From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1503
Ca2+ sparks were first detected as spontaneous, highly
localized elevations of Ca2+ indicator fluorescence in
confocal microscope images of rat cardiac myocytes
studied under resting conditions (Cheng et al., 1993 Ca2+ sparks also occur in frog skeletal muscle fibers,
both "spontaneously" in resting fibers (Klein et al.,
1996 Observations and Interpretations
Spark fluorescence and the underlying time course of SR Ca2+
release.
Before discussing the number of channels responsible for generating a Ca2+ spark, it is important to
first consider what information the spark provides concerning the underlying time course of SR Ca2+ release.
The signal actually monitored in studies of Ca2+ sparks
is the change in fluorescence of a calcium indicator, generally fluo-3, within the confocal volume in a confocal line scan image. The observed time course of fluorescence at the spatial center of a spark can be interpreted using a qualitative approach that provides a general perspective on the possible time course of the
underlying Ca2+ release and serves as a starting point
for the present considerations. During the rising phase
of a spark, the concentration of Ca2+-fluo-3 must be increasing in the confocal volume. This indicates that
Ca2+ entry into the confocal volume must exceed the
net effect of Ca2+ "removal" by binding and diffusion
out of the confocal volume. Thus, during the rising
phase of a spark, Ca2+ ions are being released from the
channel or channels responsible for generating the
spark (Fig. 1 A). In contrast, during the falling phase of
fluorescence in a spark, there is a net fall of Ca2+-fluo-3
in the confocal volume, indicating that Ca2+ binding
and Ca2+ diffusion out of the confocal volume exceed
Ca2+ entry. Since the diffusion and Ca2+ binding properties of the myofibril are unlikely to change significantly during the spark, the declining phase of a spark
must correspond to a period during which Ca2+ release
occurs at a much lower rate than during the rising
phase. In the extreme case, Ca2+ release could occur at
an approximately constant rate during the rising phase
of the spark, and then stop completely during the falling phase (Fig. 1, B-D).
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). It was immediately suggested that these events are likely
to reflect the localized release of Ca2+ from a small cluster of sarcoplasmic reticulum (SR) Ca2+ release channels or perhaps even from a single SR channel (Cheng et al, 1993). Ca2+ sparks were found to occur at increased rates during small depolarizations of rat cardiac myocytes, leading to the concept that the macroscopic [Ca2+] transient during larger depolarizations of
cardiac myocytes might be due to the spatio-temporal
summation of such events occurring at high frequencies throughout the myocyte (Cannell et al., 1993
, 1995
; López-López et al., 1994
).
) and at higher frequencies during small depolarizations that produce relatively low levels of voltage activation of Ca2+ release (Tsugorka et al., 1995
; Klein et al,
1996). As in cardiac myocytes, during large depolarizations of frog skeletal muscle fibers such events could
occur at much higher rates and spatio-temporally summate to compose the macroscopic [Ca2+] transient.
The present perspective focuses on Ca2+ sparks in frog
skeletal muscle and the possible SR Ca2+ release channel activity that underlies the generation of the observed sparks. A central unresolved issue is whether a
spark is generated by a single channel, by a small cluster of a few channels, or by a larger strip of many contiguous channels in the SR junctional face membrane.
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Fig. 1.
Hypothetical alternative patterns of SR Ca2+ release
channel activity that could underlie the Ca2+ sparks observed
in frog skeletal muscle. (A) Theoretical function representing
the relative change in fluorescence ( F/F) as a function of
time (t) in a typical Ca2+ spark.
The line was generated by the
function
F/F = 0 before the
first vertical dashed line,
F/F = A(1
exp[
k1(t
d1)] between
the two vertical dashed lines, and
F/F = A{1
exp[
k1(d2
d1)]
exp[
k2(t
d2)] + C} after the
second vertical dashed line. This
function, which has discontinuous derivatives at both its start
and peak, systematically closely
followed almost all aspects of the
time course of Ca2+ sparks recorded experimentally with video
rate recording (Lacampagne et
al., 1999
). The parameter values
obtained from a least squares fit
of this function to a Ca2+ spark
(k1 = 340 s
1, k2 = 214 s
1, and
d2
d1 = 3.3 ms) were used to
generate the theoretical record
shown here. (B-D) Three alternative hypothetical classes of activity
patterns, either of an individual
SR Ca2+ release channel (B) or
of a group of three (C) or five
(D) channels, that could underlie the observed Ca2+ spark time
course. Each individual record
represents the time course of current (i) through a single channel, with c and o denoting the
closed and open state of the channel. Note that the time course of
total Ca2+ efflux is approximately
the same for each channel activity pattern in B-D.
Ca2+ release turns on and off abruptly at the start and peak
of a spark.
Our recent studies using relatively high
speed (63 µs per line) line scan confocal imaging of
Ca2+ sparks in frog skeletal muscle provide a 30-fold increase in the available temporal resolution of spark
time course and give results that are not inconsistent
with the extreme interpretation in Fig. 1. These studies
demonstrate a very abrupt transition from rising to falling fluorescence at the peak of the spark, as shown by
the theoretical time course in Fig. 1 A, which provides a
good representation of most aspects of the observed
sparks (Lacampagne et al., 1999). The sharp peak in
the observed spark time courses indicates a large and
abrupt decrease in the rate of Ca2+ release rate at the
peak of the spark. From our results, it does not seem
implausible that release could turn off completely at the peak of a spark. In this case, the rise time of the
spark would correspond to the total time that the channel or group of channels generating the spark were
open, and the declining phase would be a time during
which the release rate were zero. Since the high time
resolution studies also demonstrate a rather abrupt transition to a high rate of rise of fluorescence at the
start of a spark, the level of Ca2+ release activity underlying the spark also seems to achieve a near maximal
rate early in the spark rising phase. In this case, the net
level of overall release channel activity in a spark would rapidly jump from zero to a constant rate early in the
rising phase, remain approximately constant throughout the rising phase, and then rapidly fall off to zero at
the peak of the spark. As indicated diagrammatically in
Fig. 1, this interpretation could correspond to a single
channel open for the entire rising phase of the spark
(B), multiple channels, each of which remain open throughout the rising phase of the spark (C), or multiple channels that open and close asynchronously and
repeatedly during the rising phase of the spark but
close within a short interval at the time of peak of the
spark (D). In a more general interpretation, the rise time
of a spark provides a lower limit for the open time of
channels responsible for generating the spark since
some channel(s) might open or remain open during the
declining phase, even though the rate of release during
the declining phase of the spark must have been markedly less than during the rising phase (not illustrated). The above types of interpretation can be made quantitative through the use of detailed modelling of Ca2+
binding and diffusion in a fiber after release from a
channel or group of channels (e.g., Pratusevich and
Balke, 1996
). These quantitative models indicate that
release could abruptly turn on and off at the start and
peak of the observed sparks.
Properties of individual sparks are not obviously incompatible with Ca2+ release from a single channel.
The Ca2+ sparks
detected in a frog skeletal muscle fiber have an average
rise time of 4.6 ms and an average decay time constant of 8.6 ms (Lacampagne et al., 1999) and an average
spatial full width at half max of ~1.4-1.5 µm (Lacampagne et al., 1996
). The peak change in relative fluorescence (
F/F) of the larger amplitude events, which are
most likely to represent events arising spatially closest
to the scan line, is ~1-2. These properties, together with the diffusion and binding properties of the fiber,
provide an indication of the amount of Ca2+ released
by the channel or channels that generate the spark. It
was already pointed out in early reports on cardiac Ca2+
sparks that rough approximations of the amount of Ca2+
released in a spark were not obviously incompatible
with channel open times and Ca2+ flux rates of single
SR Ca2+ channels in bilayers (Cheng et al., 1993
). Using detailed models of the sarcomeric distribution of
myoplasmic Ca2+ binding sites together with the diffusion properties of Ca2+ and fluo-3, it is possible to calculate the spatio-temporal distribution of Ca2+-fluo-3
and the resulting Ca2+ spark that would be produced
by an assumed time course of Ca2+ release from a point
source corresponding to the channel or group of channels generating the spark. These modelling calculations, using reasonable values for the various model parameters and reasonable values for single channel current and channel open time (e.g., 1-2 pA of current for
~10 ms) result in theoretical Ca2+ sparks that are not
obviously incompatible with the observed sparks (Pratusevich and Balke, 1996
; Jiang et al., 1998
). Thus, based on such calculations alone, it is not necessary to
exclude the possibility that a Ca2+ spark could be generated by the Ca2+ released during the opening of a
single SR Ca2+ release channel. However, it should be
noted that this finding does not imply that Ca2+ sparks
are in fact generated by the activity of a single SR Ca2+
release channel, but only establishes that the experimentally observed sparks are not obviously quantitatively inconsistent with the possibility of spark generation by a single channel.
Spark frequency increases during activation, but spark properties remain constant.
The "spontaneous" sparks observed
in frog fibers appear to be ligand-gated events triggered
by calcium-induced calcium release (CICR) since the
frequency of spontaneous events increases with increased
myoplasmic [Ca2+] and in the presence of caffeine
(Klein et al., 1996), and decreases with increased myoplasmic [Mg2+] (Lacampagne et al., 1998
), all hallmarks of CICR. The sparks initiated by fiber depolarization are voltage-activated events, presumably triggered
by activation of voltage sensors (Schneider and Chandler, 1973
), the dihydropyridine receptors (Tanabe et al., 1987
) in the transverse tubule (TT) membrane of
the fibers. One of the salient features of Ca2+ release by
both ligand- and voltage-activated Ca2+ sparks in frog
skeletal muscle fibers is that the overall level of calcium
release appears to be graded by variations of the frequency of occurrence of Ca2+ sparks, but that the individual sparks themselves have similar average properties despite marked differences in their frequency of
occurrence. For example, with protocols that use relatively brief repriming of chronically depolarized fibers,
the frequency of occurrence of voltage-activated events
can be modulated by both the extent of repriming and
by the membrane potential of the test depolarization
used to activate events after repriming, but neither of
these parameters appears to affect the average amplitude
or average rise time of the detected events (Lacampagne et al., 1996
; Klein et al., 1997
). Lowering myoplasmic free [Mg2+] increases the spontaneous frequency of ligand-gated events, but does not alter the average properties of the individual events (Lacampagne
et al., 1998
). Thus, the average properties of the individual events appear to be quite constant despite relatively large changes in their frequency.
Possible regulation of channel opening or Ca2+ release during a spark.
By analyzing sparks that occur repetitively
at particular locations at much higher rates than the average frequency over the entire fiber, the limitations of
variable spark origin and arbitrary cut-off of event amplitudes discussed in the preceding paragraph can to
a large extent be overcome. Such higher frequency events occur spontaneously in cardiac myocytes (Parker and
Wier, 1997) and both spontaneously and during depolarization in frog skeletal muscle fibers (Klein et al., 1999
)
and appear to represent a repetitive mode of spark activation. If these repetitive events are generated by the
repeated opening of a given channel or small cluster of channels (Klein et al., 1999
), they would all arise at the
same spatial site within the fiber. Thus, variation in spark
amplitude due to variation in the site of origin relative
to the scan line would not be a factor in the relative amplitude of the individual events within a given repetitive
train. Analysis of events in such trains indicates an unanticipated lack of smaller events, even when objective
procedures are employed to evaluate possible occurrence
of unidentified events between identified events (Klein et
al., 1999
). Examination of the records of repetitive events from cardiac myocytes (Parker and Wier, 1997
) also indicates a relative lack of smaller amplitude events. If the
sparks in a repetitive train were generated by the opening of a single channel having a single or multiple exponential open time distribution and if the spark amplitude were directly related to the channel open time,
then smaller events would actually be expected to occur more frequently in the train than larger events. Thus, if a
single channel is responsible for generating the repetitive sparks, the observed paucity of smaller amplitude
events in repetitive trains would indicate the possibility
of some sort of regulation of the amount of Ca2+ released by the single channel generating the spark.
Are two types of sparks activated during fiber depolarization?
In our original report of the existence of ligand- and
voltage-activated sparks in skeletal muscle, the observed
amplitude distributions of the sparks initiated by these
two types of mechanisms were different (Klein et al.,
1996). The spontaneous (i.e., ligand-gated) sparks appeared to correspond to a single population of events, whereas the voltage-activated sparks appeared to correspond to one population of events having the same amplitude distribution as the spontaneous events together
with another population of events having an amplitude
distribution corresponding to approximately twice that
of the spontaneous events. The smaller amplitude voltage-activated events were attributed to the opening of a
single SR Ca2+ channel directly by interaction with the
voltage sensor. The larger amplitude events were attributed to opening of two channels, one activated directly
by the voltage sensor and a second activated by CICR
due to the locally elevated [Ca2+] in the immediate
neighborhood of the voltage-activated channel. In this
case, at least two channels would have to be involved in
generating the population of larger voltage-activated
Ca2+ sparks. However, in subsequent studies in our laboratory, we have found no obvious differences in the
mean amplitudes of ligand- and voltage-activated sparks
in different fibers. In our initial study (Klein et al., 1996
),
we employed levels of activation that resulted in the occurrence of relatively large numbers of sparks at relatively high frequencies since we were searching for evidence for the existence of sparks. Although this strategy
may have been appropriate for an initial demonstration
of the existence of these events, it was not ideal for the
detailed characterization of the properties of individual
events due to the possibility of events randomly overlapping in space and time. Although the possibility of
chance overlap of random independent events was discounted in our original report, our estimate of the
probability of random overlap did not include possibly
overlapped events in the estimate of the total event rate
used for the calculation. Based on our subsequent experience, it now seems possible that the event rate during
the depolarizations in our initial study may have been too high to permit accurate determination of the properties of isolated individual sparks. Thus, the question of
the relative amplitudes of ligand- and voltage-activated
events in the same fibers should probably be reexamined using lower event rates during fiber depolarization.
Could a single SR Ca2+ channel trigger a multichannel release unit?
Under conditions of low average rates of occurrence of Ca2+ sparks as used in our recent experiments, it seems likely that each voltage-activated spark
is initiated by the activation of the SR channel controlled by a single TT voltage sensor and that each ligand-activated event is initiated by the opening of a
single SR Ca2+ release channel by CICR. Thus, if a
spark involves the opening of multiple SR Ca2+ release
channels, the single channel that opens to initiate the spark must activate one or more neighboring channels,
presumably by CICR. These channels could in turn activate additional neighboring channels, which in principal could continue until all the SR channels along an
entire region of continuous TT-SR junctional couplings were activated. For the typical relatively brief
sparks having a rising phase of a few milliseconds duration, it might be imagined that the propagation of activation would have to occur rapidly at the start of the rising phase, and that the rising phase would end as the
channels close in near synchrony, possibly by calcium-dependent inactivation. This general type of propagated activation scheme involving many SR Ca2+ channels has been simulated using a model of possible local
Ca2+ signalling within the TT-SR junctional region
(Stern et al., 1997). Thus, the possibility that activity of
many SR channels coupled by CICR underlies a spark is
not theoretically inconsistent with initiation of the
spark by activation of a single channel.
Could a single SR Ca2+ channel maintain prolonged release
in many other channels?
We have observed that application of Imperatoxin A (IpTxa) to a permeabilized frog
muscle fiber causes the appearance of prolonged Ca2+
sparks having durations of several hundred milliseconds or longer (Shtifman et al., 1999). Since IpTxa is
known to produce similarly prolonged subconductance
openings (about one-third conductance of normal
channel opening) of individual SR Ca2+ release channels incorporated in lipid bilayers (Tripathy et al.,
1998
), possibly by acting as an analogue of the 2-3 cytoplasmic loop of the TT dihydropyridine receptor/voltage sensor, it seems likely that the prolonged sparks observed in the presence of IpTxa were generated by the
prolonged opening of a single SR channel to a subconductance state. The very long duration sparks initiated by IpTxa were also smaller in amplitude than normal
short-duration events observed in the same fibers.
These observations raise the interesting question of
whether the prolonged toxin-induced opening of a single SR channel could maintain the opening of many
other SR channels without the other channels inactivating. Alternatively, the prolonged toxin-induced sparks
could be readily explained if toxin-induced sparks are
generated by the prolonged opening of only the single
channel interacting with the toxin. In this case, the observation that the amplitude of the toxin-induced event
is smaller then that of a brief toxin-independent spark, together with the fact that IpTxa produces a subconductance state when applied to isolated SR channels,
could indicate that the normal sparks observed in the
absence of toxin could also be generated by the opening of a single SR Ca2+ release channel, but to the full
conductance state and only for a few milliseconds.
Conclusion
Based on the various considerations presented above, it does not appear that we can yet exclude the possibilities that either one or many channels are involved in the generation of a Ca2+ spark. If a single channel is responsible for generating a spark, the single channel must have appropriate feedback regulation so as to account for the reproducible spark amplitude and relative lack of small events during the repetitive spark gating mode observed at occasional triads. On the other hand, the very prolonged small amplitude sparks observed in the presence of IpTxa are readily explained on the basis of prolonged subconductance opening of a single toxin-bound channel. If many channels are involved in the normal, short duration voltage- or ligand-activated spark, they would have to open and close in close synchrony, or burst over the same few milliseconds time interval to account for the abrupt start and peak of the observed spark time course. If many channels are involved in a short spark, it would also seem to be necessary for a single open channel to have the capability of maintaining long duration opening of at least some of the other channels involved in the short spark to account for the long duration events produced by IpTxa. It will be an interesting challenge to try to resolve these still viable important alternative possibilities as to the channel activity pattern underlying the Ca2+ sparks observed in frog skeletal muscle.
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FOOTNOTES |
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Original version received 9 December 1998 and accepted version received 20 January 1999.
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