Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method.

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.     

The changes in Ca2+ sparks associated with measured modifications of intra-store Ca2+ concentration in skeletal muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 microM on average in 10 experiments) and a high value (433 microM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The "strength" of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.     

The spark and its ember: separately gated local components of Ca(2+) release in skeletal muscle

Amplitude, spatial width, and rise time of Ca(2+) sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca(2+)]. A total of approximately 17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg(2+)] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence "ridge" and is continued by an "ember," a prolongation of width approximately 1 microm and amplitude <0.2, vanishing in approximately 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg(2+)] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg(2+)], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca(2+) release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source-presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg(2+). Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca(2+)-dependent activation (caffeine, low [Mg(2+)]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca(2+) release directly operated by voltage sensors.     

Uncontrolled calcium sparks act as a dystrophic signal for mammalian skeletal muscle

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.     

Calcium-dependent inactivation terminates calcium release in skeletal muscle of amphibians

In skeletal muscle of amphibians, the cell-wide cytosolic release of calcium that enables contraction in response to an action potential appears to be built of Ca2+ sparks. The mechanism that rapidly terminates this release was investigated by studying the termination of Ca2+ release underlying sparks. In groups of thousands of sparks occurring spontaneously in membrane-permeabilized frog muscle cells a complex relationship was found between amplitude a and rise time T, which in sparks corresponds to the active time of the underlying Ca2+ release. This relationship included a range of T where a paradoxically decreased with increasing T. Three different methods were used to estimate Ca2+ release flux in groups of sparks of different T. Using every method, it was found that T and flux were inversely correlated, roughly inversely proportional. A simple model in which release sources were inactivated by cytosolic Ca2+ was able to explain the relationship. The predictive value of the model, evaluated by analyzing the variance of spark amplitude, was found to be high when allowance was made for the out-of-focus error contribution to the total variance. This contribution was estimated using a theory of confocal scanning (Ríos, E., N. Shirokova, W.G. Kirsch, G. Pizarro, M.D. Stern, H. Cheng, and A. González. Biophys. J. 2001. 80:169-183), which was confirmed in the present work by simulated line scanning of simulated sparks. Considering these results and other available evidence it is concluded that Ca2+-dependent inactivation, or CDI, provides the crucial mechanism for termination of sparks and cell-wide Ca2+ release in amphibians. Given the similarities in kinetics of release termination observed in cell-averaged records of amphibian and mammalian muscle, and in spite of differences in activation mechanisms, CDI is likely to play a central role in mammals as well. Trivially, an inverse proportionality between release flux and duration, in sparks or in global release of skeletal muscle, maintains constancy of the amount of released Ca2+.     

SparkMaster: automated calcium spark analysis with ImageJ

Ca sparks are elementary Ca-release events from intracellular Ca stores that are observed in virtually all types of muscle. Typically, Ca sparks are measured in the line-scan mode with confocal laser-scanning microscopes, yielding two-dimensional images (distance vs. time). The manual analysis of these images is time consuming and prone to errors as well as investigator bias. Therefore, we developed SparkMaster, an automated analysis program that allows rapid and reliable spark analysis. The underlying analysis algorithm is adapted from the threshold-based standard method of spark analysis developed by Cheng et al. (Biophys J 76: 606–617, 1999) and is implemented here in the freely available image-processing software ImageJ. SparkMaster offers a graphical user interface through which all analysis parameters and output options are selected. The analysis includes general image parameters (number of detected sparks, spark frequency) and individual spark parameters (amplitude, full width at half-maximum amplitude, full duration at half-maximum amplitude, full width, full duration, time to peak, maximum steepness of spark upstroke, time constant of spark decay). We validated the algorithm using images with synthetic sparks embedded into backgrounds with different signal-to-noise ratios to determine an analysis criteria at which a high sensitivity is combined with a low frequency of false-positive detections. Finally, we applied SparkMaster to analyze experimental data of sparks measured in intact and permeabilized ventricular cardiomyocytes, permeabilized mammalian skeletal muscle, and intact smooth muscle cells. We found that SparkMaster provides a reliable, easy to use, and fast way of analyzing Ca sparks in a wide variety of experimental conditions. myocytes; sarcoplasmic reticulum; confocal microscopy     

Factors shaping the confocal image of the calcium spark in cardiac muscle cells.

The interpretation of confocal line-scan images of local [Ca2+]i transients (such as Ca2+ sparks in cardiac muscle) is complicated by uncertainties in the position of the origin of the Ca2+ spark (relative to the scan line) and by the dynamics of Ca(2+)-dye interactions. An investigation of the effects of these complications modeled the release, diffusion, binding, and uptake of Ca2+ in cardiac cells (producing a theoretical Ca2+ spark) and image formation in a confocal microscope (after measurement of its point-spread function) and simulated line-scan images of a theoretical Ca2+ spark (when it was viewed from all possible positions relative to the scan line). In line-scan images, Ca2+ sparks that arose in a different optical section or with the site of origin displaced laterally from the scan line appeared attenuated, whereas their rise times slowed down only slightly. These results indicate that even if all Ca2+ sparks are perfectly identical events, except for their site of origin, there will be an apparent variation in the amplitude and other characteristics of Ca2+ sparks as measured from confocal line-scan images. The frequency distributions of the kinetic parameters (i.e., peak amplitude, rise time, fall time) of Ca2+ sparks were calculated for repetitive registration of stereotyped Ca2+ sparks in two experimental situations: 1) random position of the scan line relative to possible SR Ca(2+)-release sites and 2) fixed position of the scan line going through a set of possible SR Ca(2+)-release sites. The effects of noise were incorporated into the model, and a visibility function was proposed to account for the subjective factors that may be involved in the evaluation of Ca(2+)-spark image parameters from noisy experimental recordings. The mean value of the resulting amplitude distributions underestimates the brightness of in-focus Ca2+ sparks because large numbers of out-of-focus Ca2+ sparks are detected (as small Ca2+ sparks). The distribution of peak amplitudes may split into more than one subpopulation even when one is viewing stereotyped Ca2+ sparks because of the discrete locations of possible SR Ca(2+)-release sites in mammalian ventricular heart cells.     

Effects of ryanoids on spontaneous and depolarization-evoked calcium release events in frog muscle

The effects of ryanoids on calcium sparks and transients were studied in voltage-clamped cut frog muscle fibers with a laser scanning confocal microscope. For each ryanoid employed, several sequential effects were observed, including: a), transient increases in spontaneous spark frequency; b), conversions of sparks to long-lasting steady glows; and c), occasional interruptions of the glows. The ratio of the amplitude of the glow induced by a ryanoid to that of the precursory spark followed the order: ryanodol > ryanodine > C(10)-O(eq)-glycyl-ryanodine > C(10)-O(eq)-beta-alanyl-ryanodol. This sequence of glow amplitudes parallels that of the subconductances induced by these ryanoids in single-channel studies, suggesting that the glows reflect Ca(2+) fluxes through semiopen calcium release channels. Ryanoids also abolished depolarization-evoked sparks elicited with small pulses, and transformed the calcium release during depolarization to a uniform nonsparking fluorescence signal. The ratio of this signal, averaged spatially, to that of the control followed the order: ryanodol < ryanodine < C(10)-O(eq)-glycyl-ryanodine < C(10)-O(eq)-beta-alanyl-ryanodol, implying an inverse relationship with the amplitudes of ryanoid-induced glows. The observation that depolarization-evoked calcium release can occur after ryanoid suppression of calcium sparks suggests the possibility of a new strategic approach for treating skeletal muscle diseases resulting from leaky calcium release channels.     

Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Properties of Ca2+ sparks revealed by four-dimensional confocal imaging of cardiac muscle

Parameters (amplitude, width, kinetics) of Ca(2+) sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-μm separation (x-y-z-t mode). In 3,000 x-y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F(0) = 0.7. The Ca(2+) release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca(2+) release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca(2+) in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca(2+)](SR)) or that different channel clusters respond to [Ca(2+)](SR) with different sensitivity.     

Ca2+ sparks and embers of mammalian muscle. Properties of the sources

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.     

Ca(2+) spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large-conductance K(+) channels, and coupling ratio between them

Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.     

Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.     

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.     

Theoretical analysis of the Ca2+ spark amplitude distribution.

A difficulty of using confocal microscopy to study Ca2+ sparks is the uncertainty of the linescan position with respect to the source of Ca2+ release. Random placement of the linescan is expected to result in a broad distribution of measured Ca2+ spark amplitudes (a) even if all Ca2+ sparks were generated identically. Thus variations in Ca2+ spark amplitude due to positional differences between confocal linescans and Ca2+ release site are intertwined with variations due to intrinsic differences in Ca2+ release properties. To separate these two sources of variations on the Ca2+ spark amplitude, we determined the effect changes of channel current or channel open time--collectively called the source strength, alpha--had on the measured Ca2+ spark amplitude histogram, N(a). This was done by 1) simulating Ca2+ release, Ca2+ and fluo-3 diffusion, and Ca2+ binding reactions; 2) simulation of image formation of the Ca2+ spark by a confocal microscope; and 3) using a novel automatic Ca2+ spark detector. From these results we derived an integral equation relating the probability density function of source strengths, f alpha (alpha), to N(a), which takes into account random positional variations between the source and linescan. In the special, but important, case that the spatial distribution of Ca(2+)-bound fluo-3 is Gaussian, we show the following: 1) variations of Ca2+ spark amplitude due to positional or intrinsic differences can be separated, and 2) f alpha (alpha) can, in principle, be calculated from the Ca2+ spark amplitude histogram since N(a) is the sum of shifted hyperbolas, where the magnitudes of the shifts and weights depend on f alpha (alpha). In particular, if all Ca2+ sparks were generated identically, then the plot of 1/N(a) against a will be a straight line. Multiple populations of channels carrying distinct currents are revealed by discontinuities in the 1/N(a) plot. 3) Although the inverse relationship between Ca2+ spark amplitude and decay time might be used to distinguish Ca2+ sparks from different channel populations, noise can render the measured decay times meaningless for small amplitude Ca2+ sparks.     

Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes

Ca²⁺ sparks are the elementary release events in many types of cells. Here we present a morphometric analysis of Ca²⁺ sparks (i.e., amplitude and kinetic parameters) using an approach that minimizes the confounding factor of the detection of out-of-focus events. By activation and visualization of Ca²⁺ sparks from Ca²⁺ release units under loose-seal patch-clamp conditions, we found that the amplitude and rising rate of in-focus sparks exhibited a broad modal distribution, whereas spark rise time and spatial width appeared to be stereotyped. Spark morphometrics were constant irrespective of the latency of spark production and the time-dependent L-type Ca²⁺ channel activation. Polymorphism of Ca²⁺ sparks in terms of variable amplitude and rising rate was evident for events from the same release units, and intra- and interrelease unit variability contributed equally to the overall variability. The rising rate, a reporter of the underlying Ca²⁺ release flux, displayed a strong positive correlation with spark amplitude, but a negative correlation with spark rise time, an index of Ca²⁺ release duration. On the basis of Ca²⁺ spark morphometrics measured here, we suggested a model in which cohorts of variable number of ryanodine receptors are activated in the genesis of Ca²⁺ sparks, and the ensuing negative feedback overrides the regenerative Ca²⁺-induced Ca²⁺ release to extinguish the ongoing Ca²⁺ spark.     

Contribution of ryanodine receptor type 3 to Ca(2+) sparks in embryonic mouse skeletal muscle.

The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.     

Effects of imperatoxin A on local sarcoplasmic reticulum Ca(2+) release in frog skeletal muscle

We have investigated the effects of imperatoxin A (IpTx(a)) on local calcium release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx(a) induced the appearance of Ca(2+) release events from the sarcoplasmic reticulum that are approximately 2 s and have a smaller amplitude (31 +/- 2%) than the "Ca(2+) sparks" normally seen in the absence of toxin. The frequency of occurrence of long-duration imperatoxin-induced Ca(2+) release events increased in proportion to IpTx(a) concentrations ranging from 10 nM to 50 nM. The mean duration of imperatoxin-induced events in muscle fibers was independent of toxin concentration and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, where IpTx(a) induced opening of single Ca(2+) release channels to prolonged subconductance states. These results suggest involvement of a single molecule of IpTx(a) in the activation of a single Ca(2+) release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductance to be the same in the fibers as in bilayer, the amplitude of a spark relative to the long event indicates involvement of at most four RyR Ca(2+) release channels in the production of short-duration Ca(2+) sparks.     

Effects of tetracaine on voltage-activated calcium sparks in frog intact skeletal muscle fibers

The properties of Ca(2+) sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K(+)] Ringer's are well described by a computational model with a Ca(2+) source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 degrees C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca(2+) current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1-5 RyR Ca(2+) release channels open during a voltage-activated Ca(2+) spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K(+)] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75-100 microM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca(2+) channels per spark, and (b) the single-channel Ca(2+) current of an RyR channel is 相似文献   

Dynamics of signaling between Ca(2+) sparks and Ca(2+)- activated K(+) channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca(2+) current

Ca(2+) sparks are highly localized cytosolic Ca(2+) transients caused by a release of Ca(2+) from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca(2+) in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca(2+) indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca(2+) released into the cytosol, and its rate of rise is proportional to the Ca(2+) current flowing through the RyRs during a spark (I(Ca(spark))). Thus, Ca(2+) currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I(Ca(spark)) in different sparks varies more than fivefold, Ca(2+) sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca(2+) current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca(2+)] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca(2+) concentration resulting from the measured range of I(Ca(spark)). At the onset of a spark, the Ca(2+) concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca(2+)](EC50) for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca(2+)] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca(2+) action on a variety of molecular targets within cellular microdomains.