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From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510-8026
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Resting sarcoplasmic reticulum (SR) Ca content ([CaSR]R) was varied in cut fibers equilibrated with
an internal solution that contained 20 mM EGTA and 0-1.76 mM Ca. SR Ca release and [CaSR]R were measured
with the EGTA-phenol red method (Pape et al. 1995
. J. Gen. Physiol. 106:259-336). After an action potential, the fractional amount of Ca released from the SR increased from 0.17 to 0.50 when [CaSR]R was reduced from 1,200 to
140 µM. This increase was associated with a prolongation of release (final time constant, from 1-2 to 10-15 ms)
and of the action potential (by 1-2 ms). Similar changes in release were observed with brief stimulations to 
20
mV in voltage-clamped fibers, in which charge movement (Qcm) could be measured. The peak values of Qcm and
the fractional rate of SR Ca release, as well as their ON time courses, were little affected by reducing [CaSR]R from
1,200 to 140 µM. After repolarization, however, the OFF time courses of Qcm and the rate of SR Ca release were
slowed by factors of 1.5-1.7 and 6.5, respectively. These and other results suggest that, after action potential stimulation of fibers in normal physiological condition, the increase in myoplasmic free [Ca] that accompanies SR Ca
release exerts three negative feedback effects that tend to reduce additional release: (a) the action potential is
shortened by current through Ca-activated potassium channels in the surface and/or tubular membranes; (b) the
OFF kinetics of Qcm is accelerated; and (c) Ca inactivation of Ca release is increased. Some of these effects of Ca on
an SR Ca channel or its voltage sensor appear to be regulated by the value of [Ca] within 22 nm of the mouth of
the channel.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Pape et al. (1995) described a method for the direct
measurement of sarcoplasmic reticulum (SR)1 Ca release and [CaSR]R in frog cut muscle fibers equilibrated with an internal solution that contains 20 mM EGTA
and phenol red. With 1.76 mM Ca in the internal solution, the value of [CaSR]R varied threefold, between
1,391 and 4,367 µM, whereas that of the fractional
amount of [CaSR]R released from the SR by a single action potential (f1) remained relatively constant, between 0.13 and 0.17. During the course of those experiments, some of the fibers were partially depleted of Ca
by decreasing the concentration of Ca in the internal
solution and by repetitive stimulation. After the value
of [CaSR]R fell below 1,000-1,200 µM, the value of f1 began to increase and, with [CaSR]R = 150 µM, it was 0.5. This increase in f1 helped stabilize the amount of Ca released from the SR. Since such stabilization may be important in helping to maintain normal contractile activation, experiments were undertaken to determine the
factors involved in the regulation of f1 by [CaSR]R.
This article describes our results. They show that three factors participate in this regulation: (a) Ca-activated potassium channels in the surface and/or tubular membranes that, when opened, can shorten the duration of the action potential; (b) the OFF kinetics of Qcm that determines the rate of removal of voltage-dependent activation during the repolarization phase of an action potential; and (c) inhibition of SR Ca release caused by an elevation of myoplasmic free [Ca] (called Ca inactivation of Ca release).
The DISCUSSION presents a comparison of the effects
on SR Ca release produced by the addition of high affinity Ca buffers to the myoplasm and by a reduction of
SR Ca content (which is expected to reduce the single-channel Ca flux). APPENDIXES B and C give derivations
of steady state and transient solutions of 
[Ca] near a
point source in the presence of different kinds of Ca
buffers. With these derivations, Appendix a shows that
some of the effects of Ca on release appear to be controlled by the concentration of Ca ions within a region
22 nm from the mouth of the channel. Such effects
of Ca could be mediated by a receptor or receptors located on the SR Ca channel (including the foot structure) and/or its voltage sensor. The appendixes also
show that, in the absence of extrinsic Ca buffers, the
concentration of Ca ions near the mouth of a channel
can contain a large contribution from Ca ions that are
released from neighboring channels and that this contribution is markedly decreased by the addition of 0.5- 1.0 mM fura-2 or 20 mM EGTA to the myoplasm. The
general features of this theoretical analysis may apply to
other nonmuscle cells.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Action potential and voltage-clamp experiments were carried out
on frog cut muscle fibers mounted in a double Vaseline-gap chamber and equilibrated with an internal solution that contained 20 mM EGTA and phenol red. The theoretical and experimental basis for the measurement of SR Ca release with EGTA-
phenol red is described in Pape et al. (1995). After stimulation by
an action potential, EGTA is expected to capture almost all
(~96%) of the Ca released from the SR, to capture it rapidly
(<0.1 ms), and to exchange it for protons with a 1:2 stoichiometry. [CaT] is calculated from the associated decrease in myoplasmic pH (which is monitored with phenol red) and the value of
myoplasmic buffering power (which was determined in a separate set of experiments with EGTA, phenol red, and fura-2). This
method for the measurement of SR Ca release has the advantage
that it is direct and little influenced by the presence of intrinsic
myoplasmic Ca buffers. Indeed, for reasons that are not completely clear, the amount of Ca that appears to be complexed by
the Ca-regulatory sites on troponin after an action potential is
negligibly small and not statistically significant, 2.9 µM (SEM, 3.8 µM) out of a total site concentration of 240 µM (column 8 in Table II in Pape et al., 1995). The expectation that the EGTA-phenol red method gives an accurate measurement of the amplitude
and time course of SR Ca release is validated by the finding that
values of the peak rate of release and time to half-peak measured
with this method are not significantly different from those estimated from Ca transients measured with low affinity Ca indicators (antipyrylazo III, tetramethylmurexide, and purpurate-3,3'diacetic acid [PDAA]) in cut fibers with only 0.1 mM EGTA
(Pape et al., 1995). On the other hand, the half-width of the rate
of SR Ca release measured with EGTA-phenol red is 0.7-0.9 ms
longer than that estimated with 0.1 mM EGTA, an increase likely
caused by the ability of 20 mM EGTA to reduce Ca inactivation of
Ca release (Pape et al., 1995). Another feature of the EGTA-phenol red method is that [CaSR]R can be monitored routinely by the
measurement of [CaT] after a train of action potentials or after
a long lasting step depolarization that releases essentially all of
the readily releasable Ca from the SR. Additional information
about the EGTA-phenol red method is given in Pape et al.
(1995).
The methods used for the determination of Qcm are described
in Chandler and Hui (1990), Hui and Chandler (1990, 1991),
and Jong et al. (1995b). For the estimation of Icm associated with a brief depolarization (see Fig. 8 A), the final level of the OFF
ITEST-ICONTROL was used for the template of the OFF ionic current, and the template of the ON ionic current was adjusted to
make the amplitudes of ON and OFF charge equal.
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In the action potential experiments, a K-glutamate internal solution was used in the end pools. It contained (mM): 45 K-glutamate, 20 EGTA as a combination of K and Ca salts, 6.8 MgSO4,
5.5 Na2-ATP, 20 K2-creatine phosphate, 5 K3-phospho(enol)pyruvate, and 5 MOPS, with pH adjusted to 7.0 by the addition of
KOH. The concentration of Ca-complexed EGTA was 0, 0.44, or
1.76 mM; at pH 7.0, the calculated concentration of free Ca was
0, 0.008, or 0.036 µM, respectively, and the calculated concentration of free Mg was 1 mM. A NaCl Ringer's solution was used in
the central pool in the action potential experiments (see Table I
of Irving et al., 1987).
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In the voltage-clamp experiments, a Cs-glutamate internal solution was used in the end pools. It was similar to the K-glutamate internal solution except that Cs replaced K and Na. The composition of the external solution used in the central pool was 110 mM tetraethylammonium-gluconate, 10 mM MgSO4, 10 mM MOPS, and 1 µM tetrodotoxin; it was nominally Ca-free and had a pH of 7.1.
In the experiments, the sarcomere length of the fibers was 3.3-
3.7 µm, the holding potential was 90 mV, and the temperature was 14-15°C. The difference between the mean values of two sets of results was assessed with Student's two-tailed t test and considered to be significant if P < 0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of a Reduction of [CaSR]R on SR Ca Release During a Train of Action Potentials
Fig. 1 A shows traces from a fiber that was initially equilibrated with an end-pool solution that contained 20 mM
EGTA and 1.76 mM Ca. The top traces show superimposed voltage records taken during four trials in which
the fiber was stimulated to give a train of action potentials with different amounts of Ca inside the SR. The
other four traces show the associated [CaT] signals.
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The first [CaT] signal (Fig. 1 A, a) was obtained 125 min after saponin treatment. The first action potential
elicited an abrupt increase in [CaT] of 445 µM. This
amount of release is similar to that obtained from other
fibers equilibrated with the same internal solution and
having a similar value of [CaSR]R (Pape et al., 1995). On
the other hand, it may be somewhat larger than that
obtained in fibers not equilibrated with EGTA because
of a reduction of Ca inactivation of Ca release caused
by the ability of EGTA to reduce myoplasmic free [Ca].
Ca inactivation of Ca release represents an inhibition
of Ca flux through SR Ca channels that is produced by
an increase in myoplasmic free [Ca] (Baylor et al.,
1983; Simon et al., 1985, 1991; Schneider and Simon,
1988). According to Schneider and Simon (1988) (see
also Jong et al., 1995a), its properties can be described
by a model in which Ca equilibrates rapidly with a receptor on the release channel, after which the Ca receptor-channel complex is able to undergo a slower transition to an inactivated state.
The amplitude of the [CaT] signal elicited by the
second action potential in Fig. 1 A, a, was only half that
elicited by the first action potential. Part of the reduction can be attributed to a decrease in [CaSR] after the
first response. Most of the reduction, however, is probably caused by Ca inactivation of Ca release produced by
the first response, even though the internal solution
contained 20 mM EGTA (Jong et al., 1995a). The responses elicited by the third and subsequent action potentials progressively decreased, and, after 20-30 action
potentials, [CaT] had reached a maximal value of
2,452 µM, which is taken for the value of [CaSR]R.
4 min after Fig. 1 A, a, was taken, Ca was removed from the end-pool solution. b-d were obtained after the SR Ca content had been reduced by repeated stimulation with a train of action potentials, delivered first every 5 min and then every 2 min. During this period, the value of [CaSR]R decreased from 2,452 µM in a to 1,760 µM in b, 1,224 µM in c, and 708 µM in d.
Fig. 1 B shows voltage and [CaT] during the first 24 action potentials in A; the [CaT] signals have been
normalized by [CaSR]R to give a maximal value of unity
and have been plotted on expanded horizontal and
vertical gains. f1 was 0.181 in a and 0.194 in b. These values are slightly larger than the mean value of 0.144 (SEM, 0.004) found by Pape et al. (1995) in 12 fibers
with [CaSR]R = 1,391-4,367 µM. The normalized increase in [CaT] produced by the first few action potentials became progressively larger from a to d, indicating a more rapid fractional depletion of Ca from the
SR. In addition, the stepwise appearance of the [CaT]
signal after each action potential became progressively
more rounded from a to d. The experiments described
below were undertaken to study possible causes of
these changes, such as a reduction of Ca inactivation of
Ca release associated with partial SR Ca depletion.
Changes in the Action Potential when [CaSR]R Is Varied between 150 and 1,200 µM
The purpose of the experiment described in Figs. 2-4 was to reduce [CaSR]R to values smaller than those used in Fig. 1 and to study the effect on the action potential, as described in this section, and on SR Ca release, as described in the following section. The value of [CaSR]R was varied reversibly between 140 and 1,154 µM by the use of 0.44 mM Ca in the end-pool solution and by variation of the recovery period between successive trials between 0.5 and 10 min.
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Fig. 2 A, top, shows superimposed voltage records
from four trials. A single action potential was followed
by a 150-ms recovery period. Then, a train of action
potentials was used to deplete the SR of its readily releasable Ca so that the value of [CaSR]R could be determined. During the first part of each trace, the interval
of time between data points was 0.12 ms, which is sufficiently brief to resolve the time courses of the first action potential and of the associated rate of SR Ca release. After the recovery period, the interval of time
between data points was increased to 0.6 ms, which allowed resolution of the relatively slow changes in
[CaT], but not the time courses of the individual action potentials in the train.
Fig. 2 A, a-d, show the [CaT] signals associated with
the four trials. The value of [CaSR]R was decreased from
512 µM in a to 296 µM in b to 146 µM in c by the use of
progressively shorter recovery periods between trials
(see legend). The recovery period was increased after c,
and, when d was taken, the value of [CaSR]R was 876 µM. More information about the [CaT] signals is given in the legend of Fig. 2 and in the following section.
In Fig. 2 B, top, superimposed traces show the first action potential of each trial, plotted on expanded vertical and horizontal gains. Action potentials a and d are similar to each other and have a shorter duration than b, which has a shorter duration than c.
The difference between action potentials is seen more clearly in Fig. 2 C, c and d, where the time scale has been expanded. The top pair of traces shows that, during the first 2 ms after stimulation, the two action potentials were virtually identical. During the next millisecond, the traces started to diverge as the slope of d became more negative than that of c.
For a membrane action potential in an axon with
constant surface capacitance (C), a more negative slope
would be expected to have been caused by an increase
in outward ionic current equal to CdV/dt (Hodgkin
and Huxley, 1952). In a muscle fiber, however, the situation is complicated by the presence of the transverse tubular system. As a consequence, if C represents the
capacitance of the surface membrane of the fiber,
CdV/dt would equal the sum of the ionic current
through the surface membrane and the current from
the mouths of the transverse tubules where they invaginate from the surface membrane. Another complication of the muscle fiber experiment is that the ideal of
a membrane action potential is only approximately realized in a fiber mounted in a double Vaseline-gap
chamber. In spite of these complications, however, the
idea that the more negative slope of d is caused by an
increase in outward ionic current across the surface
and/or tubular membranes is still expected to apply.
During the period when c and d diverged, the maximal
value of the difference between the derivatives of the
traces (not shown) occurred 3.2 ms after stimulation
and was 17 mV/ms. This indicates that the outward
ionic current at this time was larger in d than in c by
~17 mV/ms = 17 µA/µF. With the internal and external solutions used in this experiment, such an outward
ionic current could have been carried by potassium ions
leaving the fiber or chloride ions entering the fiber.
At about the time that action potentials c and d in Fig.
2 started to diverge, the associated d[CaT]/dt signals
became noticeably different; d[CaT]/dt represents the
estimated rate of SR Ca release. A possible explanation
for the extra outward ionic current in action potential
d is that SR Ca release produced an increase in myoplasmic free [Ca], which is expected to be approximately proportional to the rate of SR Ca release (Pape
et al., 1995), and that this, in turn, activated ionic channels permeable to potassium or chloride. A comparison
(not shown) of the differences between the two dV/dt
signals and the two d[CaT]/dt signals in Fig. 2 C indicates that such channel activation by Ca must have been rapid, with a delay no greater than 1-2 ms.
Fig. 3 shows the amplitude of the first action potential of a stimulation (A) and its half-width (B) plotted as a function of [CaSR]R, from the experiment in Fig. 2. The half-width is taken to be the interval between the times to half-peak on the rising and falling phases of the signal. In this figure, filled circles (which include a-c) denote values obtained during the first part of the experiment, when the value of [CaSR]R was decreased from 1,154 to 146 µM by a progressive decrease in the recovery period between successive trials from 5 to 0.5- 0.6 min. Open circles (including d) denote values obtained thereafter, when the recovery period was progressively increased to 10 min, decreased to 0.5 min, and finally increased again to 10 min.
In Fig. 3 A, the first stimulation occurred when
[CaSR]R = 1,154 µM and the amplitude of the first action potential was 135.0 mV (
at extreme right). The
amplitude showed little change during the experiment,
with a small progressive decrease, 3 mV, that can be
reasonably attributed to fiber run down.
In contrast, Fig. 3 B shows that the half-width of the
action potential was increased when the value of
[CaSR]R was decreased from its initial value of 1,154 to
146 µM (Fig. 3 B, ). The increase in half-width was
most pronounced for [CaSR]R 500 µM. Most of this
increase in half-width was reversed when the value of
[CaSR]R was allowed to increase (
).
The tentative conclusions of this section are that (a) an increase in the value of [CaSR]R from 150 to 800 µM produces a reversible 1-2-ms decrease in the half-width of the action potential, (b) the outward ionic current responsible for this decrease flows through Ca-activated potassium or chloride channels, and (c) the activation of these channels by Ca is normally rapid, developing within 1-2 ms after SR Ca release begins.
Changes in SR Ca Release Elicited by a Single Action Potential when [CaSR]R Is Varied between 150 and 1,200 µM
Fig. 2 A, a-d, shows four [CaT] signals obtained with
[CaSR]R = 146-876 µM. The value of [CaSR]R and the
amplitude of the [CaT] signal after the first action potential progressively decreased from a to b to c, and
then increased in d to values larger than those in a. The
initial segments of the [CaT] traces are shown in Fig. 2
B, plotted on expanded horizontal and vertical gains. These signals show that, as the value of [CaSR]R was decreased, the [CaT] signal became more rounded, indicating a longer period of SR Ca release.
Fig. 4 A shows the value of [CaT] after the first action potential in each trial, plotted as a function of
[CaSR]R. The concave curvature of the relation between
[CaT] and [CaSR]R indicates that the reduction in
[CaT] was less marked than that in [CaSR]R. For example, a threefold decrease in [CaSR]R from 1,200 to 400 µM resulted in a reduction of [CaT] of only 30%.
Thus, under these conditions, the amount of Ca released by an action potential is relatively insensitive to
SR Ca content. On the other hand, a steeper dependence was observed for values of [CaSR]R < 400 µM.
The open and filled circles track the same relation between [CaT] and [CaSR]R, showing that the effect of
[CaSR]R on [CaT] was reversible.
Fig. 4 B shows the corresponding dependence of f1 on [CaSR]R. The value of f1 increased threefold from 0.165 at [CaSR]R = 1,154 µM to 0.51-0.52 at [CaSR]R = 140-150 µM. Remarkably, at the smallest values of [CaSR]R obtained in this experiment, 140-150 µM, slightly more than half of the readily releasable Ca inside the SR was released by the first action potential.
Fig. 4 C shows the peak value of d[CaT]/dt plotted
as a function of [CaSR]R. The relation is slightly convex,
indicating that the increase in f1 associated with decreasing [CaSR]R (Fig. 4 B) is not caused by an increase
in the fractional rate of SR Ca release; rather, it occurs
in spite of a small decrease in the peak fractional rate
of release.
Since the d[CaT]/dt signals are noisier than the
[CaT] signals (compare, for example, Fig. 2, B and C),
the data in Fig. 4 C have more fractional scatter than
those in Fig. 4 A, especially at small values of [CaSR]R.
Noise reduces the reliability of estimates of the half-width of d[CaT]/dt and its final time constant when the
value of [CaSR]R is small (Fig. 2 C; but see Fig. 7, C and D). In this situation, the time constant associated with
the final half of the [CaT] signal (
[CaT]) can be used as
an estimate of the duration of SR Ca release. Fig. 4 D
shows [CaT] plotted as a function of [CaSR]R. From
[CaSR]R = 1,154 to 140-150 µM, the value of [CaT] increased by an order of magnitude, from 0.9 to 11-13 ms.
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The value 11-13 ms is similar to the time constant expected for Ca dissociation from the Ca-regulatory sites
on troponin, estimated to be 8.7 ms (column 5 in Table
I, model 2, in Baylor et al., 1983). This similarity raises
the possibility that, with [CaSR]R = 140-150 µM, the Ca
complexed by EGTA (which determines [CaT]) came
from Ca that had just dissociated from troponin rather than from Ca that had just left the SR. This seems unlikely for the following reason. In fibers with [CaSR]R 
1,391 µM, the increase in [CaEGTA] that accompanies
Ca dissociation from troponin after an action potential
appears to be negligibly small and not statistically significant, 2.9 µM (SEM, 3.8 µM) (Table II in Pape et
al., 1995). Since a decrease in [CaSR]R would be expected to produce a decrease, not an increase, in the
amount of Ca complexed by troponin, it seems unlikely
that Ca that dissociated from troponin made a significant contribution to the Ca that was complexed by
EGTA during the 140-150 µM [CaT] signals that had
the 11-13-ms time constant.
These results show that, when the SR is partially depleted of Ca, the amount of Ca released by a single action potential does not decrease in proportion to the
value of [CaSR]R (Fig. 4 A). Rather, its value is partially
stabilized by an increase in f1 (Fig. 4 B) that is caused by
a prolongation of Ca release (increase in [CaT], Fig. 4
D). This prolongation of Ca release may be due, at least
in part, to the accompanying prolongation of the action potential (Figs. 2 C and 3 B). The order-of-magnitude increase in [CaT], however, suggests that some
other effect may also be involved (next section).
The Effect of Partial SR Ca Depletion on Action Potential-stimulated SR Ca Release Can Be Mimicked by a Brief Voltage-Clamp Depolarization
To study further the effect of [CaSR] on the rate of turn-off of SR Ca release, experiments were carried out on voltage-clamped fibers. One advantage of this method is that, unlike experiments with action potential stimulation, the voltage waveform is constant and does not depend on the value of [CaSR]R; thus, any changes in release that are observed cannot be attributed to changes in voltage. Another advantage is that measurements can be made of intramembranous charge movement (Qcm), which is thought to arise from movement of the voltage sensors for SR Ca release. Such information might help determine whether changes in the turn-off of SR Ca release are caused by changes in the voltage sensor. Before describing the effects of [CaSR] on Qcm, however, it is important to establish that the effect of SR Ca depletion on Ca release is similar with voltage-clamp and action potential stimulation.
Fig. 5 shows the results of a voltage-clamp experiment
that was designed to mimic the action potential experiment illustrated in Fig. 2. For this purpose, the same
concentration of Ca, 0.44 mM, was used in the end-pool solution. A 10-ms pulse to 20 mV was used to release a small fraction of the readily releasable Ca from
the SR, similar to that released by the first action potential in a trial in Fig. 2. This pulse was followed by a 200-ms repolarization to 90 mV, and then a 420-ms depolarization to 40 mV to deplete the SR of its remaining
Ca so that the value of [CaSR]R could be determined.
Fig. 5 A, a, shows the [CaT] signal that was obtained
78 min after saponin treatment. After the first depolarization, [CaT] reached a value of 229 µM. By the end
of the second depolarization, [CaT] had increased to
a quasi-steady value of 1,152 µM, which is taken for the
value of [CaSR]R. Thus, the first depolarization released
229/1,152 = 0.199 of the readily releasable Ca from
the SR, similar to the fraction f1 released by the first action potential in Fig. 2 d (0.221 with [CaSR]R = 876 µM).
Fig. 5 A, b and c, were obtained later in the experiment
after the value of [CaSR]R had decreased to 430 and 213 µM, respectively. The [CaT] traces in Fig. 5, A and B,
are similar to those in Fig. 2, A and B.
Fig. 6 shows the effect of [CaSR]R on [CaT] (A), f1
(B), peak d[CaT]/dt (C), and [CaT] (D), from the experiment illustrated in Fig. 5. It is plotted with the same
format used in Fig. 4. Filled circles represent measurements made when the recovery period between successive trials was progressively decreased from 5 to 1 min.
After the value of [CaSR]R had decreased to 141 µM by
three successive stimulations with recovery periods of 1 min (three at extreme left in each panel), the recovery period was increased to 10 min for two trials (),
and then, 35 min later in the experiment, to 5 min for
two trials (
). The relations between release parameters and [CaSR]R in Fig. 6 are similar to those in Fig. 4.
In the experiment in Fig. 6, after the value of [CaSR]R
had been decreased to 141 µM, its value was increased
to only 205-212 µM by increasing the recovery period
to 10 min (). A similar small increase in [CaSR]R, from
148 µM after two 1-min recovery periods to 174 µM after a 10-min recovery period, was observed in the other
voltage-clamp experiment in which 0.44 mM Ca was used in the end-pool solution (fiber O08912). This
small recovery of [CaSR]R is unlike the large recovery
observed in the action potential experiment in Fig. 2,
from 146 µM in c to 876 µM in d. Although the reason
for this difference is unknown, an important factor may
be the presence of 1.8 mM Ca in the external solution in the action potential experiments and the absence of
external Ca in the voltage-clamp experiments. Whatever the reason, the poor recovery of [CaSR]R in the
voltage-clamp experiments makes it difficult to study
the reversibility of the effect of [CaSR]R on SR Ca release in these experiments, as was done in the action potential experiments (Fig. 2).
Experiments similar to the one in Figs. 5 and 6 were
carried out on three other voltage-clamped fibers in
which a 10-12-ms pulse to 20 mV was used for the
first stimulation. In one of the experiments (O08912),
0.44 mM Ca was used in the end-pool solution, and the
value of [CaSR]R was reduced by progressively decreasing the recovery period between successive trials from 5 to 1 min, as was done in the experiment in Figs. 5 and
6. In the other two experiments (N14911 and N15911),
the fibers were first equilibrated with an end-pool solution that contained 1.76 mM Ca. Then, Ca was removed from the end-pool solution and the value of
[CaSR]R was reduced by successive stimulations every 1.5 min. The results of all four experiments were similar; the mean value of f1 was 0.147 (SEM, 0.022) with
[CaSR]R = 1,000-1,200 µM and 0.267 (SEM, 0.082) with
[CaSR]R = 140-300 µM; the mean value of the ratio
f1([CaSR]R = 140-300 µM)/f1([CaSR]R = 1,000-1,200 µM) was 1.71 (SEM, 0.33), which is not significantly different from unity. The mean value of [CaT] was 3.63 ms (SEM, 0.54 ms) with [CaSR]R = 1,000-1,200 µM and
16.9 ms (SEM, 4.0 ms) with [CaSR]R = 140-300 µM; the
mean value of the ratio [CaT]([CaSR]R = 140-300
µM)/[CaT]([CaSR]R = 1,000-1,200 µM) was 4.51 (SEM, 0.59), which is significantly different from unity.
The results in Fig. 6 and those described in the preceding paragraph are qualitatively similar to those in
Fig. 4. This suggests that the effect of [CaSR]R on
[CaT] signals elicited by action potential stimulation
cannot be explained primarily by the effect of [CaSR]R
on the duration of the action potential.
The Effect of Partial SR Ca Depletion on d[CaT]/dt
Further analysis of the [CaT] signals in Fig. 5 is shown
in Fig. 7. Fig. 7 A, middle, shows d[CaT]/dt expressed
in units of micromolar per millisecond. As the value of
[CaSR]R decreased from 1,152 (a) to 430 (b) to 213 (c) µM
(only a and c are labeled), the amplitude of d[CaT]/dt
was decreased and its duration was increased.
Fig. 7 A, bottom, shows d[CaT]/dt corrected for SR
Ca depletion. At each moment in time, the value of
d[CaT]/dt in units of micromolar per millisecond was
divided by [CaSR]R [CaT] and multiplied by 100 to
give units in percent per millisecond (Jacquemond et al.,
1991). Because of the division by [CaSR]R [CaT],
each trace becomes progressively noisier with time, and
the noise increases from a to b to c. Within the noise of the signals, the initial time courses are similar. After the
peak value was reached, however, the duration became
progressively longer from a to b to c.
Fig. 7 B shows the peak amplitude of d[CaT]/dt in
units of percent per millisecond, plotted as a function
of [CaSR]R. The values increased slightly from [CaSR]R = 1,152 to 300-400 µM, and then decreased as the value
of [CaSR]R became smaller than 300 µM. In 25 measurements of d[CaT]/dt in four fibers, the mean peak
value of d[CaT]/dt with [CaSR]R = 140-300 µM was
0.861 (SEM, 0.016) times that measured in the same fiber with [CaSR]R = 600-1,200 µM; the value 0.861 is significantly different from unity.
Fig. 7 C shows the half-width of d[CaT]/dt (, ,
and ) and its time to half-peak (
), plotted as a function of [CaSR]R. Fig. 7 D shows the final time constant
of d[CaT]/dt (d[CaT]/dt). As the value of [CaSR]R
decreased from 1,152 to 141 µM, the half-width of
d[CaT]/dt (C) and the value of d[CaT]/dt (D) progressively increased; in both panels, the open symbols and filled circles superimpose within the scatter of the
points. On the other hand, the relative constancy of
Fig. 7 C, , shows that the rising phase of d[CaT]/dt
was little affected by [CaSR]R.
It is clear from Fig. 7 A, bottom, that the d[CaT]/dt
signal became noisier as the value of [CaSR]R became
smaller, making the determinations of the half-width of
d[CaT]/dt (Fig. 7 C) and of the value of d[CaT]/dt (Fig.
7 D) more difficult. For this reason, the filled circles at
the left-hand side of Fig. 7, C and D, with small values of
[CaSR]R, show considerable scatter. In spite of this, the
relation between d[CaT]/dt and [CaSR]R in Fig. 7 D is
similar to that between [CaT] and [CaSR]R in Fig. 6 D,
which suggests that the relation in Fig. 7 D is reliable
within the noise of the d[CaT]/dt signals. This being
the case, it seems likely that the relation between the half-width of d[CaT]/dt and [CaSR]R in Fig. 7 C is also
reliable.
In four fibers, the mean value of d[CaT]/dt was 2.48 ms
(SEM, 0.53 ms) with [CaSR]R = 1,000-1,200 µM and 16.2 ms (SEM, 4.0 ms) with [CaSR]R = 140-300 µM; the mean
value of the ratio d[CaT]/dt ([CaSR]R = 140-300 µM)/ d[CaT]/dt([CaSR]R = 1,000-1,200 µM) was 6.47 (SEM,
0.76), which is significantly different from unity.
The results in this section, obtained from voltage-clamped fibers, confirm those obtained with action potential stimulation (Fig. 4). In particular, when the value of [CaSR]R is decreased from 1,000-1,200 to 140-300 µM, the observed increase in f1 (Fig. 6 B) is caused by a prolongation of SR Ca release (Fig. 7, C and D), and not by an increase in the fractional rate of release (Fig. 7 B).
The Effect of Partial SR Ca Depletion on Qcm
The next question to consider is whether partial SR Ca depletion affects the voltage sensor for SR Ca release. Fig. 8 A, middle, shows the currents from intramembranous charge movement (Icm) associated with Fig. 5, a-c (only a and c are labeled). The ON waveforms of a and b superimpose, with an amplitude that is slightly larger than that of c, owing to a small decrease in the amount of charge in c (see below). On the other hand, the OFF waveforms of Icm are clearly different. From a to b to c, as the value of [CaSR]R decreased from 1,152 to 430 to 213 µM, the amplitude of the OFF response became smaller and its duration became longer.
Fig. 8 A, bottom, shows Qcm, the running integral of Icm, which is expected to be more closely related than Icm to the state of activation of the SR Ca channels. Except for a small reduction in the amplitude of c, the initial time courses of the three traces are similar until the time to peak; thereafter, the return to baseline became progressively slower from a to b to c.
Fig. 8 B, , shows the peak value of Qcm plotted as a
function of [CaSR]R. As expected from Fig. 8 A, bottom,
the peak value of Qcm was similar in a and b and was
slightly smaller in c. The 10-15% decrease in Qcm that
was associated with the decrease in [CaSR]R from 600 to
141 µM may have been caused by a direct effect of the
reduction in [CaSR]R or by the decrease in the recovery period between successive trials that was used to reduce
[CaSR]R from 4 to 1 min. Since the decrease in Qcm was
reversed by increasing the recovery period to 5 or 10 min ( and , respectively), which increased [CaSR]R
only slightly, the decrease in recovery period is the
more likely explanation. Such a decrease in Qcm could
have been caused by slow inactivation of charge movement (Chandler et al., 1976). According to this idea, a
small amount of slow inactivation would develop during the 10-ms pulse to 20 mV and the subsequent
420-ms pulse to 40 mV. This inactivation would then
be removed during the recovery period at 90 mV, and
removal would be more complete with a 3-4-min recovery period than with a 1-min period.
Fig. 8 C shows the half-width of Qcm (, , and )
and the time to half-peak of Qcm () plotted as a function of [CaSR]R. The filled circles show that the half-width progressively increased when the value of [CaSR]R
was decreased from 1,152 to 141 µM ( and are discussed in the following section). The increase in half-width is caused by a prolongation of the falling phase
of the signal since the time to half-peak of the rising
phase of the signal was constant ().
The falling phase of Qcm was analyzed by fitting an exponential function to the final half of the signal, starting at the time from half-peak and ending 164 ms after
repolarization (not shown). Fig. 8 D, , shows the values of the time constant of the exponential function
that were obtained in this manner (Qcm). The value of
Qcm increased from 12 to 27 ms as the value of [CaSR]R was decreased from 1,152 to 141 µM.
Results similar to those shown in Fig. 8, , were obtained in four fibers in which the value of [CaSR]R was
progressively decreased from 1,000-1,200 to 140 µM.
In the two fibers with 0.44 mM Ca in the end-pool solution (O08911 and O08912), this depletion was accomplished by decreasing the recovery period between trials from 5 to 1 min. In the two fibers with 0 mM Ca in
the end-pool solution (N14911 and N15911), the recovery period was 1.5 min throughout. The mean value
of Qcm was 10.0 ms (SEM, 1.1 ms) with [CaSR]R = 1,000-1,200 µM and 17.7 ms (SEM, 3.7 ms) with
[CaSR]R = 140-300 µM; the mean value of the ratio
Qcm([CaSR]R = 140-300 µM)/Qcm([CaSR]R = 1,000-
1,200 µM) was 1.71 (SEM, 0.20), which is significantly
different from unity.
The Effect of the Duration of the Recovery Period on OFF Qcm
Fig. 8, B-D, , denotes measurements made when the
value of [CaSR]R was decreased by reducing the duration of the recovery period between successive trials.
With the shortest recovery period used, 1 min, the
value of [CaSR]R was 141-159 µM (three left-most in
each panel). When the recovery period was then increased to 5 or 10 min, the value of [CaSR]R was increased to 180-185 µM () or 205-212 (), respectively. The associated values of the half-width of Qcm
(Fig. 8 C) and of the value of Qcm (D) were reduced by
the longer recovery periods so that the open symbols lie below the relation defined by the filled circles; the
reduction was larger with a 10-min recovery period ()
than with a 5-min period (). Recovery periods >10
min were not studied.
In two fibers, a 10-min recovery period was used after
the value of [CaSR]R had decreased to 141 (O08911) or
148 (O08912) µM. The mean value of Qcm was 11.9 ms
(SEM, 0.1 ms) with [CaSR]R = 1,000-1,200 µM and 17.4 ms (SEM, 0.2 ms) with a 10-min recovery period and
[CaSR]R = 140-300 µM; the mean value of the ratio
Qcm([CaSR]R = 140-300 µM)/Qcm([CaSR]R = 1,000-
1,200 µM) was 1.47 (SEM, 0.03), which is significantly
different from unity.
This dependence of the half-width of Qcm and of the
value of Qcm on recovery period was not observed in fibers with [CaSR]R > 1,200 µM. (In two experiments,
not shown, fibers N14911 and N15911 were first equilibrated with an end-pool solution that contained 1.76 mM Ca and had initial values of [CaSR]R that were >2,000 µM. Later in the experiment, Ca was removed
from the end-pool solution and intramembranous charge
movement and SR Ca release were monitored with 10-ms pulses to 20 mV, as was used in Fig. 5. In fiber
N14911, the values of Qcm half-width and Qcm were 9.66 and 7.37 ms, respectively, after a 6.9-min recovery period ([CaSR]R = 2,442 µM) and were 9.94 and 7.12 ms after a 1.57-min recovery period ([CaSR]R = 1,729 µM).
In fiber N15911, the values of Qcm half-width and Qcm
were 10.02 and 6.64 ms, respectively, after a 13-min recovery period ([CaSR]R = 2,268 µM) and were 10.88 and 8.01 ms after a 1.5-min recovery period ([CaSR]R = 1,289 µM). These small changes in Qcm half-width and
Qcm are probably within the error of measurement.)
The results described above suggest that, as the value of [CaSR]R is reduced from 1,200 to 140-210 µM, the OFF kinetics of Qcm is slowed in a use-dependent manner. With [CaSR]R >1,200 µM, either the slowing effect does not occur or, if it does, it is removed by a 1.5-min recovery period. Unfortunately, recovery periods <1 min could not be studied with our voltage-clamp method owing to the time required to take and process data during the CONTROL and TEST pulses of each trial.
The main conclusion from this and the preceding section is that a reduction in [CaSR]R from 1,000-1,200 to
140-300 µM is able to prolong the duration of OFF Qcm
after a brief depolarization in a use-dependent manner
with little, if any, effect on ON Qcm. In four fibers in
which a recovery period as brief as 1-1.5 min was used
to determine Qcm with [CaSR]R = 140-300 µM, the final time constant of OFF Qcm was increased by the factor
1.71 (SEM, 0.33) (see preceding section). In the two fibers in which a 10-min recovery period was also used, the
factor was 1.47 (SEM, 0.03) (this section). As mentioned above, recovery periods >10 min were not studied.
A Comparison of the Effect of Partial SR Ca Depletion on the
Time Courses of Qcm and d[CaT]/dt
Fig. 9 A shows three pairs of Qcm and d[CaT]/dt traces,
replotted from Figs. 8 A and 7 A, bottom, respectively;
the same calibration factors, 4 nC/µF and 1%/ms, apply, respectively, to all the Qcm and d[CaT]/dt traces.
Within each pair of traces, the rising phase of the noisier d[CaT]/dt signal lagged that of the Qcm signal by 2-3
ms so that its peak value was reached after that of Qcm.
|
Simon and Hill (1992) measured the time course of
charge movement and SR Ca release during and after
100-ms depolarizations to potentials between 60 and
20 mV. A depolarizing prepulse was used to produce
Ca inactivation of Ca release so that the time course of
d[CaT]/dt would, according to them, correspond to
the time course of voltage activation of the noninactivating component of d[CaT]/dt. They found that the
time course of d[CaT]/dt matched that of Qcm4. Based
on this result, they proposed that four identical voltage sensors, which move independently, act in concert to
gate the SR Ca release channel. In our experiments,
with 10-15 ms depolarizations, the time courses of Qcm4
and of d[CaT]/dt were clearly different (not shown).
For example, the peak value of d[CaT]/dt occurred after that of Qcm4 or, for that matter, Qcm raised to any
positive power (see Fig. 9 A). Moreover, the later time
to peak of d[CaT]/dt cannot be attributed to the development of Ca inactivation of Ca release since this
would be expected to decrease, not increase, the time to peak of d[CaT]/dt. Thus, if four voltage sensors act
in concert to gate the SR Ca channel, as proposed by Simon and Hill (1992), the opening of the channel does
not occur immediately with the movement of the voltage sensors, but after a 2-3-ms delay.
After the peak values of d[CaT]/dt in Fig. 9 A were
reached, both Qcm and d[CaT]/dt returned to zero
with rates that progressively decreased from a to b to c.
In a, the final return of d[CaT]/dt to zero was more
rapid than that of Qcm. The relative difference in the final time courses of Qcm and d[CaT]/dt was less marked in b. In c, the final time courses of the two signals were
similar, with the Qcm trace lying within the noise of the
d[CaT]/dt trace.
Since, with small values of [CaSR]R, the value of Qcm
with a 1.5-min recovery period was larger than that with
a 10-min recovery period (Fig. 8 D, c and ), whereas
the corresponding values of d[CaT]/dt were within the
scatter of the points (Fig. 7 D), it was of interest to compare Qcm and d[CaT]/dt signals with a 10-min recovery
period. Within the noise of the [CaT]/dt signal, the final
time courses of the two signals were similar (not shown).
Fig. 9 B shows the ratio Qcm/d[CaT]/dt plotted as a
function of [CaSR]R. Its value decreased from 4.5 with [CaSR]R = 1,152 µM (a) to values that fluctuated
around unity with values of [CaSR]R < 400 µM. With
[CaSR]R < 400 µM, the mean value of Qcm/d[CaT]/dt
was 1.07 (SEM, 0.05) for the filled circles and 0.91 (SEM, 0.07) for the open symbols; these two values are
not significantly different from each other or from
unity. The comparisons described in this and the preceding paragraph show that, with [CaSR]R < 400 µM,
the noise in the d[CaT]/dt signals makes it difficult to
assess quantitatively the effect of increasing the recovery period from 1-2 to 10 min on the relation between
Qcm and d[CaT]/dt.
Results similar to those in Fig. 9 B were obtained in
three other fibers, and the combined data are shown in
Fig. 9 C. The mean value of Qcm/d[CaT]/dt with [CaSR]R = 1,000-1,500 µM was 4.66 (SEM, 0.35) and, with [CaSR]R
= 140-300 µM, was 1.06 (SEM, 0.04). The first, but not
the second, value is significantly different from unity.
Jong et al. (1995a) also measured Qcm and d
