From the Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médecine, Sherbrooke, Québec J1H5N4, Canada
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ABSTRACT |
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Cut muscle fibers from Rana temporaria (sarcomere length, 3.5-3.9 µm; 14-16°C) were mounted in a
double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium-
gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were
stimulated with a voltage-clamp pulse protocol that consisted of pulses to 70,
65,
60,
45, and
20 mV, each
separated by 400-ms periods at
90 mV. The change in total Ca that entered into the myoplasm (
[CaT]) and the
Ca content of the SR ([CaSR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and
W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). Fibers were stimulated with the pulse protocol, usually every
5 min, so that the resting value of [CaSR] decreased from its initial value of 1,700-2,300 µM to values near or below
100 µM after 18-30 stimulations. Three main findings for the voltage pulses to
70,
65, and
60 mV are: (a)
the depletion-corrected rate of Ca release (release permeability) showed little change when [CaSR] decreased
from its highest level (>1,700 µM) to ~1,000 µM; (b) as [CaSR] decreased below 1,000 µM, the release permeability increased to a maximum level when [CaSR] was near 300 µM that was on average about sevenfold larger than
the values observed for [CaSR] > 1,000 µM; and (c) as [CaSR] decreased from ~300 µM to <100 µM, the release
permeability decreased, reaching half its maximum value when [CaSR] was ~110 µM on average. It was concluded
that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.
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INTRODUCTION |
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In vertebrate fast twitch skeletal muscle, contraction is
initiated when depolarization of the surface and transverse (t)-tubular1 membranes causes calcium ions (Ca)
to move from the lumen of the sarcoplasmic reticulum
(SR) into the myoplasm, where it complexes with the
Ca regulatory sites on troponin C. Schneider and
Chandler (1973) described a nonlinear capacitative
current in skeletal muscle in response to step depolarizations termed intramembranous charge movement.
This signal started to become prominent at voltages near the threshold for contraction and it was suggested
that it arises from movement of voltage sensors in the
t-tubular membrane from a resting state to a state that
activates Ca release from the SR. Later, Adrian and
Peres (1979)
identified two components of the movable charge, an early Q
component followed by a delayed or "hump" component called Q
. Subsequent
work employed various approaches to try to evaluate
the relative contributions of Q
and Q
(reviewed by
Huang, 1988
; Ríos and Pizarro, 1991
; Schneider, 1994
).
In one of these approaches, Hui and Chandler (1991)
found a statistically better fit to the overall charge (Q)
vs. voltage (V) data with the sum of two Boltzmann
functions as opposed to just one. The Boltzmann distribution function is given by
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(1) |
where qmax is the maximum amount of charge, is the
voltage at which half the charge is moved, and k is the
voltage-steepness factor. The value of k identified with
the Q
component was 3.3 mV on average, which was
similar to the voltage steepness of Ca release and tension near the mechanical threshold (Baylor et al., 1979
,
1983
; Miledi et al., 1981
; Maylie et al., 1987a, 1987b).
This correlation and others have led to suggestions that the Q
component is responsible for activating SR Ca
release.
The voltage steepness of Ca release was also determined with small voltage pulses with the EGTA/phenol
red method, a sensitive method for measuring Ca release developed in the laboratory of Knox Chandler
(Pape et al., 1995). With this method, fibers are equilibrated with 20 mM EGTA and Ca release is estimated
from the pH change caused by the release of protons
when EGTA complexes Ca. With small voltage pulses,
the density of Ca release sites is low (<1 in 104 channels
are open at
75 mV) so that, in the presence of 20 mM EGTA, it is very unlikely that the open state of a Ca release site could have been influenced by Ca coming
from a neighboring release site (Pape et al., 1995
). A Ca
release site here and throughout this article is considered to be a single SR Ca release channel activated via
depolarization of the transverse tubule or a cluster of
Ca release channels composed of a single such voltage-activated release channel together with any neighboring "slaved" channels activated via calcium-induced Ca
release (CICR) or by some other mechanism. The rate
of Ca release increased on average e -fold for 3.48 mV in
the voltage range between
80 and
57 mV (Pape et
al., 1995
), a value close to the steepness factor of 3.3 mV for Q
(Eq. 1 predicts that Q
varies exponentially with voltage when
is several k values more negative
than V). These results were taken as evidence that the
voltage steepness of release is not due to Ca-dependent
processes. They support the idea that SR Ca release is
controlled by voltage sensors in the t-tubular membrane whose movement also gives rise to the Q
signal.
Measurements of the voltage steepness of Ca release at small voltages in addition to measurements of intramembranous charge movement were used to assess
whether or not the voltage activation process remained
stable during the long experiments in this article.
The experiments in this article were designed to establish whether or not Ca-dependent processes occur
within a voltage-activated Ca release site (as defined
above). Ca release was measured with the EGTA/phenol red method at small voltages (70 to
60 mV) and at different levels of SR Ca content ([CaSR]). The idea
of varying [CaSR] was to vary the free [Ca] at possible
Ca binding sites on the myoplasmic side of SR Ca release channels. Solutions of the diffusion equation in
the presence of 20 mM EGTA (Neher, 1986
; Stern,
1992
; Pape et al., 1995
) indicate that the change in free [Ca] in the vicinity of an open Ca release channel
should be proportional to the flux of Ca ions through
the channel. Since the change in free [Ca] is predicted
to be much greater than the resting free [Ca], it follows
that free [Ca] in the vicinity of an open channel is approximately proportional to the Ca flux, which in turn
should be proportional to the free [Ca] in the SR. The
free [Ca] in the SR is expected to be approximately
proportional to the SR Ca content during Ca release
since calsequestrin complexes Ca with low affinity (MacLennan and Wong, 1971
) and with rapid kinetics (Prieto et al., 1994
). Therefore, the free [Ca] at a Ca binding site, (either on the same or an immediately adjacent
release channel), should be approximately proportional
to [CaSR].
One advantage of the EGTA/phenol red method is that it can give a direct estimate of [CaSR] at any time during a stimulation. This allows a determination of the depletion-corrected rate of Ca release that is expected to be approximately proportional to the Ca permeability of the SR. (For conciseness, the term "release permeability" is substituted for "depletion-corrected rate of Ca release" in this article.) The experimental goal in this article was to determine the release permeability as a function of [CaSR] at small voltages. Providing that the extent of voltage activation was unmodified, a nonconstant dependence on [CaSR] would be indicative of a Ca-dependent process acting at the level of a single Ca release site.
A major stimulus for the experiments in this article
was the possibility of obtaining evidence for or against
CICR in skeletal muscle. CICR plays an important role
in the activation of SR Ca release in cardiac muscle, a
process that requires external Ca. Although external
Ca is not required for excitation-contraction coupling
in skeletal muscle (Armstrong et al., 1972), various studies have suggested that some type of CICR process
might still be involved. Addition of Ca has been shown
to cause or enhance Ca release in skinned muscle fibers (Endo et al., 1968
; Ford and Podolsky, 1968
), in
small microdissected sections of fibers (Fabiato, 1984
),
in heavy SR vesicles (e.g., Meissner, 1984
), in Ca release channels reconstituted into planar lipid bilayers (Smith
et al., 1986
), in skinned muscle fibers stimulated by depolarization of sealed t-tubules (Lamb and Stephenson,
1991
), and in depolarized cut skeletal muscle fibers
(Klein et al., 1996
). Experimental evidence in support
of a role of CICR in normal voltage-activated Ca release
has been less clear. In support of CICR, Jacquemond et
al. (1991)
reported that the rate of Ca release is reduced by the addition of 2-3 mM of the fast Ca buffer
fura-2. In contrast to these results, Baylor and Hollingworth (1988)
, Hollingworth et al. (1992)
, Pape et al.
(1993)
, and Jong et al. (1993)
found that 2-3 mM fura-2
increased Ca release, consistent with a removal of Ca
inactivation of Ca release. A reason for the different
findings was never found, though it was suggested that
they might be due to some unexplored difference(s) in
the experimental conditions (Jong et al., 1993
). In
Pape et al. (1993)
and Jong et al. (1993)
, a large decrease in Ca release was observed when [fura-2] increased from 2-4 to 6-8 mM, a finding that could be
due to a decrease in CICR or to some pharmacological
effect of fura-2. Based on measurements of Ca sparks
with confocal microscopy, Klein et al. (1996)
recently
suggested that multiple channels (two to three) can
open at a Ca release site in fibers held at
70 mV.
Their results and the possibility that neighboring channels are activated via CICR are discussed in the DISCUSSION.
In the experiments in this study, an increase in release permeability with increasing [CaSR] would be consistent with the presence of CICR in normal voltage-activated Ca release. Ca inactivation of Ca release, another Ca-dependent mechanism acting on the SR Ca
release channel, is an important and well-studied mechanism that serves to limit the amount of Ca that is released from the SR (Baylor et al., 1983; Simon et al.,
1985
, 1991
; Schneider and Simon, 1988
; Jong et al.,
1995a
). Another aim of this article was to evaluate
whether Ca inactivation operates at the level of an isolated Ca release site. A decrease in release permeability with increasing [CaSR] would be indicative of a reduction in Ca inactivation of Ca release.
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MATERIALS AND METHODS |
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The experiments were carried out at 13-14°C at sarcomere
length of 3.5-3.9 µm on cut twitch fibers (Hille and Campbell,
1976) from semitendinosus or ileo-fibularis muscles isolated
from Rana temporaria that were adapted to 6°C. The fiber preparation, mounting procedures, and electrical connections were
identical to those described in Pape et al. (1995)
. One of the end
pools, denoted 1, was used to measure potential and the other
was used to inject current, denoted I2. Current was collected by a
bath clamp that maintained the central pool at earth potential
and converted the current to a voltage signal for detection. The
potential in end pool 1, denoted V1, was maintained at
90 mV
at rest by passing a small holding current. V1 was controlled with
a voltage-clamp set-up made by the Yale University Physiology
Electronics Lab (New Haven, CT) using conventional feedback
electronics. The command pulses were rounded by a 0.5-ms time
constant.
The temperature of the chamber was maintained by its contact with a copper block cooled by water from a regulated water bath equipped with a cooling condenser. This arrangement produced a stable temperature (fluctuations < ±0.2°C for periods >1 h).
Optical Measurements and Data Acquisition
The optical methods were similar to those described in Irving et al.
(1987). Fig. 1 shows a diagram of the chamber mounted in a Zeiss
Axiovert-100 inverted microscope (Carl Zeiss, Inc., Oberkochen,
Germany). Briefly, when the shutter (S ) was opened, the fiber was
illuminated with white light (~400-850 nm) from a tungsten-halogen light source (TH ). The light emerging from the fiber
was collected and split into three beams with two beam-splitting cubes (BSC ). The three wavelengths for the measurements were determined by three interference filters (F1, F2, and F3). A lens (L3) focused a beam onto one of the three photodiodes (PD).
Each photodiode was connected to an operational amplifier circuit (not shown) made by the Yale University Physiology Electronics Lab, which had a 10 M
feedback resistor.
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The V1 and I2 signals passed through separate voltage followers (VF-4; World Precision Instruments, Sarasota, FL) and, together with the three photodiode signals, passed through separate instrumentation amplifier/filter channels constructed in the University of Sherbrooke's Electronics Lab. Each of the signals was filtered with a four-pole Bessel filter (824L8L-4; Frequency Devices Inc., Haverhill, MA) with its cutoff frequency set at 1 kHz. Separate switch-selectable amplifiers were placed before (gain of 1, 10, or 100) and after (gain of 1, 2, 5, or 10) the filter in each channel. The five signals were multiplexed and sampled by the 16-bit A/D converter in an ITC16-MAC computer interface device (Instrutech Corp., Great Neck, NY), which was connected to a Power Macintosh 7100 computer. D/A ports of the interface were also used to provide the command voltage to the voltage-clamp instrument and to control the shutter. Stimulation and sampling protocols written in C were downloaded into the ITC16-MAC interface, which then controlled the timing of the stimulation and the data acquisition during an experimental run.
Composition of the Internal and External Solutions
The Cs-glutamate solution that was used in the end pools contained 45 mM Cs-glutamate, 20 mM EGTA as a combination of
the Cs salt and, if Ca was present, the Ca salt, 6.8 mM MgSO4,
5 mM Cs2-ATP, 20 mM Cs2-creatine phosphate, 5 mM Cs3-phospho(enol)pyruvate, and 5 mM 3-[N-morpholino]-propanesulfonic
acid (MOPS). The solution with no Ca present is called the Ca-free Cs-glutamate solution. In the other internal solution used,
the concentrations of Ca-complexed and Ca-free EGTA were 1.76 and 18.24 mM, respectively. The pH was adjusted to 7.0 by the
addition of CsOH; at this pH, the calculated concentrations of
free Ca and free Mg were 36 nM and 1 mM, respectively. The Cs
salts of ATP, creatine phosphate, and phospho(enol)pyruvate were prepared as described in Pape et al. (1995).
The tetraethylammonium hydroxide (TEA)-gluconate solution that was used in the central pool contained 110 mM TEA-gluconate, 10 mM MgSO4, 1 µM tetrodotoxin (TTX), and 10 mM MOPS. Its pH was 7.1 and it was nominally Ca free.
Tetraethylammonium hydroxide was obtained from Aldrich
Chemical Co., (Milwaukee, WI), creatine phosphate and TTX
were obtained from Calbiochem-Novabiochem Corp. (La Jolla,
CA), and all other compounds were obtained from Sigma Chemical Co. (St. Louis, MO). Phenol red was purified by a method
similar to that used by Kendrick (1976) to purify Arsenazo III.
Phenol red was dissolved in a solution containing n-butanol, pyridine, acetic acid, and water in the volume ratios 3:1:1:3, respectively, and passed through an anion-exchange column (Dowex 1-8 resin, 100-200 mesh size; Bio-Rad Laboratories, Richmond, CA)
equilibrated with the same solution. Early samples from the column contained impurities as identified by their brown to amber
color and their absorbance spectra, which differed from those of
phenol red. Later samples were pooled together and the solvent
was removed by freeze drying, followed by ether-water extractions
to remove remaining pyridine as judged by the loss of a scent associated with pyridine. There were several indications that the
product obtained from this procedure was not altered and that it
was in fact purified phenol red. Absorbance spectra of aqueous
solutions of the phenol red at different pH values were essentially
the same as previously published spectra. The compound showed
no apparent toxicity; all seven experiments in this article lasted
for several hours. In addition, the rate of diffusion and extent of
binding in the muscle were not significantly different from those
determined previously (see RESULTS).
Estimation of Myoplasmic pH with Phenol Red
The method used to estimate the myoplasmic pH and changes in
myoplasmic pH, denoted pH, with phenol red was essentially
the same as that described in Pape et al. (1995)
. The main differences in this article are that unpolarized light and different interference filters were used. In this article, a 10-nm bandpass interference filter centered at 480 nm, the isosbestic wavelength of
phenol red, was used at position F3 in Fig. 1; a green filter with a bandpass range of 510-590 nm, a pH-sensitive range of phenol
red, was used in position F1; and a long pass filter (Schott No.
RG665) was used in position F2 to monitor the intrinsic absorbance of the fiber. All of these filters were obtained from Edmund Scientific (Barrington, NJ).
The concentration of phenol red at the optical site was estimated from the value of the indicator-related absorbance at 480 nm, denoted A ind(480), and the molar extinction constant of
phenol red at 480, which was taken to be 1.1 × 104 M1 cm
1 (Lisman and Strong, 1979
). The fractional amount of the phenol red
in the nonprotonated form, f (see Irving et al., 1989
), was estimated from
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in which r = A ind(green)/A ind(480). The values of rmax and rmin determined with cuvettes mounted on the set-up were 3.21 and 0.0541, respectively. The value of pH was calculated from the usual expression,
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with a value of 7.7 assumed for pK (Lisman and Strong, 1979).
Estimation of pH and SR Ca Release
The indicator-related absorbance change during activity at the
wavelength of the green filter, A ind(green), was given by
A(green)
1.23 ·
A(RG665). The factor 1.23 was the same
factor used to correct the active signals obtained with a 570-nm-centered filter for the intrinsic fiber absorbance estimated with a
690-nm-centered filter in Pape et al. (1995)
. The change in pH
during activity (
pH) was estimated from
A ind(green) and the
values of A ind(480), f, rmax, and rmin above as described in Pape et
al. (1995)
. The noise in the
A(green) and
A(RG665) signals
were first reduced with a 0.05 kHz digital Gaussian filter (Colquhoun and Sigworth, 1983
) before the intrinsic correction was
done.
The EGTA-phenol red method (Pape et al., 1995) was used to
estimate the total amount of Ca released from the SR into the
myoplasm (
[CaT]). Briefly, the two predominant forms of
EGTA are H2EGTA2
and CaEGTA2
under the conditions of
these experiments. When Ca binds to EGTA, two protons are released, producing a pH change that is measured with phenol red.
[CaT] is given by 0.5
(the buffering power of myoplasm) ×
pH.
was assumed to be 22 mM/pH unit (Pape et al. 1995
).
With 20 mM EGTA present in the myoplasm, almost all of the Ca
that is released is rapidly captured by EGTA. The total amount of
Ca in the SR (referred to as myoplasmic volume) is denoted as
[CaSR] and is given by [CaSR]R
[CaT], where [CaSR]R is the value of [CaSR] at the start of a stimulation (the subscript R refers
to resting). [CaSR]R is estimated by the maximum value of
[CaT]
during a stimulation that releases essentially all of the Ca from
the SR. The rate of Ca release is given by d
[CaT]/dt. The depletion-corrected rate of release {100 × d
[CaT]/dt
([CaSR]R
[CaT])}, in units of percent of SR Ca content released per millisecond, is expected to be approximately proportional to the Ca
permeability of the SR. The depletion-correction method is similar to one that has been employed in Schneider's laboratory ( Jacquemond et al., 1991
), though the method for estimating
[CaSR]R is different. It is also the same method that has been used
in Knox Chandler's laboratory (e.g., Jong et al., 1993
; see Fig. 13 in Pape et al., 1995
; Jong et al., 1995a
). For conciseness, the term
"release permeability" is substituted for "depletion-corrected rate
of Ca release" in this article.
Intramembranous Charge Movement
The Icm currents were obtained by subtracting off small ionic
components from the Itest-Icontrol signals as described in Hui and
Chandler (1990) and Jong et al. (1995b)
. Other aspects of the
measurements are described in Chandler and Hui (1990)
and Hui and Chandler (1990
, 1991
).
Statistical Tests of Significance
Two sets of results were considered to be significantly different if the Student's two-tailed t test parameter P was <0.05.
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RESULTS |
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Voltage Protocol
The aim of this study was to determine the release permeability (see MATERIALS AND METHODS for definition)
over a range of voltages and at different levels of
[CaSR]. In cut-fiber experiments, a long time is required to equilibrate the internal solution at the optical
recording site with the end pools (several tens of minutes) and a long time (5 min or more) is required to reaccumulate Ca released in the presence of 20 mM
EGTA, the amount present in these experiments. For
these reasons, it was impractical to try to equilibrate a
fiber at different levels of [CaSR] followed by a series of
voltage-clamp measurements at each [CaSR] level. Fig. 2
shows the protocol adapted to obtain information at
several voltages during a single experimental run. The
top trace shows the voltage, which started and ended at
the holding potential of 90 mV. Pulses to
70,
65,
60,
45, and
20 mV with durations of 400, 300, 300, 800, and 400 ms, respectively, were interspersed by 400-ms periods at
90 mV. The middle trace shows
[CaT]
obtained with the EGTA-phenol red method. One purpose of the final pulses to
45 and
20 mV was to release all of the Ca from the SR to estimate [CaSR]R, the
amount of Ca present in the SR at the start of the stimulation. The value of [CaSR]R is estimated as the maximum level of
[CaT], which was 2,043 µM in this case.
The bottom trace shows the same trace plotted with an
expanded vertical scale up until the start of the pulse to
45 mV.
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The main rationale for the five different pulses in the
voltage protocol shown in Fig. 2 was to obtain information to help assess whether possible changes in release
permeability could be due to changes in the voltage-sensing mechanism of E-C coupling. Since most of the
charge at the small voltages (70 to
60 mV) has been
associated with Q
, measurements of charge movement (which contain both Q
and Q
) would not be sensitive to changes in Q
, the component of charge thought to
reflect voltage activation of SR Ca release (see INTRODUCTION). The main purpose for using three small
voltages (
70,
65, and
60 mV) was to determine
whether the voltage steepness of Ca release in this
range was changed by varying [CaSR]R. Monitoring the
amount of intramembranous charge moved by the
pulses to
45 and
20 mV provided a rough method
for assessing possible changes in the maximum amount
of movable charge (Qmax) and/or a shift in the Q-V
curve along the voltage axis or a change in the voltage
steepness of charge movement.
One concern with using multiple pulses is that the response of a later pulse is influenced by the earlier
pulses. Jong et al. (1995a) used a two-pulse protocol to
study properties of Ca inactivation of Ca release produced by a 10-15-ms pulse to
20 mV. Under conditions similar to those employed here, they found that
most of the Ca inactivation recovered with an exponential course with a time constant of ~50 ms. The use of
400-ms intervals at
90 mV between each of the pulses
should therefore be sufficient time for essentially complete recovery from this type of inactivation.
[CaSR]R Versus Time
Fig. 3 shows a plot of [CaSR]R vs. time after the saponin
treatment for the experiment in Fig. 2. Point a is from
the experimental run shown in Fig. 2. There was a significant decrease in [CaSR]R during the first three runs
even though Ca was present in the internal solution.
This type of initial rundown is typical of most fibers that
have Ca in the internal solution, though some do not
show any rundown. The reason for this rundown is unknown, though it might be related to the fact that
[Ca2+] in the end-pool solution was estimated to be 36 nM; a value that is likely to be less than the resting myoplasmic [Ca2+] before the fiber was cut. After the first
three stimulations, Ca was removed from the internal
solution and the fiber was stimulated with the same
voltage protocol applied, usually every 5 min. There was a progressive decrease in [CaSR]R with time as Ca
diffused from the inside of the fiber to the end pools.
The reason that the progressive decrease in [CaSR]R was
not more rapid was that most of the released Ca was recaptured by the SR within 5 min so that only a fraction
of the Ca escaped to the end pools. After point c (Fig. 3
) was obtained, the delay until the next stimulation (
) was reduced to <1 min. The decrease in [CaSR]R
was mostly reversed with the following stimulation (
),
which was obtained 5 min later. 200 min after saponin
treatment, Ca was reintroduced into the end pools and,
with the exception of the
symbols, [CaSR]R showed a
progressive increase. This indicates that the decrease in
[CaSR]R with no Ca present could be reversed, at least
partially, by reintroducing Ca into the end pools.
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SR Ca Release Signals at Different [CaSR]R Values
Fig. 4 shows signals associated with points a-e in Fig. 3.
Fig. 4 A, top, shows the voltage. The bottom traces show
the [CaT] signals. As seen in Fig. 3 and by the maximum value of the traces in Fig. 4 A (except for a, which
does not show the signal after the start of the pulse to
45 mV), the value of [CaSR]R progressively decreased
from a to d and increased in e after Ca had been reintroduced into the end pools. One important thing to
note is that the fractional decrease in the amount of Ca
released by the first three pulses from Fig. 4 A, a-b, is
approximately equal to the fractional decrease in [CaSR]R
from a-b. Another important thing to note is that the first three pulses in b, c, and e released about the same
amount of Ca and had similar rates of Ca release
(d
[CaT]/dt) even though [CaSR]R was much greater
in b than in c and e. As indicated later, this could be due
to Ca inactivation of Ca release associated with the
greater [CaSR] values in b. Interestingly, d
[CaT]/dt during the pulse to
45 mV is much greater in b than
in c and e despite the similarity of the signals during the
pulses to the smaller voltages. Since one would have expected relatively more Ca inactivation of Ca release to
occur at
45 mV compared with the smaller voltages,
one possibility is that some type of extra activation component is present at
45 mV when the amount of Ca in
the SR is relatively large. The possibility that this extra component is related to CICR is currently being explored.
As mentioned in the INTRODUCTION, the aim in this
study was to assess the effect of [CaSR] on the release
permeability, which is calculated as 100 · d[CaT]/dt
([CaSR]R
[CaT]) (see MATERIALS AND METHODS) to
give units of percent of SR Ca content released per unit
of time (percent/millisecond). If SR Ca release channels have a single conductance with a Ca flux that is
proportional to the Ca content of the SR, then this release permeability should be proportional to the fraction of SR Ca release channels that are open. Because
of noise in the signals at small values of [CaSR], the Ca
release signals in Fig. 4 B are shown as 100 · ln{[CaSR]R
([CaSR]R
[CaT])}. The slope at any time during one
of these traces gives the corresponding release permeability. (This is easily demonstrated by taking the derivative of this relationship with respect to time, noting
that [CaSR]R is constant during a stimulation.) These
traces show that, for all three small voltage pulses, the
release permeability was relatively unchanged when [CaSR]R decreased from 2,043 µM in a to 1,029 µM in b,
increased when [CaSR]R decreased from 1,029 µM in b
to 267 µM in c, and then decreased when [CaSR]R decreased further to 74 µM in d. This latter decrease was
mostly reversed after Ca was added back to the internal
solution and [CaSR]R increased to 347 µM in e. (The time courses of the release permeability signals are discussed later with Fig. 9.)
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One possible explanation for the dramatic changes
in the calculated release permeability signals could be
that there are large relative errors in the estimated values of [CaSR]R. For example, the greater release permeability signal in Fig. 4 B, c, relative to those shown in b
and d, could occur if only a fraction of the Ca was released from the SR in c while essentially all of the Ca
was released in b and d. As seen in Fig. 4 A, however,
the [CaT] trace c approached a maximum level well
before the end of the series of pulses, consistent with
the idea that essentially all of the Ca was released from
the SR. In all of the other traces in the experiment of
Fig. 4, and in the other experiments reported in this article, the time course of the
[CaT] signals approached a maximum level before the end of the series of pulses.
This indicates that the results in Fig. 4 B and similar results from the other experiments are not attributable to
an underestimation of [CaSR]R resulting from an incomplete release of Ca from the SR.
Fig. 5 shows plots of release permeability vs. [CaSR]
for pulses to 60,
65, and
70 mV (A-C, respectively). The data were determined from the traces in
Fig. 4 B (for a-e) and from similar traces from other
stimulations from the same experiment. Each value is
the difference of the slope during the pulse minus the average of the slopes during the periods at
90 mV before and after the pulse. Each slope was determined
from the line fit to all points from 100 ms from the start
of the interval to the end of the interval; the starting
and end points are plotted as crosses in Fig. 4 B. The results for the three voltages had several features in common. As [CaSR] decreased from ~2,300 to ~1,000 µM,
there was little if any significant change in the release
permeability. As [CaSR] decreased further (Fig. 5, filled
symbols), the release permeability increased significantly, reaching a maximum level when [CaSR] was
200-400 µM. As [CaSR] decreased further to below 100 µM, there was a significant decrease in release permeability. After Ca was added back to the internal solution
(Fig. 5, open symbols), the release permeability increased
as [CaSR] increased. This indicates that the decrease in
release permeability with decreasing [CaSR] was at least
partially reversible in this experiment.
As shown in Fig. 3, [CaSR] was also decreased by decreasing the interval between stimulations from 5 min
to ~50 s. In Figs. 3 and 5, the upside-down triangles,
squares, and triangles, respectively, show the results
from the stimulations just before the decreased interstimulus period, just after the short period, and 5 min
later. In Fig. 5, there was a clear decrease in release permeability as [CaSR] was decreased by the decrease in
the interstimulus recovery period, both in the absence
of Ca in the internal solution () and after Ca was
added back to the internal solution (
). (Note that
there were two decreases in interstimulus periods after
Ca was added back. In each panel, the open square at
the smallest [CaSR] value corresponds to the pair of triangles with the smallest [CaSR] values. Also note that
there is a filled square and an open square in the cluster of low points for the results at
60 mV.) The
squares are close to values obtained later (Figs. 3 and 5,
) or earlier (
) in the experiment when the values of
[CaSR] were similar. The values of release permeability
shown by the triangles are close to those shown by the
upside-side down triangles, indicating that the effect of
decreasing [CaSR] in this way was reversible. Similar maneuvers when [CaSR]R was between 100 and 350 µM
were done in five other experiments when no Ca was
present and in four other experiments after Ca had
been reintroduced into the internal solution. In each
case, results similar to those in Fig. 5 were obtained.
Summary of Results from All Experiments
Table I summarizes the release permeability versus
[CaSR] data in Fig. 5 and similar data from the six other
experiments. All of the experiments had an approximately constant release permeability when [CaSR] was
varied from its greatest value (1,800-2,200 µM) down
to ~1,100 µM, at which point it started to increase. Column 2 gives this plateau level, which was obtained by
averaging all of the values with [CaSR]R between 1,200 and 1,900 µM. Column 3 gives the maximum release
permeability, which was obtained by a least-squares fit
of a quadratic function to points spanning the largest
value obtained when no Ca was present in the end
pools. Column 4 gives the ratio of the maximum value
to the plateau value. Column 5 gives the value of [CaSR]
at the maximum. Column 6 gives the value of [CaSR]
when the release permeability was midway between the
maximum and the plateau level. Column 7 gives the
value of [CaSR] when the release permeability had decreased to half of its maximum value. A main conclusion from the mean and SEM values in Table I is that
the release permeability versus [CaSR] data had essentially the same shape at all three voltages (60,
65, and
70 mV). The relevant features that were not significantly different at the three voltages were: (a) a
maximum in release permeability when [CaSR] was
~300 µM on average (column 5), which was about sevenfold greater on average (column 4) than the plateau level, (b) a half-maximal effect of decreasing release
permeability with increasing [CaSR] when [CaSR] was
~520 µM on average (column 6), and (c) a half maximal effect of increasing release permeability with increasing [CaSR] when [CaSR] was ~110 µM on average
(column 7).
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Test of the Reversibility of the Effect of Varying [CaSR] between 1,100 and 350 µM by Decreasing the Interstimulus Interval
As discussed above, the increase of release permeability
with increasing [CaSR] from 100 to 350 µM was shown
to be reversible by decreasing the interstimulus interval
from 5 min to ~1 min. Fig. 6 shows the effect of this
maneuver carried out at greater values of [CaSR]. Fig. 6
A (top) shows the voltage pulses to 70,
65, and
60
mV, and the bottom three traces show 100 · ln{[CaSR]R
([CaSR]R
[CaT])}. (Recall from Fig. 4 B that the
slopes of these latter signals give the release permeabilities.) The value of [CaSR]R for Fig. 6 f was 1,087 µM.
Fig. 6 g was measured 43 s later and had a [CaSR]R value
of 384 µM. There was a clear increase in the release
permeability from f to g. This increase was mostly reversed in h, which was obtained 5 min later and had a
[CaSR]R value of 874 µM. (The smaller signal to noise
ratio of the signals in Fig. 6 A compared with those in
Fig. 4 B is attributable to a smaller release permeability
in Fig. 6 A. The large variation between fibers of the release permeabilities at similar values of [CaSR] is discussed below.)
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Fig. 6 B shows a plot of release permeability vs. [CaSR]
at 60 mV from Fig. 6 A (f-h) and from other stimulations in the same experiment. g and h agree reasonably
well with the relationship between release permeability
and [CaSR] established by other points in the experiment. Results very similar to these were obtained in the
one other experiment (fiber reference 514972) in
which [CaSR] was reversibly decreased from ~1,000 to
~350 µM by decreasing the interstimulus interval.
These results indicate that the increase in the release
permeability when [CaSR] decreases from ~1,000 to
300-400 µM is reversible.
During the course of an experiment when no Ca is present in the internal solution, Ca is gradually lost from the fiber due to diffusion out of the ends of the fibers and, as a result, the free Ca concentration decreases. The results with decreased interstimulus intervals rule out the possibility that the effects of decreasing [CaSR] on release permeability are due to a decrease in resting free [Ca]. Decreasing the interstimulus period from 5 to 1 min should increase the free [Ca] before the stimulation, since more Ca is present in the myoplasm compared with just before the first stimulation. Likewise, the free [Ca] should return to a smaller value before the stimulation 5 min later, since most of the released Ca should have been pumped back into the SR.
The above results also indicate that the apparent effects of [CaSR] on release permeability are not due to some other type of long-term change in the fiber or in the experimental apparatus, since the stimulations just before and after the shortened interstimulus period and the stimulation 5 min later are all done within 6 min. One type of long-term change of particular concern would be a drift in the electrodes. Because of the steep voltage dependence of Ca release, a change in error of even 1 mV in the measurement of V1 could produce a significant change in SR Ca release.
Reversibility after Ca Was Introduced into the Internal Solution
In all experiments, Ca was introduced into the internal
solution at some point in the experiment after [CaSR]R
had decreased to <100 µM. In all experiments, the release permeability increased with increasing [CaSR] after this point. In the experiment of Figs. 2-5 and Table
I (fiber reference 510971) and one other experiment (fiber reference 516972), there was a significant, continual increase in holding current that started within
four stimulations after introducing Ca into the end
pools. In these experiments, the release permeability
values were similar to, though somewhat less than, the
values at corresponding [CaSR] values during the period with 0 Ca in the internal solution when [CaSR] was
declining from 300 to <100 µM (compare open symbols and filled symbols with [CaSR] <400 µM in Fig. 5).
In the other five experiments, the holding currents
were relatively stable for longer periods. In all of these
latter experiments, the release permeability values after introducing Ca were significantly greater than the values at corresponding [CaSR] values during the period
with 0 Ca when [CaSR] was declining from 300 to <100
µM (compare open symbols and filled symbols in Fig. 6
B). In two of the experiments (fiber references 513971 and 514972), plots (not shown) of the release permeability at 60 mV vs. [CaSR] showed maximums; these
maximums occurred at similar values of [CaSR] (near
300 µM), but were about two and three times greater,
respectively, than the maximums observed with 0 Ca. In
the other experiments, [CaSR] values were not increased beyond ~250 µM before significant increases
in the holding currents occurred. The important conclusion from this section is that all experiments showed
a reversal of the decline in the release permeability with decreasing [CaSR] when Ca was introduced into
the internal solutions and [CaSR] was allowed to increase.
Order of Pulses Changed to 60,
65,
70,
45, and
20 mV
One problem with these experiments is that the signal
to noise ratio of the Ca signals in response to the pulses
to 70 mV was often very low when the [CaSR] values
were small. Because of this, the signals were too small
in several experiments to obtain meaningful values for
several parameters listed in Table I. As a result, some of
the conclusions from the results in Table I depend
mainly on the results at
65 and
60 mV. Since it is possible that the results at
60 mV (
65 mV) were influenced by the earlier pulses to
70 and
65 mV (earlier pulse to
70 mV), an experiment (fiber reference
724972) was carried out in which the order of the first
three pulses was reversed in several stimulations during
the experiment. Therefore, the pulse to
60 mV was
first and was not preceded by any depolarization. This
protocol was carried out at several points during the experiment marked by the vertical line segments in Fig. 6
B. As seen in Fig. 6 B, the release permeability at
60
mV for these points matched well the release permeability vs. [CaSR] relationship established by the other
stimulations in which the pulse to
60 mV came after
the pulses to
70 and
65 mV. This indicates that the
results at
60 mV listed in Tables I and III are not due
to some influence of the earlier pulses to
70 and
65
mV in the stimulation.
|
Variability in Release Permeability between Fibers
One curious finding is the large spread in release permeabilities between the different fibers in columns 2 and 3 in Table I; there was an ~11-fold (0.03529/
0.00325) difference between the largest and smallest
plateau levels of release permeability values at 60 mV
and a 22-fold difference for the maximum rates (0.34860/
0.01562). Interestingly, a similar large spread was not
observed for the corresponding release permeability
values at
45 mV, which had a less than threefold difference between the largest and smallest values at both
the plateau levels and maximum levels of the release
permeability vs. [CaSR] data (data not shown). As will
be seen in Table III, the large spread at the small voltages cannot be attributed to differences in intramembranous charge movement or to differences in the voltage steepness of SR Ca release. To assess whether there
could be other explanations, various experimental parameters are summarized in Table II. As mentioned in
earlier studies (e.g., Pape et al., 1995
), phenol red concentration vs. time data can be well fitted by a solution of the one-dimensional diffusion equation with the assumption that the concentration of bound phenol red
is linearly related to the concentration of free phenol
red in the myoplasm. Column 2 gives the apparent diffusion constant of phenol red and column 3 gives the
value of R + 1, where R is the ratio of bound to free phenol red. Column 4 gives the time after saponin
treatment for the results tabulated in Table I and for
columns 5-9 in Table II; there was no Ca in the internal solution during this period in all of the fibers. Columns 5-9 give the first and last values of the fiber diameter, concentration of phenol red, resting pH, holding
current, and apparent capacitance of the fiber, respectively. There was no apparent correlation between any
of the parameters listed in Table II and the release permeability values in columns 2 and 3 of Table I.
One possible explanation for the large spread in the
release permeabilities is that the condition of the fibers
differed somehow. The condition of the frogs was probably similar, however, since the first six fibers were studied during a 1-wk period and the frogs were all treated
in the same way. Since the large spread in release permeability values was not observed at 45 mV, one possibility is that there is some fiber to fiber variability in some activation and/or inactivation process(es) that is
more pronounced at small voltages than at more positive voltages. Alternatively, there could be some process(es), perhaps Ca-feedback mechanism(s), that tend
to equalize the release process at
45 mV, but not at the smaller voltages. Whatever the explanation, the effects of [CaSR] on release permeability summarized in
Table I were similar for all of the fibers.
Effect of [CaSR] on Intramembranous Charge Movement
Fig. 7 A (top) shows voltage and Fig. 7 A (bottom) shows
intramembranous charge movement (Icm) signals for
the stimulations a-e in Figs. 3-5. The Icm signals are similar to those observed previously under essentially the
same experimental conditions with gluconate as the
principal external anion. In particular, the complex
time courses of the ON Icm signals at 45 mV are consistent with two effects of SR Ca release on the kinetics
of I
. One effect is a speeding up of the kinetics of I
with increasing rates of Ca release that were half maximal at
45 mV when [CaSR]R was 172 µM on average
(Table III in Jong et al., 1995b
). The other is a slowing
down of I
with increasing rates of Ca release that were
half-maximal when [CaSR]R was 500-1,000 µM (Pape et al., 1996
). The early decrease of the ON Icm signals at
45 mV in Fig. 7, a and b, compared with c -e, is due to
this latter effect. Another thing to note is that there is
not a clear distinction between an early I
component
followed by a delayed I
hump component as is usually
observed with other external anions. This result is consistent with the findings of Hui and Chen (1991)
and
Huang (1994)
who concluded the Q
component is
suppressed when gluconate is the external anion.
Although it is possible that [CaSR] also influences the
kinetics of I at the small voltages (
70 to
60 mV),
the release permeabilities were determined when most
of the steady state charge is expected to have moved
(points ranging from 100 to 300-400 ms after the start
of the pulse; see Figs. 4 B and 6 A). The main purpose
of the charge movement signal in this study, therefore, was to assess whether changes in the amount of steady
state intramembranous charge occurred during the experiments. Results from one fiber given in Fig. 6 of
Pape et al. (1996)
showed that the amount of steady
state charge that moves is essentially independent of
[CaSR]R for voltage pulses ranging from
80 to
10
mV. Since the experiments in this study are long, it
seemed important to confirm that the steady state
charge also did not change during the course of these experiments.
As expected for an intramembranous charge movement signal, Huang (1994) confirmed that the amount
of charge that moves during the ON pulse (Q ON) is essentially the same as the amount that moves during the
OFF pulse (Q OFF) when gluconate is the principal external anion. Because the ON Icm signal is slower than
the OFF signal leading to more uncertainty in the correction for the nonlinear ionic component, it is generally considered more reliable to estimate the amount of
charge that moves from the integral of the OFF Icm signal. Fig. 7, B and C, shows the OFF Icm signals at
45
and
20 mV, respectively, on expanded time scales.
The time courses of the OFF Icm signals are similar in
both B and C. Fig. 7 D plots the
Q OFF values vs. time
after saponin treatment for the pulses to
60 (asterisks),
45 (open symbols), and
20 mV (filled symbols). There is some scatter in the data, particularly for the results at
20 mV, which is probably due to uncertainty
in the correction for ionic currents. Assuming this is
the case, it appears that there was not a significant
change in the amount of charge that moves during the
experiment at any voltage.
Fig. 8 A shows a semi-logarithmic plot of the release permeability vs. voltage for stimulations a-e in Figs. 3-5 and 7. The lines show the least squares best fits of an exponential function to the data. Fig. 8 B shows the voltage-steepness factor of the exponential function for the fits in A and for the other stimulations in the experiment. The values are approximately constant except for noise in the data close to when Ca was reintroduced into the end pools and the presence of a local minimum between b and c. Such a minimum was not observed in other fibers, and overall there were no consistent trends in the different experiments to indicate that the voltage steepness of Ca release varied with [CaSR] or with time during the experiment.
Table III summarizes charge movement and voltage-steepness results shown in Figs. 7 and 8, respectively,
and analogous results from the other six experiments.
The three sections of the table correspond to the following [CaSR] levels: (a) when [CaSR] was 1,100-1,900
µM (release permeability was at a plateau level in this
range of [CaSR] values in all experiments; see Figs. 5
and 6 B), (b) when the largest release permeabilities
were obtained, and (c) when [CaSR] was near or below
100 µM. Columns 5-7 show Q OFF at
45 mV,
20
mV, and the ratio of these values, respectively. Column
8 shows the e -fold voltage-steepness factor of Ca release between
70 and
60 mV. The mean of the e -fold factor when [CaSR] was between 1,100 and 1,900 µM was
3.43 mV. This value is not significantly different than
the value of 3.48 mV (SEM, 0.16 mV; n = 4) given in
Pape et al. (1995)
and determined under similar conditions, except that 1.76 mM Ca was present in the internal solution in their experiments.
As mentioned previously, the purpose of monitoring
Q OFF values at 45 and
20 mV and the voltage steepness of release was to assess in a rough manner whether
there might have been a change in the overall Q vs. V
curve during the course of an experiment. An increase
in the maximal amount of charge that could move
(Q max) would be expected to increase the Q OFF values
at both voltages (columns 5 and 6 in Table III). A shift
in the Q vs. V relationship along the voltage axis would
be expected to change the ratio of the Q OFF values (column 7). A shift in the voltage steepness of charge
movement would also be expected to show up as a shift in the voltage steepness of Ca release measured with
small voltage steps (column 8). An asterisk in the second (third) section indicates an average value that is
significantly different from the corresponding average
for the same fiber in the first (second) section. There was not a significant change in most of the values for
columns 5-8 from the first to the second section and
from the second section to the third. In addition, there
were no significant changes in the mean of the averages (given at the bottom of the sections) between any
of the sections for columns 5-8. In contrast, all of the
changes in the release permeability in column 4 were
significant between the first and second and between
the second and third sections.
The conclusion from these results is that significant
changes in intramembranous charge movement responsible for SR Ca release in the voltage range of interest, 70 to
60 mV, probably did not occur during
the course of most of these experiments. Therefore, the effects of [CaSR] on release permeability (Fig. 5,
Table I, and column 4 of Table III) cannot be attributed to long-term changes in intramembranous charge
movement (associated either with the time of the experiment or with the decrease in [CaSR]).
Time Course of Release Permeability at Different [CaSR]R Values
A potential problem is that the time course of voltage activation at the small voltages changed during the course of these experiments. For reasons stated earlier, the intramembranous charge movement signal is not useful for assessing possible changes in voltage activation at small voltages. The aim of this section is to evaluate whether changes in the time course of the release permeability signal occurred and, if so, whether they reflect changes in the time course of voltage activation or some other process.
Fig. 9 A (top) shows a voltage pulse to 60 mV; the
bottom traces show, in chronological order from top to
bottom, release permeability signals obtained at different [CaSR] values. In Fig. 9 A, a and b correspond to
points in the plateau region, c to the peak, and d to a
minimal (to the left of the peak) value of the release
permeability vs. [CaSR] curve in Fig. 5 A. The trace between b and c corresponds to a point about midway
down the falling phase (right of the peak) in Fig. 5 A,
and the trace between c and d corresponds to a point
about midway up the rising phase (left of the peak).
The cursors show the points during the pulse used in
the estimate of the release permeability and the line is
the average of the points in these signals. a and b rise
rapidly to their maximal levels, where they remain unchanged for the remainder of the pulse. (Note that the
final cursor is smaller than the maximum, because of
the additional 0.01 kHz Gaussian filter that was applied
to these traces because of noise in the bottom two traces.) In contrast, Fig. 9 A, c, continues to rise after
100 ms from the start of the pulse (first cursor) and appears to approach a maximum near the end of the
pulse. A similar slow-terminating increase after 100 ms
was observed in signals near the peak of the release permeability vs. [CaSR] curves in four of the other six experiments. In the other two, the release permeability
signal approached its maximum close to 100 ms after
the start of the pulse, though there was still a slowing of
the time course when [CaSR] decreased from >1,000 to
~300 µM.
The second and third release permeability signals from the top in Fig. 9 A are shown in B superimposed with c. All of the signals are plotted at the same gain. These pairs of traces indicate that the initial rising phases of the signals are approximately the same but that the signals at the larger [CaSR] values terminate early, whereas c continues to increase. In all experiments, the change in the time course from a fast to a slower terminating rising phase appeared to correspond with the increase in release permeability as [CaSR] decreased from >1,000 to ~300 µM. Although changes in the time course of voltage activation cannot be ruled out, it seems more likely that the time course of voltage activation is the same but that the onset of Ca inactivation somehow prevents the later increase in the release permeability signal.
As illustrated by the bottom three traces in Fig. 9 A, there was no detectable change in the time course of the release permeability when [CaSR] decreased from ~300 to <100 µM. Similar results were obtained in all of the other experiments, though changes could not always be ruled out at the smaller values of [CaSR] due to noise in the signals. These results suggest that the decrease in release permeability when [CaSR] decreased from ~300 to <100 µM is not attributable to a change in the time course of voltage activation.
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DISCUSSION |
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Evidence of Ca-dependent Regulatory Processes Operating on Isolated Voltage-activated Ca Release Sites
This article describes the effect of [CaSR] on the release
permeability (depletion-corrected rate of Ca release) at
three voltages, 70,
65, and
60 mV. The following
main findings were observed for all three voltages (illustrated in Figs. 5 and 6 B). (a) The release permeability did not vary much when [CaSR] decreased from the
highest level (1,700-2,300 µM) down to ~1,000 µM.
(b) As [CaSR] decreased below 1,000 µM, the release
permeability increased and reached a maximum that
was 5-12× larger than the plateau level when [CaSR] was
>1,000 µM; the maximum occurred when [CaSR] was
near 300 µM (columns 4 and 5 in Table I). (c) The average [CaSR] value when the release permeability was
midway between the plateau level and the maximum
was ~520 µM (column 6 in Table I). (d) There was a
significant decrease in the release permeability when
[CaSR] decreased below 300 µM; the release permeability was half of the maximum value when [CaSR] was
~110 µM (column 7 in Table I).
There was little, if any, change in the amount of intramembranous charge moved at 45 or
20 mV or in
the voltage steepness of Ca release (Figs. 7 and 8 and
Table III). These results suggest indirectly that the effects of [CaSR] were not due to a change in the activating charge movement. Effects b and d above were shown to be reversible. Long term changes during the
experiment, including changes in resting free Ca concentration or a drift in the electrodes, were also ruled out.
It seems reasonable to conclude, therefore, that findings
a-d above were due to effects of [CaSR] on the activation state of SR Ca release channels.
Tripathy and Meissner (1996) recently reported effects of lumenal [Ca2+] on the open probability (Po) of
SR Ca release channels from mammalian muscle reconstituted into planar lipid bilayers. There was a significant increase in Po when lumenal [Ca2+] was increased
from <0.1 to ~250 µM, and a decrease in Po when lumenal [Ca2+] was increased to millimolar levels. By
varying the Ca flux through the channel independently
of lumenal [Ca2+], they concluded that the effects were
due to the Ca flux rather than a direct effect of lumenal
[Ca2+]. The effects of varying Ca flux on Po were mainly
due to modulation of the mean open times of the channels, as opposed to the frequency of channel openings.
Even though Ca flux was initiated by t-tubular depolarization in this study and by ATP in their study, it seems reasonable to suppose that the bimodal dependence of
Ca release on lumenal Ca in this study may also have
been due, at least in part, to modulation by Ca of the
mean open time of the Ca release channels. If the single channel results apply to the intact system, then the
additional conclusion of Tripathy and Meissner (1996)
that the activation and inactivation Ca binding sites are
on the myoplasmic side of the channels rather than the
lumenal side should also apply to the effects described
in this article.
As mentioned in the INTRODUCTION, the principal aim of this study was to evaluate whether Ca feedback mechanisms act at the level of a single Ca release site activated by voltage. A Ca release site was defined to be a single SR Ca release channel activated via t-tubular depolarization or a cluster of Ca release channels composed of a single such voltage-activated release channel together with any immediately neighboring "slaved" channels activated via CICR or by some other mechanism. The results in this article are consistent with the regulation of a voltage-activated SR Ca release site by both Ca inactivation and by some type of CICR mechanism acting at Ca binding sites accessible to Ca released at the site.
It is not possible to determine from the results in this article whether these regulatory processes act at the level of a single SR Ca release channel or whether multiple channels are involved.
Possibility of Multiple Channel Openings at a Calcium Release Site
With confocal microscope imaging of muscle fibers
containing a fluorescent Ca indicator, it has become
possible in recent years to observe small release events
termed calcium sparks. Klein et al. (1996) recently reported that calcium spark events were often larger
when fibers were held at
70 mV compared with
90 mV. Assuming that the calcium sparks at
90 mV corresponded to the opening of a single Ca release channel, they showed that the larger calcium sparks at
70
mV could have been produced by multiple (two or
three) channel openings within a single Ca spark. If
this is in fact the explanation for the larger sparks, then
multiple open channel events would also be expected
to have occurred at individual calcium release sites at
small voltages in the experiments in this article. Solutions of the diffusion equation indicate that the buffering action of 20 mM EGTA is not fast enough to produce a significant decrease in
[Ca2+] 30 nm from an
open channel (Fig. 15 A in Pape et al., 1995
; see also
Neher, 1986
; Stern, 1992
). Therefore, the activation of
neighboring channels via CICR, if it occurs, should not
have been significantly affected by the presence of 20 mM EGTA in the experiments in this article.
One question (relevant to E-C coupling in both skeletal and cardiac muscles) is how would it be possible to
activate neighboring channels via CICR without producing an all-or-none response. An all-or-none response would appear inevitable since Ca from a channel opened by CICR would then open its neighbor by
CICR and so on. Stern et al. (1997) recently addressed
this problem with a Monte-Carlo simulation of the Ca
release process in skeletal muscle. Although several of
the assumptions in their model are debatable, the results do suggest that the spread of release into an all-or-none response could be terminated by Ca inactivation
of Ca release.
Based on the background in the last two paragraphs,
the following scenario gives one possible explanation
for the results at small voltages in this article. Under
normal physiological conditions ([CaSR] > 1,000 µM),
two to three channels open in about half of the Ca release sites and only one channel in the other sites
(from Klein et al., 1996). When [CaSR] decreases to 300 µM, termination via Ca inactivation of Ca release is reduced so that there is an increased probability of multiple channel openings, perhaps with as many as 5-15
channels open at a site. When [CaSR] decreases further
to below 100 µM, there is a decrease in multiple channel openings due to a decrease in the ability to activate
neighboring channels via CICR.
To have acceptance of this scenario, it seems important to have additional evidence to support the conclusions of Klein et al. (1996) given above. One problem
was that their conclusions were based on three of six fibers that happened to show a much greater frequency
(by a factor of ~45 in one fiber) of Ca sparks at
90
mV than expected from activation of the voltage sensors. Since the process responsible for the initiation of
the Ca sparks was apparently different at
90 and
70
mV in those fibers, the termination of the sparks may
also have occurred via different processes, thereby accounting for differences in the magnitude of the
sparks. Even if the termination of the sparks was due to a return of the voltage sensor to a nonactivating state at
both
90 and
70 mV, it is possible that the larger
spark size at
70 mV was due to a slower transition rate
between the activating and nonactivating state of the
voltage sensor. Another problem is that there does not
seem to be an easy explanation of why Ca from an open
channel would be able to open neighboring channels via CICR at
70 but not
90 mV.
Possibility that Only a Single Channel Opens at a Calcium Release Site
It is not necessary to postulate multiple channel openings at a Ca release site to explain the results in this article. It is possible that Ca release channel opening in an
intact muscle requires a direct connection with an activated t-tubular voltage sensor so that no channel can be
opened by CICR alone. As with the single channel results of Tripathy and Meissner (1996), Ca bound to the
activation site would somehow increase the mean open time of the Ca release channel while Ca bound to the
inactivation site closes the channel. The channel would
still be under voltage control, consistent with the observation that SR Ca release terminates when a fiber is repolarized to the holding potential. If this scenario is
correct, it would explain how it is possible to have CICR
without producing an all-or-none response.
This scenario would not be in agreement with the
proposal of Ríos and Pizarro (1988) (also see Shirokova
et al., 1996
; Shirokova and Ríos, 1997
) that half the Ca
release channels are activated by voltage and the other
half by CICR in frog muscle. This proposal was stimulated by the observation of Block et al. (1988)
and Franzini-Armstrong and Kish (1995)
that alternate foot proteins (SR Ca release channels) are not coupled to dihydropyridine receptors (voltage sensors). If multiple
channel openings at Ca release sites did not occur, these uncoupled Ca release channels would have to
have been silent; i.e., not opened by CICR.
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FOOTNOTES |
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Address correspondence to Dr. P.C. Pape, Département de physiologie et biophysique, Université de Sherbrooke Faculté de Médecine, 3001, 12e Avenue Nord, Sherbrooke, Québec J1H5N4, Canada. Fax: 819-564-5399; E-mail: p.pape{at}courrier.usherb.ca
Original version received 15 December 1997 and accepted version received 16 June 1998.
We thank the staff of the Biomedical Instrumentation Laboratory of the Yale Department of Cellular and Molecular Physiology for the design and construction and W.K. Chandler for the design of the voltage-clamp electronics. We also thank the Atelier Central Electronique-Mécanique of the Université de Sherbrooke Faculté de Médecine for help with the design and construction of equipment. We thank Drs. S.M. Baylor, W.K. Chandler, and E. Rousseau for reading the manuscript and providing useful criticism.
This work was supported by Medical Research Council of Canada grant MT-12552 and a grant from Fonds de la Recherche en Santé du Québec.
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Abbreviations used in this paper |
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CICR, calcium-induced Ca release; Q, overall charge; SR, sarcoplasmic reticulum; t-tubule, transverse tubule; V, voltage.
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