
P retained on the filter were simultaneously measured
by scintillation. [
-
P]ATP or
[
Ca]Ca
contained in the wet
volume was subtracted from the total
P or
Ca
counts to evaluate the phosphoenzyme and the Ca
bound
to the ATPase.
Ca]Ca
accumulation in the vesicles was measured in the presence of
[
Ca]Ca
,
[
H]glucose, ATP, and Mg
as
specified. The reaction was started by addition of vesicles at 0.2
mg/ml. After various periods of time, 1 ml of the reaction mixture was
deposited on a filter (Millipore HA 0.45), and the radioactivity
retained on the filter was counted.
Steady-state ATPase Activity
Steady-state ATPase
activity was measured spectrophotometrically by coupling ATP hydrolysis
to NADH oxidation in the presence of 0.4 mg/ml pyruvate kinase, 0.2
mg/ml lactate dehydrogenase, 1 mM phosphoenolpyruvate, 0.45
mM NADH, 1-3 mM Mg,
0.1-1 mM ATP, 300 mM KCl, at pH 8 and
6-10 °C. NADH absorbance variations were followed at 350 nm
by an HP 8452A diode array spectrophotometer.
ions from the phosphorylated ATPase is
intrinsically sequential, the fact that Inesi (5) found that
the two ions dissociate sequentially, whereas Hanel and Jencks (9) and Orlowski and Champeil (10) found that the two
ions could not be distinguished can simply indicate that this
assumption was difficult to prove under their approximately similar
conditions. Keeping this in mind, we looked for experimental conditions
that would allow measuring an effect of cold Ca
on
the lumenal dissociation of radioactive Ca
bound to
the phosphorylated ATPase. Alkaline pH has been shown to increase the
affinity of the phosphorylated ATPase for Ca
(12) and to be even more effective at low
temperatures(13) . Also high KCl concentrations are known to
favor Ca
E-P, the ADP-sensitive
phosphoenzyme(14) . Thus, 300 mM KCl, pH 8 and 5
°C, were the conditions chosen for these experiments, as we
expected to have all the ATPase in its Ca
E-P form,
with a low ATPase activity and a high enough affinity for lumenal
Ca
to see an effect of cold Ca
on
the dissociation of radioactive Ca
.
Ca
CaAccumulation into the Vesicles and
Steady-state ATPase Activity
accumulation in the vesicles and steady-state ATPase activity
were measured under the conditions of the Ca
dissociation experiments, i.e. 300 mM KCl, pH 8
at 5 °C, to check the activity of the enzyme under such conditions.
accumulation into the
vesicles during 30-min incubation in the presence of 100 µM ATP. With 3 mM Mg
, tight vesicles
accumulated 70 nmol/mg, a value that is comparable with the maximum
amount of Ca
accumulated into the vesicles under
standard conditions (i.e. 80 nmol/mg at pH 6 and 20 °C),
and the so-called leaky vesicles did not accumulate
Ca
, as was intended. In the absence of
Mg
, accumulation was lower, as expected from slower
turnover in the presence of CaATP(15) .
accumulation into
the vesicles. Vesicles were incubated for various times with 100
µM [
Ca]Ca
and 100
µM ATP and no added Mg
(tight vesicles,
) or 3 mM Mg
(tight vesicles,
,
leaky vesicles, ▪).
and 100 µM ATP was 240 nmol/mg/min at 8 °C.
Ca
Once two radioactive
CaDissociation from the
Nonphosphorylated ATPase
coming from the cytoplasm have been bound to the
transport sites of the nonphosphorylated ATPase, excess EGTA induces
rapid dissociation of both ions, whereas excess nonradioactive
Ca
induces rapid dissociation of only one of the two
bound Ca
(2) . This rapidly exchangeable
Ca
has been identified as the last Ca
bound to ATPase(5, 6) . As the dissociation of
the other Ca
is impaired by the binding of cold
Ca
at the exchangeable site, it has been identified
as the first Ca
bound to ATPase(6) .
dissociation experiments under the
present conditions, namely using leaky vesicles at pH 8 and 5 °C,
in the presence of 300 mM KCl. Ca
E was
formed by perfusing the vesicles with 10 µM [
Ca]Ca
plus 3 mM Mg
, and dissociation was initiated by perfusing
1 mM EGTA or 1 mM
Ca
,
plus 3 mM Mg
. EGTA induced biphasic
dissociation of Ca
ions with rates of 20 and 1
s
, whereas
Ca
induced
dissociation of only half the radioactive Ca
with the
rate of 9 s
. This experiment was repeated using
tight vesicles that yielded similar rates for cytoplasmic
Ca
dissociation (data not shown), indicating that the
Ca
transport sites are not modified by the incubation
in EDTA and Tris.
Ca
Phosphorylated ATPase was formed by
perfusing tight vesicles with 100 µM
[Occlusion by the Phosphorylated
ATPase (Tight Vesicles)
Ca]Ca
, 100 µM
[
-
P]ATP, 3 mM
Mg
, 300 mM KCl for 5 s. It was then perfused
with 1 mM EGTA plus 3 mM Mg
(Fig. 3, open symbols). The amounts of bound
[
Ca]Ca
and phosphoenzyme were
unchanged even after 10 s, indicating that Ca
was no
longer accessible from the cytoplasmic side.
occlusion by the
phosphorylated ATPase (tight vesicles). ATPase was first phosphorylated
by manual perfusion with 100 µM
[
Ca]Ca
, 100 µM
[
-
P]ATP, 3 mM Mg
as described and then perfused with 1 mM EGTA plus 3
mM Mg
(
,
) or 1 mM EGTA plus 300 µM ADP (
, ▪) for various
times. Bound [
Ca]Ca
(
,
), phosphoenzyme (
, ▪). Cartoons illustrating the
experiment: A, initial state; B, final state for EGTA
plus Mg
(
,
); C, final state
for EGTA plus ADP (
, ▪).
can be either occluded in the membrane in the
Ca
E-P form of ATPase as the Ca
bound to this form is not accessible from the cytoplasmic side or
it can be occluded inside the vesicles, if ATPase turnover is such that
some Ca
has been transported during the
phosphorylation step(16, 17) . The existence of these
two types of occluded Ca
is demonstrated by the
effect of ADP, as shown in Fig. 3(closed symbols).
was trapped in the
membrane in the Ca
E-P form of ATPase.
Phosphorylated ATPase, prepared as described above, was perfused with a
mixture of 300 µM ADP and 1 mM EGTA. At variance
with the perfusion with 1 mM EGTA alone, with ADP present
there was fast dephosphorylation together with Ca
dissociation. This experiment showed that all the phosphoenzyme
was ADP sensitive, as expected from the use of 300 mM KCl at
low temperature, and that there was 8 nmol/mg calcium accumulated in
the vesicle lumen during the phosphorylation step.
Ca
Phosphorylated
ATPase was formed as above, but using leaky vesicles. It was then
perfused with 1 mM EGTA plus 3 mM MgDissociation from the
Phosphorylated ATPase (Leaky Vesicles)
(Fig. 4) or 1 mM
Ca
plus 3 mM Mg
(Fig. 5). The
perfusion with EGTA induced biphasic dissociation of all the bound
[
Ca]Ca
, together with
dephosphorylation of ATPase at rates of 0.6 and 0.06
s
, whereas the perfusion with
Ca
induced biphasic dissociation of
[
Ca]Ca
at 0.4 and 0.03
s
and slow dephosphorylation at 0.03
s
. To determine whether the phosphoenzyme measured
during these perfusions was still the Ca
E-P form
of ATPase, its ADP sensitivity was tested at time 0 and after 19 s of
perfusion. That is the perfusions with EGTA or
Ca
were followed by manual perfusion of
a mixture of 300 µM ADP and 1 mM EGTA for 5 s.
These measurements are represented by filled symbols in Fig. 4and Fig. 5. They confirm that after perfusion with
EGTA or
Ca
, the phosphoenzyme was
ADP-sensitive, as it was before perfusion, and that all the
Ca
bound to the vesicles was bound to the
ADP-sensitive phosphoenzyme, as expected from leaky vesicles.
dissociation from the
phosphorylated ATPase (leaky vesicles). ATPase was first phosphorylated
as in Fig. 3and then perfused with 1 mM EGTA plus 3
mM Mg
for various times (
,
).
ADP sensitivity of the phosphoenzyme was evaluated by perfusing a
mixture of 300 µM ADP and 1 mM EGTA (
,
▪).
,
, Bound
[
Ca]Ca
;
, ▪,
phosphoenzyme. Cartoons illustrating the experiment: A,
initial state; B, final state for EGTA (
,
).
exchange from the
phosphorylated ATPase (leaky vesicles). ATPase was first phosphorylated
as in Fig. 3and then perfused with 1 mM
Ca
plus 3 mM Mg
for various times (
,
). ADP
sensitivity of the phosphoenzyme was evaluated by perfusing a mixture
of 300 µM ADP and 1 mM EGTA (
, ▪).
,
, Bound [
Ca]Ca
;
, ▪, phosphoenzyme. Cartoons illustrating the experiment: A, initial state; B, final state for
Ca
(
,
).
sites are not
accessible from the cytoplasm (Fig. 3), they are accessible from
the lumen (Fig. 4). The use of leaky vesicles enabled EGTA (Fig. 4) and
Ca
(Fig. 5)
to induce Ca
dissociation, and moreover,
Ca
impaired dephosphorylation. The
stability of the ADP-sensitive phosphoenzyme during the perfusion with
Ca
suggests that half the bound
[
Ca]Ca
was replaced by
Ca
on the Ca
E-P
form of ATPase. This appears clearly in Fig. 6which shows that
the [
Ca]Ca
/E-P ratio
varies from 1.6 to 1 during the perfusion by
Ca
, whereas it remains around 1.8 during
the dephosphorylation induced by EGTA.
Ca]Ca
to phosphoenzyme ratio
calculated from Fig. 4and Fig. 5. The ratio was
calculated after correction for the small amount of Ca
and phosphoenzyme remaining after the perfusion with a mixture of
ADP and EGTA.
, 1 mM EGTA; ▴, 1 mM
Ca
.
Sequential Dissociation or
In the experiment
reported in Fig. 4and Fig. 5, 3 mM MgCa
/Mg
Exchange at the Catalytic Site?
were present, together with EGTA or
Ca
, in the perfusion buffers. Recalling
that the phosphorylation step was done in the presence of 3 mM Mg
versus 100 µM Ca
, ATPase was phosphorylated by MgATP, so that
we can assume the presence of Mg
at the catalytic
site of the phosphoenzyme. Nevertheless, during the perfusion with 1
mM
Ca
and 3 mM
Mg
, partial substitution of
Ca
for Mg
at the
catalytic site cannot be excluded. ATPase turnover with CaATP as
substrate is known to be much slower than with MgATP, possibly because
of slower dissociation of Ca
from the
Ca
E-P form when it has Ca
at its
catalytic site(15) . Such a substitution at the catalytic site
during the perfusion could induce a slow phase in the Ca
dissociation kinetics that would be difficult to distinguish from
a slow phase due to sequential dissociation from the transport sites.
This possibility was tested by repeating the experiment described in Fig. 5with various Mg
concentrations.
Ca
and either no added Mg
or 1, 3, or 5 mM Mg
(Fig. 7).
The concentration of Mg
did not significantly modify
the Ca
dissociation kinetics, as
Ca
impaired dissociation of half the
bound [
Ca]Ca
and
dephosphorylation. The rate of the fast phase was 0.2-0.5
s
, whereas the rate of the slow phase was the same
as that of the phosphoenzyme, 0.02-0.03 s
. If
a Mg
/Ca
exchange was to take place
at the catalytic site during the perfusion, one would expect the
amplitudes and the rates of the fast and slow phases to be modified by
the Mg
/Ca
ratio in the perfusion
buffer. It is therefore more likely that the observed slow phase
corresponds to a non-exchangeable Ca
.
on
Ca
dissociation from the phosphorylated ATPase (leaky
vesicles). ATPase was first phosphorylated as in Fig. 3and then
perfused with 1 mM
Ca
and no
added Mg
(
,
), 1 mM Mg
(
, ▪), 3 mM Mg
(
, ▴), 5 mM Mg
(
, ▾) for various times.
,
,
,
, bound
[
Ca]Ca
;
, ▪,
▴, ▾, phosphoenzyme.
is transported from
the cytoplasm to the SR lumen requires some knowledge of the
Ca
binding sites on the different forms of ATPase. In
the absence of ATP, when there is no turnover, the cytoplasmic
Ca
binding reaction (step 4 in Fig. S1) has
been well characterized, as it can be studied both kinetically and at
equilibrium (1, 8, 18, 19, 20, 21) .
The situation is different for the Ca
binding sites
on the phosphorylated ATPase (step 2 in Fig. S1), which should
be studied during turnover. Knowledge of the lumenal affinity for
Ca
can be obtained with tight vesicles loaded with
Ca
. For instance, the study of phosphoenzyme
formation from P
in the presence of various lumenal
Ca
concentrations has yielded information on the
lumenal affinity for Ca
, which was found in the
millimolar range at pH 7 and 20 °C(22) . Direct access to
the Ca
binding sites on E-P requires working
with leaky vesicles. In this case, as the affinity of the lumenal sites
is lower than that of the cytoplasmic sites, addition of Ca
to E-P induces dephosphorylation and Ca
binding to E(23) . Nevertheless, de Meis et
al. (13) have shown that working on leaky vesicles ATP
could be synthesized in the absence of a Ca
gradient,
provided that a mixture of Ca
and ADP was added to E-P. In this paper, ATP synthesis was studied as a function of
the Ca
concentration under various conditions that
yielded other estimations of the luminal affinity for
Ca
. Of particular interest is that at pH 8 and 0
°C, half the maximal amount of ATP synthesized was obtained with 10
µM Ca
, instead of 300 µM at
pH 8 and 30 °C or pH 7 and 0 °C. Although these numbers should
not be taken as absolute values for Ca
affinity, it
is likely that alkaline pH and low temperature are conditions that
increase the lumenal affinity for Ca
.
Lumenal Ca
In the absence of any known
possibility to measure CaDissociation from
Phosphorylated ATPase
binding to the
phosphoenzyme directly, we have studied Ca
dissociation from phosphorylated ATPase toward the vesicle lumen
using the rapid filtration technique. The experimental conditions were
chosen to yield (i) as high as possible lumenal affinity for
Ca
, i.e. an affinity high enough to use
reasonable concentrations of
Ca
to
compete with bound [
Ca]Ca
,
(ii) as much as possible Ca
-bound phosphoenzyme,
(iii) as low as possible ATPase activity, in order to have enough ATP
in the 30-µl wet volume of the filter to maintain the ATPase
phosphorylated during the few seconds necessary to start the rapid
filtration experiment. All three requirements were achieved while
working at pH 8, 5 °C, 300 mM KCl and using 100
µM ATP for phosphorylation.
( Fig. 1and Fig. 3)
showing that the ATPase cycled normally. After phosphorylation, the
phosphoenzyme was ADP-sensitive, and there were two Ca
bound per phosphoenzyme ( Fig. 3and Fig. 4). Thus,
the vast majority of ATPase was in the Ca
E-P form,
in which Ca
is said to be occluded, because it cannot
be released on the cytoplasmic side unless the ATPase has bound ADP.
When this occluded Ca
was formed in leaky vesicles,
perfusion with EGTA simultaneously induced dissociation of the two
Ca
ions and dephosphorylation, as expected from the
fact that hydrolysis of E-P, the Ca
-deprived
phosphoenzyme, is fast in the presence of KCl(14) .
dissociation, i.e. the
EGTA-induced dissociation of Ca
from
Ca
E-P (Fig. 4) was slow compared with the
cytoplasmic dissociation of Ca
from
Ca
E (Fig. 2). It is likely that this slow
rate illustrates Ca
deocclusion from the
Ca
-bound phosphoenzyme. Keeping in mind that
hydrolysis of E-P is fast with KCl present and that the ADP
sensitivity is lost together with Ca
dissociation,
the fact that both the dephosphorylation and the Ca
dissociation are biphasic suggests that there is an equilibrium
between two forms of Ca
E-P. One form, denoted as
[Ca
]E-P, would bear occluded
Ca
, and the other form would be able to exchange
Ca
with the lumen. Thus, biphasic dissociation of
Ca
and biphasic dephosphorylation would be due to a
fast dissociation of Ca
from the non-occluded form
and the slow conversion of the occluded form into the non-occluded
form. At this point, more details on the luminal dissociation of
Ca
come from the Ca
exchange
experiments.
Sequential Dissociation from the Phosphorylated
ATPase
Comparison of the Ca exchange
experiments performed on the phosphorylated ATPase (Fig. 5) and
on the nonphosphorylated ATPase (Fig. 2) shows that cytoplasmic
Ca
exchange was faster than lumenal Ca
exchange. On the cytoplasmic side, this exchange is explained by
the following sequence,
Ca
is
followed by the binding of one
Ca
. The
same sequence on the phosphorylated ATPase suggests the existence of an
intermediate phosphoenzyme having bound only one
Ca
and able to bind one
Ca
. Taking into account the occluded
state, Ca
exchange on the phosphorylated ATPase can
be described by the following sequence.
. In the absence of lumenal
Ca
, sequential dissociation of both Ca
ions would thus occur following.
impairs the dissociation of one of the two
Ca
bound.
ions could not be distinguished once the ATPase was
phosphorylated. As stated above, this discrepancy is probably due to
the difference in the experimental conditions. For instance, at room
temperature the luminal affinity for Ca
is low and
the ATPase activity is high, so that it is necessary to phosphorylate
ATPase and to exchange cold Ca
for radioactive
Ca
simultaneously, i.e. to perfuse at least
10 mM
Ca
and ATP together. In
this case, an ATP/ADP-mediated Mg
/Ca
exchange can occur in the filter, leading to various populations
of phosphoenzymes displaying different rates at step 2 in Fig. S1, as discussed by Orlowski and Champeil (10) .
Under our conditions, there was only 1 mM
Ca
perfused, and ATP and ADP were washed
out of the filter after the first 25 ms of perfusion; thus, there was
very little possibility to have an ATP/ADP-mediated
Mg
/Ca
exchange at the catalytic
site. There still remains the possibility that the effect of cold
Ca
is due to a direct substitution of Ca
for Mg
at the catalytic site. Fig. 7shows that this was not the case, because varying the
Mg
concentration during the Ca
exchange did not change its kinetics.
whether they monitored internalization of both
ions or each individual ion into empty or Ca
-loaded
vesicles, they suggested that they could only measure a slow
deocclusion rate preceding the Ca
dissociation steps.
This slow deocclusion step was also seen in our experiments, but at
variance with Hanel and Jencks(9) , we observed different
effects when EGTA or
Ca
was used. Thus,
the comparison between their results and ours suggests that the
experimental conditions modify the equilibrium between the occluded and
deoccluded states of Ca
on the phosphoenzyme. Under
our conditions, there would be enough Ca
in the
deoccluded state to observe the dissociation of the first ion and thus
the impairment of the dissociation of the second ion in the presence of
cold Ca
.
dissociation from the phosphorylated enzyme is sequential, as is
Ca
binding to the nonphosphorylated enzyme, suggests
at first sight that during the transport cycle, the Ca
ions cross the membrane sequentially via a channel-like structure
such as the one sketched here. Nevertheless, a few points should be
emphasized. First, a channel-like structure is obviously the simplest
way to describe the interactions between the two Ca
sites, but it is not the only one. Second, a channel-like
structure also suggests that the first ion bound to the cytoplasmic
sites is the first ion to dissociate toward the lumen. Such a
first-in-first-out model has been suggested by Inesi(5) .
Third, there is a controversy about the possibility to have a
channel-like structure crossing the membrane and including the putative
Ca
sites. Site-directed mutagenesis has yielded
information on the location of the Ca
sites, as six
charged residues from the membrane helices M4, M5, M6, and M8
(Glu
, Glu
, Asn
,
Thr
, Asp
, and Glu
) were shown
to be crucial for Ca
transport(24) , and
among them, the five residues belonging to M4, M5, and M6 were shown to
be crucial for Ca
occlusion(25, 26) . According to Inesi (27) , M4, M5, M6, and M8 could form a channel providing the
residues coordinating the two Ca
ions at its inner
surface. According to Andersen(28) , the three residues
belonging to M6 cannot at the same time coordinate the two
Ca
ions and be part of the same
-helix.
ions is compatible with a channel-like structure, because it is
sequential. However, we did not try to obtain any information about
what happens in the occluded state, as we did not follow a specified
Ca
ion, i.e. the first or the second, from
the cytoplasmic side to the lumenal side.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.