(Received for publication, April 25, 1995; and in revised form, June 26, 1995)
From the
There is a considerable controversy in the literature concerning
the effects of higher concentrations of calcium chelators (e.g. BAPTA
(1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid) or fura-2) on the intracellular Ca transients
in muscle. We induced calcium release from sarcoplasmic reticulum (SR)
in the triad preparation by chemical depolarization of the T-tubule in
the presence of various concentrations of BAPTA-calcium buffer
([Ca
] = 0.1 µM) and
investigated the effects of the BAPTA concentration on the time courses
of conformational changes in the junctional foot protein (JFP) and
calcium release from SR. Upon stimulation, the JFP underwent biphasic
conformational changes, as determined by stopped-flow fluorometry of
the JFP-bound conformational probe. The first phase of protein
conformational change, which preceded calcium release from SR, was
virtually unaffected by the BAPTA concentration. However, the magnitude
of the second phase increased in an inversely proportional fashion to
the BAPTA concentration. An abrupt increase in
[Ca
] from 0.1 µM up to 1.0
µM (
Ca
), concurrently with T-tubule
depolarization, produced biphasic protein conformational changes: a
Ca
-independent first phase and a
Ca
dependent second phase. Similar Ca
jump experiments under non-depolarizing conditions produced a
slow monophasic conformational change equivalent to the second phase
described above. These results suggest that the first phase of protein
conformational change represents the activation of JFP by T-tubule
depolarization to induce calcium release, and the second phase the
secondary activation by the released Ca
. Activation
of the JFP by the released Ca
resulted in an
acceleration of both (i) the rate of initial calcium release, and (ii)
the subsequent attenuation of calcium release. The acceleration of both
was suppressed by higher concentrations of BAPTA. These results provide
a reasonable explanation for both of the apparently contradictory views
in the literature; high concentrations of calcium buffer (a)
suppress the initial activation and (b) prevent the subsequent
attenuation of calcium release.
The activity of the SR ()calcium release channel is
controlled by the cytoplasmic Ca
concentration in a
biphasic fashion, as shown in efflux studies with SR vesicles (1, 2, 3) and channel conductance
measurements of purified JFP incorporated into bilayers(4) .
Thus, the channel is activated at lower
[Ca
] with AC
0.5
µM and inhibited at higher
[Ca
] with IC
0.15
mM, suggesting that there are two classes of calcium sites
involved in the channel regulation. An abrupt increase of
[Ca
] in the activating range of
[Ca
] induces SR calcium release (5) as well as transient increase in channel open
probability(6) .
In the case of the voltage-dependent
Ca transient in the skeletal muscle fiber system,
however, the modes of regulation of SR calcium release by the
cytoplasmic Ca
are rather complex as seen in the
considerable controversy existing in the
literature(7, 8, 9) . According to one view,
an abrupt increase in the concentration of the sarcoplasmic free
Ca
(a Ca
jump) resulting from the
depolarization-induced SR calcium release leads to a rapid attenuation
of release
flux(10, 11, 12, 13, 14) .
According to the widely referred model originally proposed by Schneider
and Simon(12) , rapid binding of the released calcium to the
JFP is followed by a slow conformational change, leading to an
inactivation of the channel. According to the opposing view, the
Ca
jump activates SR calcium release probably by a
Ca
-induced Ca
release
mechanism(15, 16, 17) . Such a positive
feedback mechanism would be important for the activation of the group
of JFPs which is not physically coupled with the T-tubule voltage
sensor(18) . A considerable amount of evidence also suggests
that the binding of the released Ca
to the T-tubule
voltage sensor re-activates the group of JFPs that has been stimulated
by mediation of the voltage
sensor(19, 20, 21, 22, 23, 24, 25) .
The most frequently used method in the literature to distinguish
Ca
-induced inactivation versus Ca
-induced activation of calcium release is to
try to suppress the changes in the cytoplasmic
[Ca
] with rapidly reacting high affinity
calcium buffers such as BAPTA or fura-2. In this test, a decrease of
the release rate suggests Ca
activation, while an
increase suggests Ca
attenuation. Such experiments
have been carried out by several laboratories with controversial
results. For example, the intracellular fura-2 (2-3 mM)
activated the release rate in both voltage-clamped and action
potential-stimulated fibers, in favor of the view of Ca
inactivation(26, 27, 28) . Conversely,
an equivalent concentration of BAPTA or fura-2 virtually eliminated
depolarization-induced Ca
transient under
voltage-clamped conditions in favor of Ca
activation(15) . A similar blocking effect by
4 mM BAPTA was also reported in E-C coupling in
cell homogenates of rabbit skeletal muscle(29) .
The
isolated triad system, which mimics physiological E-C coupling (30) and is freed from cytoplasmic calcium-binding proteins,
would permit straightforward analysis of the calcium release time
course. The main purpose of this study is to investigate how the
changes in the concentration of BAPTA-calcium buffer
([Ca] = 0.1 µM) affect
the kinetics of the JFP conformational change and SR calcium release
induced by T-tubule depolarization, using the isolated triad system. As
shown here, there was an alteration in the kinetics of both protein
conformational change and calcium release when the concentration of
BAPTA was varied. Thus, stimulation of the JFP at lower
[BAPTA] produced biphasic protein conformational changes, as
evidenced by a biphasic increase of the fluorescence intensity of the
JFP-bound probe, MCA. The second phase, but not the first phase, was
reduced upon increasing the BAPTA concentration. Conversely, the
Ca
jump, applied concurrently with T-tubule
depolarization, from 0.1 µM up to 1.0 µM
increased the magnitude of the second phase of protein conformational
change without affecting the first phase, suggesting that the second
phase of protein conformational change represents a secondary
activation of JFP by the released Ca
. At lower
concentrations of BAPTA, under conditions in which an increase in the
[Ca
] in the vicinity of the Ca
channel (
Ca
) was sufficiently large,
calcium release was accelerated in an early phase and rapidly reached a
maximal level. Analysis of the d(calcium release)/dt curves
suggested that both initial increase and subsequent decrease in the
calcium release rate by
Ca
occurred in a coupled
manner. Thus, it appears that calcium release involves two sequential
reaction cycles as follows. In the first cycle, the triggering signal
applied to the JFP moiety of the triad via the T-tubule produces
conformational changes in the JFP to open the calcium release channel
and to induce calcium release from the SR. The released Ca
stimulates the JFP again, inducing the second cycle of the
reaction involving protein conformational change
SR calcium
release.
The present results suggest that the released
Ca accelerates both initial activation and subsequent
attenuation of calcium release in a synchronized fashion. This
mechanism provides a reasonable explanation for both of the two
controversial views in the literature described above.
The time courses of SR calcium release were
monitored with a stopped-flow fluorometer (BioLogic SFM-3 with MOS-200
optical system) using fluo-3 as a Ca indicator
(excitation at 437 nm, emission at 530 nm with a 510-nm cut-off
filter). Approximately 30 traces of the fluo-3 signal were averaged for
each experiment.
In some experiments, a Ca jump
was applied concomitantly with T-tubule depolarization or in
non-depolarizing conditions. For this purpose, the triads were
incubated in the priming solution for 6-7 min, and
[Ca
] was adjusted to 0.1 µM by
adding 10 mM BAPTA-calcium buffer. Then, 15 µl of solution
of the primed triads was mixed with 135 µl of depolarizing solution
or non-depolarizing control solution containing 10 mM BAPTA-calcium buffer adjusted to various concentrations of
Ca
in a range of 0.1-1.0 µM.
The time courses of fluorescence change of the protein-bound MCA (excitation at 368 nm, emission at 440 nm using an interference filter with 70-nm bandwidth) induced by depolarization were monitored with the same stopped-flow fluorometer. Approximately 60 traces of the MCA fluorescence signal were averaged for each experiment.
To calculate
the net calcium release (nmol/mg) from the fluorescence intensity of
fluo-3 in the presence of various concentrations of BAPTA-calcium
buffer, each of the 1000 data points of the fluo-3 trace was calculated
by using appropriate association constants for all ligands and metals
present in the reaction solutions. In this calculation, we assumed that
the rates of binding of the released calcium to the buffer (35) and to fluo-3 (36) were significantly higher than
the rate of calcium release itself. The coefficient of (fluo-3
signal)/(the change in [Ca]), required for
the above calculation, was determined at each
[Ca
] in the range of 0.01-1.0
µM adjusted with the BAPTA-calcium buffer.
Figure 1:
A, top of panel, time
courses of the changes in the fluorescence intensity of the MCA probe
attached to the JFP moiety of the triad (namely conformational changes
in JFP) upon chemical depolarization in the presence of various
concentrations of BAPTA at 0.1 µM
[Ca]
: a, 0.1 mM; b, 0.2 mM; c, 0.4 mM; d, 10 mM. Bottomofpanel,
Ca
-dependent
portion of conformational change calculated by subtraction of the curve
obtained at 10 mM BAPTA from those obtained with lower
concentration of BAPTA. B, time courses of the MCA
fluorescence change lack the second phase when T-tubule was depolarized
in the presence of 0.1 mM BAPTA at 0.1 µM
[Ca
]
without loading the SR moiety with calcium: see curvea(-Ca). Curvesa and d are the same experiments as those in A. Each trace
was obtained by signal-averaging a total of 180-300 traces
originating from three to five different experiments. The curves were
traced by fitting a double-exponential model: y = A
(1
-e
In the experiment shown in Fig. 1B (curvea(-Ca)), we carried out the same experiment as in Fig. 1A (curvea, 0.1 mM BAPTA), but without calcium loading of the SR moiety. Under these
conditions, T-tubule depolarization would induce JFP protein
conformational change but no calcium release. As seen, protein
conformational change showed a monophasic time course, which was
essentially identical to the curve obtained with calcium-loaded triads
in the presence of 10 mM BAPTA (Fig. 1B, curved). This is again consistent with the notion
that the second phase of protein conformational change is produced by
the released Ca.
In order to assess the magnitude
of Ca
that is required to produce the comparable
size of the second phase of protein conformational change observed in Fig. 1A, we generated, concurrently with T-tubule
depolarization (Fig. 2A) or dilution with the
non-depolarizing control solution (Fig. 2B), various
levels of Ca
jump from 0.1 µM up to 1.0
µM using strong BAPTA-calcium buffers. As shown in Fig. 2A, the Ca
jump together with
T-tubule depolarization produced the second phase of protein
conformational change, with virtually no effect on the first phase. The
Ca
jump without depolarization produced a monophasic
slow conformational change (Fig. 2B); there was no
voltage-dependent rapid conformational change in this case.
Interestingly, the curves obtained without depolarization (Fig. 2B) show a surprising similarity to those curves
representing the
Ca
-dependent portion of the
depolarization-induced conformational change shown in the lower
half of Fig. 1A.
Figure 2:
Time courses of the changes in the
fluorescence intensity of the MCA probe attached to the JFP moiety of
the triad upon application of various levels of Ca
jump concomitantly with dilution with the depolarization solution (A) or the non-depolarizing solution (B). The
Ca
jump was from 0.1 µM to various
levels (in µM) as indicated. The MCA-labeled triads were
incubated in a priming solution containing loading calcium as described
under ``Experimental Procedures,'' and the solution
[Ca
] was adjusted to 0.1 µM by
adding 10 mM BAPTA-calcium buffer. The primed triads were then
mixed with the depolarizing (A) or non-depolarizing control (B) solutions buffered at various
[Ca
]s as indicated. Each trace was obtained
by signal-averaging a total of 240 traces originating from four
different experiments.
Figure 3:
Time courses of calcium release (A), d(calcium release)/dt (B), and the
changes in [Ca] induced by chemical
depolarization in the presence of various concentrations of BAPTA at
0.1 µM [Ca
]
(C): a, 0.1
mM; b, 0.2 mM; c, 0.4 mM. Inset to panelA, the same data shown in a
longer time scale. Each trace was obtained by signal-averaging a total
of 90 traces originating from three different experiments. To obtain
the calcium flux curves shown in B, first derivatives of the
Ca
curves (A) were calculated. The resultant
d(calcium release)/dt curves were fitted by the following
equation, which is the solution of Model 1 describing
R
,
Figure S1: Scheme 1.
Fig. 3C illustrates the time course of the changes
in the free Ca concentration during the
depolarization-induced calcium release reaction in the presence of
three different concentrations of BAPTA. As seen, the maximum limit of
Ca
produced in the reaction solution was not
more than 30 nM. If we consider the changes of Ca
during the earlier phase of the reaction where sharp changes
occurred in the d(calcium release)/dt curve, the
Ca
value affecting the release kinetics would
have been in a range of several nM. From the results of the
Ca
jump experiment shown in Fig. 2, we
postulate that the
Ca
which is actually
affecting the release kinetics is not a several nM
Ca
in the reaction solution (Fig. 3C) but a submicromolar Ca
jump (Fig. 2), which presumably is occurring in a compartmentalized
region within the JFP (see ``Discussion'').
One of the important unsettled questions in muscle physiology
is as to whether the transient increase in the sarcoplasmic free
Ca concentration due to voltage-dependent activation
of skeletal muscle fiber (i.e.
Ca
)
produces positive or negative feedback effects on SR
calcium release, as outlined in the ``Introduction.'' In the
present study we addressed this question using our in vitro E-C coupling assay system, which offers several advantages as
follows. Chemical depolarizationinduced calcium release in this system
mimics physiological E-C coupling(30) , so that it provides a
simplified model useful for the analysis of physiological E-C coupling
at the molecular level. In particular, the purified vesicles are freed
from cytoplasmic Ca
binding proteins and permit an
unambiguous control of the ionic milieu of the reaction solution, e.g.
Ca
. The system also permits
straightforward studies of the calcium release time course.
The most
important aspect of this study is that we could deduce several novel
features concerning the feedback effects of released Ca on the kinetics of conformational changes in the JFP and SR
calcium release induced by T-tubule depolarization. The present studies
on the protein conformational change have permitted clear insight into
the modes of regulation of Ca
channel by the released
Ca
. The triggering signal applied to the JFP via
T-tubule induces biphasic changes in its protein conformation. Only the
second phase of protein conformational change seems to represent the
portion of conformational change that is induced by the Ca
released from SR, because as shown here the effects of varying
the BAPTA concentration are manifested primarily on the second phase,
and the secondary conformational change occurs usually after an
appreciable latent period. Furthermore, increasing the levels of the
Ca
jump applied concurrently with T-tubule
depolarization produced larger protein conformational change in the
second phase with no appreciable effect on the first phase. Moreover,
the Ca
jump applied without depolarization produced
only a
Ca
-dependent slow conformational change
equivalent to the second phase of the depolarization-induced
conformational change described above. All of these suggest the concept
that the protein conformational change in the first phase represents
the initial voltage-dependent activation of JFP, while that in the
second phase the secondary activation by the released
Ca
.
The effective size of Ca jump for the production of protein conformational change in its
second phase was in the submicromolar range, as determined in the
experiment of Fig. 2. In contrast, the actual changes in the
Ca
concentration during the initial phase of calcium
release, determined in the reaction solution (Fig. 3C),
are in the range of a few nM, which seems to be too small to
account for the large feedback effects on the calcium release kinetics
observed here. This would indicate that the
Ca
that is actually affecting the release kinetics is not the
Ca
in the reaction solution, but a submicromolar
Ca
jump that is occurring in some compartmentalized
region, the most likely location of such a compartment being in the
cytoplasmic portion of the JFP. As a matter of fact, the recently
published three-dimensional images of the JFP tetrameric complex show a
discernible internal pocket in the T-tubule-side view (see Fig. 3b in (37) and Fig. 6a in (38) ). We
postulate that the released calcium will generate a large
Ca
jump within this pocket because of its small
internal volume, and that it effectively activates the JFP. This would
be followed by dilution of the transiently compartmentalized
Ca
into the large volume of the reaction solution;
the
Ca
value determined in the reaction solution
was therefore very small. According to Stern(39) , (i) it would
be extremely difficult to buffer [Ca
] in
the vicinity of a channel pore, even with a fast buffer like BAPTA,
because the diffusion of the released calcium away from the pore is a
very rapid non-equilibrium process, and (ii) even very high
concentrations of BAPTA (e.g. 100 mM) would not
perform a sufficient control of the [Ca
] in
the close vicinity of the pore. The first point is consistent with the
discussion concerning the compartmentalized calcium we have just made.
However, the present observation that submillimolar to millimolar
concentrations of BAPTA appear to be effectively controlling the
Ca
feedback effect ( Fig. 1and Fig. 3)
is incompatible with the second point. Clearly, more work is required
along these questions regarding, in particular, the distance of the
effective calcium activation site from the channel (29) and its
relation to the diffusion dynamics of released Ca
as
well as BAPTA.
Turning to the kinetics of calcium release, a
decrease in the BAPTA concentration enhanced both the initial
activation and the subsequent attenuation of calcium release, as shown
here. This suggests that the released Ca re-activated
SR calcium release via the protein conformational change mechanism
(namely the second phase described above), which in turn was followed
by spontaneous attenuation of calcium release. Interestingly, the
faster the initial activation was, the faster the subsequent
attenuation became, suggesting that the initial activation and the
subsequent attenuation are tightly coupled processes.
All of the above observations may be explained by a relatively simple model shown in Fig. S1.
The main framework of the reaction sequences shown in the top and
bottom rows is based upon the concept derived from our recent studies.
According to it, the signal-induced calcium release from SR is mediated
by a conformational change in the JFP, which is a prerequisite
mechanism for activation of the Ca channel, and this
mechanism is commonly operating for various types of calcium release (e.g. release induced by T-tubule depolarization(33) ,
polylysine(40, 41) , and
Ca
(32) ). The model implies that upon
applying T-tubule depolarization the JFP undergoes a rapid
conformational transition from a resting state (R
)
to another state with higher MCA fluorescence
(*R
), which in turn is followed by a
third state (*R
) to open the Ca
channel, as shown in the top portion of the scheme (cf.
Reaction 2 in (33) ). The bottom portion of the scheme implies
that the resultant
Ca
of various magnitudes
(occurring presumably in the internal pocket of the JFP) stimulates the
JFP, which following the first activation phase induces a second phase
of calcium release by mediation of further protein conformational
change, forming **R
state with an even higher MCA
fluorescence level.
The scheme also contains additional reaction
steps involving isomerization of the channel protein from the open
state (*R or **R
) to an
``attenuated'' state (*R
or **R
). The
states *R
and **R
are suggested from the
present observation that the fluorescence intensity of the JFP-bound
MCA remains high at least during an early phase of calcium release
attenuation (compare Figs. 1A and 3B), although it
eventually returns to the original low fluorescence state. (
)Since the higher MCA fluorescence state (*, **) represents
an active conformation of the
JFP(32, 33, 41) , this suggests that the
observed attenuation of SR calcium release is probably due to some
mechanisms other than inactivation of the release channel per
se. Depletion of the releasable calcium in the SR store during the
initial phase of SR calcium release is widely regarded as one of the
major factors responsible for release attenuation (e.g.(11) ). In this case, the faster calcium release would
result in a sooner depletion of the releasable calcium, and in turn
result in a faster attenuation. We also propose that the faster calcium
release will cause a faster Ca
re-uptake by the SR
Ca
ATPase(42) , which in turn will lead to an
increase in the rate of attenuation of calcium release. Thus, at least
these two factors would account for the present observation that the
initial activation and the subsequent attenuation of calcium release
occur in a coupled manner. Further studies are required to characterize
the mechanisms responsible for the release attenuation.
To test the
above reaction model, the time courses of protein conformational change (Fig. 4A), calcium release (Fig. 4B),
and d(calcium release)/dt (Fig. 4C) curves
were constructed by simulation of the model. These simulated curves
represent reasonably well the overall features of the effects observed
in the present experiments. Note that in this simulation all of the
parameters were kept unchanged, except for the concentration of BAPTA
buffer. The fact that we can still see the acceleration of both initial
activation and subsequent attenuation (cf. Fig. 4C) even without changing any reaction constants
suggests that the initial activation and the subsequent attenuation of
calcium release are controlled solely by the feedback
Ca. An interesting consequence of this mechanism is
that the control of both initial activation and subsequent attenuation
by
Ca
occurs in a synchronized fashion.
Figure 4:
Simulation of the feedback reaction model (Fig. S1) produces curves that are qualitatively similar to
those obtained in the present stopped-flow experiments: A,
protein conformational change; B, calcium release curve; C, d(calcium release)/dt curve. The arbitrary rate
constants used for simulation were: k = 30, k
= 0.3; k
= 5, k
= 0.05; k
= 3, k
= 0.3; k
= 200, k
= 2.0; k
= 5, k
= 0.05; k
= 3, k
= 0.3. Note that a variation only in the BAPTA
concentration (i < ii < iii < iv) resulted in major changes in the curve shape observed in
the present experiments.
According to the above mechanism, suppression of the initial
Ca
-dependent acceleration by using high
concentration of calcium-chelating agents should inevitably lead to the
suppression of the rate of subsequent attenuation. Thus, the present
study provides a reasonable explanation for both of the apparently
contradictory observations in the muscle fiber literature concerning
the effects of high concentrations of calcium-chelating agents on the
voltage-dependent Ca
transient as outlined in the
Introduction. However, these studies in the literature have been done
in frog muscle fibers, while the present study is in the vesicular
system isolated from rabbit. Therefore, the present information may not
be immediately applicable to the fiber system at least in some
quantitative details.