1Biomedical Imaging Group, 2Department of Physiology, and 3Department of Information Services, University of Massachusetts Medical School, Worcester, Massachusetts 01655; and 4Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
Submitted 19 March 2004 ; accepted in final form 27 July 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
wide-field imaging; spontaneous transient outward current; microdomain
In recent studies of Ca2+ sparks and STOC mediated by BK channels, investigators at our laboratory (28) developed a new imaging-based methodology to measure the mean intracellular Ca2+ current flowing from the SR into the cytosol through the RyR [ICa(spark)] in groups of Ca2+ sparks of differing amplitude. Two of the major findings of that study were as follows. First, the mean ICa(spark) varied by more than fivefold for different groups of sparks. This finding indicated that large-amplitude sparks were due to the opening of a greater number of RyR rather than simply to a single RyR opening for a longer duration. Second, there was variability in the ratio of active RyR to activated BK channels, i.e., the coupling ratio, when sparks elicited STOC. In that study, signal averaging techniques were used that merged data from multiple spark sites. Hence the characteristics of individual spark sites could not be examined systematically, and a number of questions about individual sites remained unanswered. For example, did the magnitude of a spark and a STOC at a given site tend to be constant? Did those spark sites that failed to elicit STOC always fail to do so, and did the coupling ratio differ from site to site? In the present study, we have quantitatively examined the properties of individual sparks sites using high-speed florescence Ca2+ imaging in combination with the patch-clamp technique. The main findings of the present study were reported previously in preliminary form (27).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bufo marinus were killed by rapid decapitation in accordance with Institutional Animal Care and Use Committee guidelines. Single smooth muscle cells from the stomach muscularis were enzymatically dispersed as described previously (8). Membrane currents were recorded using the tight-seal, whole cell patch-clamp recording configuration. The extracellular solution contained (in mM) 130 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, with pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 137 KCl, 3 MgCl2, 10 HEPES, 3 Na2ATP, and 0.05 fluo-3, with pH adjusted to 7.2 with KOH. All experiments were performed at room temperature. Whole cell currents were recorded at a holding potential of 0 mV and low-pass filtered using the Axopatch 1D amplifier (200 Hz cutoff), and then they were digitally sampled at 1 kHz and stored for analysis. Events were counted as STOC if they exceeded a threshold of 10 pA as detected using a custom automated algorithm. The events were then checked by visual inspection to eliminate anomalies such as multiple events overlapping in time or excessively noisy traces. The data for this study were drawn from 326 sparks sites in 25 cells from 13 animals.
Imaging and Measurement of Ca2+ Sparks
Fluorescent images were obtained using fluo-3 as the Ca2+ indicator and a custom-built wide-field, high-speed digital imaging system, which was described in detail elsewhere (31). Rapid imaging was made possible by using a cooled, high-sensitivity, charge-coupled device camera (128 x 128 pixels) developed in conjunction with the Massachusetts Institute of Technology Lincoln Laboratory (Lexington, MA). Imaging was performed at 100 Hz with 10-ms acquisition times, and there were 200 images/sequence (2 s). Multiple sequences of 2 s were taken, and the interval between sequences was 30 s. The camera was interfaced with a custom-made inverted microscope equipped with a x40 oil-immersion lens (NA 1.3), and each pixel covered a 333 x 333-nm area of the cell. The 488-nm line of a multiline argon laser provided fluorescence excitation of the indicator fluo-3, and a laser shutter controlled the exposure duration. Emission of the Ca2+ indicator was monitored at wavelengths >500 nm. To obtain a constant [Ca2+] indicator, fluo-3 (50 µM) was delivered through the patch pipette, and measurements were not commenced until 1015 min after disruption of the patch. After that time, no significant change in background fluorescence was detected. Subsequent image processing and analysis were performed offline using a custom-designed software package running on a Silicon Graphics workstation.
Because the localization of sparks within a cell was a critical feature of the present study, we examined the lateral resolution of the imaging system by measuring the point spread function of the system with the same objective used during the study using 190-nm-diameter fluorescent beads. The point spread function shows that we approached the theoretical limit of resolution for this system, 0.3- to 0.4-µm full width at half-maximum intensity (FWHM), while achieving axial resolution of
1-µm FWHM (see, e.g., Ref. 11, Table 2.1). Because in the present study we were interested in determining the location of the epicenter pixels of sparks that were both spatially and temporally distinct, the resolution of the imaging system was more than sufficient for our purposes.
Two measures of Ca2+ sparks were used: 1) the conventional fluorescence ratio, F/F0, within a restricted volume; and 2) the change in total fluorescence, F F0, over a larger volume, also designated as the Ca2+ signal mass, which is proportional to the total quantity of Ca2+ released into the cytosol. These measurements were described in detail previously (28, 29), so only a brief description follows. For the fluorescence ratio measure, the fluo-3 images, with pixel size 333 x 333 nm, were first smoothed by convolution with a 3 x 3-pixel approximation to a two-dimensional (2-D) gaussian curve (
= 1). Fluorescence ratios were then calculated and expressed as a percentage on a pixel-to-pixel basis from the following equation:
![]() | (1) |
![]() | (2) |
![]() | (3) |
![]() |
Statistical Analysis
Variability in the parameters of Ca2+ sparks and STOC within and between sites was evaluated by performing analysis of variance after log transformation (see Table 1 and Figs. 3 and 4).
|
|
|
All chemicals except fluo-3 (Molecular Probes, Eugene, OR) were purchased from Sigma Chemical (St. Louis, MO).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The evolution of a Ca2+ spark is shown in Fig. 1, along with the simultaneously recorded STOC that it elicited. Figure 1A shows one spark produced using the fluorescent Ca2+ indicator dye fluo-3. Figure 1Ba shows the STOC, and Fig. 1Bb shows the conventional method of measuring a spark as a fraction of the background fluorescence (F/F0) in a relatively small region. In the present study, the region was a single 333 x 333-nm pixel that lies at the spark's center and displayed the brightest fluorescence, i.e., the epicenter pixel. This measure is often considered proportional to the average [Ca2+] in the region, but there are difficulties with this view. Because the indicator dye and Ca2+ do not come into equilibrium in the microdomain of the Ca2+ release site where the BK channels reside (29), calculations of [Ca2+] there are problematic. Consequently, another approach is needed.
|
Two features of the signal mass trace merit attention. First, as noted in an earlier study (28), the point in time at which the signal mass reaches its peak, i.e., when ICa(spark) terminates, corresponds to the time that the STOC begins to decay. Hence, the time from STOC to TTD provides an approximate measure of the duration of ICa(spark). Second, the signal mass trace remains in a plateau phase for a considerable time after reaching its peak, i.e., its decay is very much slower than its rise. This indicates that diffusion accounts for almost all the removal of Ca2+ from the vicinity of the release site once the peak is reached and suggests that sequestration or extrusion processes do not substantially diminish the magnitude of the measured signal mass. These features of the spark and STOC shown in Fig. 1 are representative of the spark population as a whole in these smooth muscle cells. Similarly, in cardiac cells, diffusion away from the release site appears to account for 80% of the fall in [Ca2+] during a spark (9).
Localizing Ca2+ Sparks and STOC in the Cell
It is a straightforward task to construct a map of spark locations in a cell from images such as those shown in Fig. 2A. Such a map is shown in two ways, in Fig. 2, B and C, which show about one-third of the cell. In Fig. 2B, each circle again indicates the occurrence of a single spark, with the center of the circle lying at the spark's epicenter pixel and the diameter of the circle proportional to the magnitude of F/F0 at that pixel. In Fig. 2C, each occurrence of a spark is shown simply by a small, closed circle at the epicenter pixel, i.e., the brightest pixel during the spark. Figure 2B suggests that the amplitude of a spark at a given site is relatively constant, described earlier in MATERIALS AND METHODS. Visual inspection of such maps also reveals that there are many spark sites per cell, considerably more than the 14 sites cited in earlier studies (12, 19). In the results below, we make a quantitative estimate of the number of sites and describe how occasionally a brief burst of 24 sparks (Fig. 2A, bottom) is generated at one location.
|
An Individual Spark Site Can Be Characterized by the Amount of Ca2+ Released Into the Cytosol During a Spark
We define a single spark site in the following explicit way to determine properties of individual sites in an objective fashion. Two or more sparks occur at the same site if they are centered on the same 333 x 333-nm pixel, that is, if the epicenter pixels for each spark are identical in location. To determine the consistency of spark properties at a given site, we examined those sites that produced two or more sparks. Figure 3Aa shows the total Ca2+ released (i.e., the signal mass) for individual sparks arising from this group of sites. The data are arranged by individual sites and ranked in ascending order along the abscissa according to the mean amount of total Ca2+ released per spark at each site. Figure 3, Ab and Ac, shows the F/F0 and FWHM as determined from the
F/F0 image for the same group of sites, in each case following the same order shown in Fig. 3Aa, that is, according to the ascending order of the signal mass. Hence, data from the same spark site appear vertically above and below one another in all the plots shown in Fig. 3A.
Examination of Fig. 3Aa conveys the impression that a given spark site releases a characteristic amount of Ca2+ into the cytosol. To address this question quantitatively, we asked whether the variation in signal mass from spark to spark within a site was significantly different from the variation between sites, using an analysis of variance measure. If all sites released the same amount of Ca2+ on average, the within-site variance likely would not differ from the between-site variance. This was not the case; that is, the between-site variance was significantly greater than the within-site variance (Table 1). Table 1 shows the data for all sites with multiple sparks and the results for those sites that generated STOC, which were 76% of the total in this data set. Sparks from 11 sites were excluded from the analysis because of uncertainty in measuring the corresponding STOC. In a larger data set (28), 21% of sparks were STOC-less. As shown in Table 1, the between-site variance was significantly greater than the intrasite variance for two other measures of the spark: F/F0 and FWHM.
A Given Spark Site Elicits a STOC of Characteristic Amplitude
A second way to assess possible heterogeneity among spark sites is to examine the properties of the STOC that are generated at a given site. This approach has the advantage of being independent of the fluorescence measure of the spark amplitude, because the spark serves only to locate the STOC in the cell. The amplitude of STOC and TTD are shown in Fig. 3B for those sites that generated two or more STOC. In each case, the sites are ranked according to increasing signal mass, that is, in the same order as shown in Fig. 3A; hence data from the same site are in vertical register. Figure 4A shows the same data as Fig. 3Aa but ranked by site according to increasing STOC amplitude. As was the case with signal mass data (Fig. 3Aa), this plot suggests heterogeneity in the STOC sites. Again, using analysis of variance, we tested whether the intrasite variance in STOC parameters was different from intersite variance.
As is evident in Table 1, intrasite variance was significantly less than the intersite variance for STOC amplitude, but not for STOC TTD. The fact that the TTD of the STOC is not significantly different from site to site is of interest because the TTD should reflect the duration of ICa(spark) (29). This finding indicates that there is no variability in the mean duration of ICa(spark) from site to site, and it suggests that there are not major differences in the open times and/or burst durations of RyR at different sites.
Spark Sites Differ in Number of Active RyR
The variation in signal mass among spark sites evident in Fig. 3Aa could result from either a difference in the number of RyR underlying a spark or a difference in spark duration. Because the number of RyR is proportional to the magnitude of ICa(spark), we examined the variation in ICa(spark) within and between sites. ICa(spark) was calculated by dividing the signal mass of the spark by the TTD of the elicited STOC and converting the quotient into units of current. These values for current cannot be corrected for nonmobile buffers, because the buffer kinetics are unknown. Thus, in their uncorrected sate, the values provide a minimum value for the peak ICa(spark) (29). The results are shown in Fig. 4B, where the sites are ranked in ascending order of the mean ICa(spark) at a site and in Table 1. These data indicate that ICa(spark), and hence the number of active RyR, varies from site to site. Note that Fig. 4A shows the data in ascending order of STOC amplitude and Fig. 4B shows the data in ascending order of ICa(spark) amplitude.
At Some Sites Sparks Consistently Elicit STOC, and at Others They Consistently Fail
RyR-BK coupling ratio varies from site to site. The coupling ratio is defined for the purposes of this study as the ratio of the magnitude of the STOC, taken at an isochronic point that lies at or before the peak of all the STOC studied, to that of the eliciting ICa(spark) measured at the same isochronic point. For the purposes of this study, the isochronic point was 5 ms after the onset of the STOC. The coupling ratio provides a measure of the number of active BK channels to active RyR during each spark. The usefulness of the 5-ms isochronic point was described in a previous study (28). Briefly, this isochronic point ensures that neither spark nor STOC has gone beyond its peak value. Figure 4C shows these values for each site in ascending order of mean coupling ratio; the points lying on the abscissa represent sparks that failed to elicit STOC. Strikingly, the sites that failed to produce STOC failed without exception, and the sites that produced STOC also did so without exception. Moreover, examination of all spark sites showed that the within-site variance for the coupling ratio is significantly different from the between-site variance (Table 1). Hence, the coupling ratio varies from site to site. The results described in this section and the preceding three subsections indicate that the choice of a single pixel, 333 nm on each side, is valid within limits. That is, if our definition of a site were excessively large, then it would be possible to obtain results that showed all sites are equivalent; i.e., the intrasite variation would be no different from the intersite variation. For the intrasite variation to be less than the intersite variation, as we found, then the definition must have validity, although, of course, it is entirely possible that the definition could be refined further.
There are numerous spark sites in a single smooth muscle cell.
Simple visual inspection of Fig. 2C discloses many more spark sites than the average of 14 reported in previous studies (12, 19). This is true even when only those sparks that are far removed from one another are considered to occur at different sites. This result is also strikingly evident in videotaped imaging of sparks, even for rather short periods of time, an example of which is shown in Supplemental Fig. 1. Please refer to the Supplementary Material for this article to view movies.1 To determine the number of spark sites in quantitative fashion, it is necessary to define a spark site for this purpose. A difficulty in doing so is apparent from the inset of Fig. 2C, in which a number of epicenter pixels belonging to different sparks lie close together in a cluster. However, such clusters comprise only a relatively small (20%) proportion of the total number of sites observed and make little difference in the overall count. When we count such clusters as a single site, we find an average of 13 different spark sites per observed field (i.e.,
cell), based on observations over a 10-s period (n = 9 cells).
Another approach to defining a spark is to rely on the knowledge we have acquired about the BK channels at the sites that give rise to a STOC. The site can be defined on the basis of the region over which BK channels are activated by a spark as follows. We demonstrated previously that BK channels underlying a STOC are exposed to a mean [Ca2+] that is 10 µM. Using this finding, we were able to determine, using reaction-diffusion simulations, the extent of the membrane area containing the BK channels activated by a spark, as shown in the plot of Fig. 5A. To produce this plot, the Ca2+ release site was placed 25 nm from the plasma membrane in accordance with the observed distance between the SR and plasma membranes (22). The x-axis in the plot indicates the profile of the [Ca2+] extending laterally along the membrane from the site of release. Depending on the buffer, the mean concentration of 10 µM is found at distances 150300 nm from the release site. Because the pixel size in our experiments was 333 nm/side, we counted separately those sites that were separated by two intervening pixels (i.e., a site being defined as an area of 3 pixels/side). With this criterion, we estimated that, on average, there were 14 spark sites per field of observation (Fig. 5B).
|
|
The rate of spark generation at a single site.
Because the spark sites are heterogeneous as outlined above, it was of interest to know whether they were heterogeneous in their rate of spontaneous discharge. If the sites were independent and all discharged at a uniform rate constant at a low probability, then the observed frequency of discharge likely would conform to a Poisson distribution. In constructing such a distribution of observations, it is critical to estimate the number of sites that fail to discharge during the period of observation. Each of the nine cells included in the data described in There are numerous spark sites in a single smooth muscle cell was observed for a total of 10 s. The total record for a cell comprised five 2-s periods separated by 30-s intervals during which the data buffers holding the images were read out. We used these 2-s intervals to measure the number of failures at an observed site. By "failures," we mean the number of 2-s intervals during which no spark was observed at that site, given that we observed at least one spark at some time during the course of the five 2-s intervals. (For example, if a site generated 1 spark in one 2-s period and failed to generate sparks in the other four 2-s periods, we then credited this site with 1 spark and 4 failures.) The results are shown in Fig. 7, and the values plotted on the ordinate axis are the totals for the portion of all nine cells in the field of observation. The number of failures obtained simply by counting is 734 (hatched bar). However, this number leaves out the silent sites (i.e., sites that failed to spark even once in 10 s). On the basis of the results of the experiments with a low dose of caffeine (see There are numerous spark sites in a single smooth muscle cell), we estimate, for the nine cells, a total of 1,719 additional silent sites [additional silent sites = 5 x (no. of observed sites x 1.8)]. This number must be added to 734, giving a total of 2,453 failures (Fig. 7, open bar) for all cells over the entire 10 s. (Note that this is a minimum count, because we do not know the fractions of silent sites revealed by caffeine application.) A 2 test shows that this is not consistent with a Poisson distribution (P < 8.6 x 107 that observed and expected distributions are the same). However, it is only a small number of observations (those sites yielding 2 or more sparks) that cause a deviation from a Poisson distribution.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a previous study (28), investigators at our laboratory found evidence of differences in the number of RyR in groups of sparks of different amplitude, in the number of BK channels in groups of STOC of different amplitude, and in RyR-BK coupling ratios in different groups of sparks. However, those results were derived from averaged data derived from groupings of sparks and STOC from different sites. Hence the differences found might indicate either that different sites varied differently in their properties or that all sites were uniform but had different functional characteristics at different points in time. For example, all sites might contain a constant number of RyR, and activation of a different fraction at different times might account for the apparent variation observed. The present study indicates that at least a part of the variation is due to differences in the properties of different sites.
Variation in Coupling Ratio and STOC-less Spark Sites
One of the most striking findings in the present study is that all spark sites that failed to generate STOC did so consistently and that spark sites that generated STOC also did so without exception. In a previous study, investigators at our laboratory (28) found that sparks with STOC differed from those without STOC, respectively, in the following ways: signal mass, 6.2 ± 0.3 x 1020 M Ca2+ vs. 4.0 ± 0.2 x 1020 M Ca2+ (P = 0.001); F/F0 (%), 12.6 ± 0.5 vs. 10.5 ± 0.5 (P = 0.004); FWHM, 2.7 ± 0.1 µm vs. 2.2 ± 0.1 µm (P = 0.001); the t
of decay, however, was not different: 16.0 ± 0.5 vs. 15.4 ± 0.91 (P = 0.269). Without STOC, values for ICa(spark) could not be calculated. On the basis of these previous results together with the present demonstration that STOC-less sparks arise from unique sites, we conclude that STOC-less sites differ from those that generate STOC in signal mass,
F/F0 and FWHM.
There are several possible explanations for the existence of STOC-less spark sites. First, the RyR might be located so far from the plasma membrane that BK channels are not activated by the Ca2+ discharged from the SR. However, this does not appear to be the case, for two reasons. First, 3-D imaging of Ca2+ sparks in feline esophageal smooth muscle preparation showed that STOC-less sparks are not located farther from the cell membrane than sparks that generate STOC, and they may even be somewhat closer, on average (13). Second, ultrastructural studies indicate that the SR and plasma membrane are quite closely positioned in smooth muscle, so that the RyR and BK channels are likely to lie within 1220 nm of one another (22). A second possible explanation for STOC-less sites is that the BK channels there may lack the 1-subunit, resulting in a decreased sensitivity to Ca2+. Although this possibility cannot be excluded definitively, it seems to be unlikely, for three reasons. First, in an earlier study, the BK channels from the cell type used in the present study were examined in excised patches and found to have a Ca2+ sensitivity that indicated the presence of the
1-subunit (23). Second, with the exception of cells from transgenic mice with the
1-subunit knocked out, smooth muscle cells seem to be characterized by the presence of BK channels that incorporate the
1-subunit (4, 20). Third, sparks in
1-knockout smooth muscle cells generate STOC, albeit at smaller amplitude; the sparks are not completely STOC-less at 0 mV (4, 20). Hence, although this explanation seems unlikely, it would be of considerable interest if there were sites characterized by the absence of
1-subunits.
At the moment, it seems most likely that the STOC-less spark sites lie at one extreme of the variation in coupling ratio, with BK channels absent or not activatable at these sites. If the RyR and BK channels were in physical contact, either directly or via an anchoring protein, then a constant coupling ratio might be expected. The variability in coupling ratio makes this sort of structural arrangement less likely.
The possible existence of spark sites without BK channels is of interest, because it leads to speculation that there may be other targets for Ca2+ released during sparks in the microdomains of these sites. These targets might require a [Ca2+] in excess of 1 µM, and thus they would have to lie close to the RyR in the spark microdomain. The usefulness of this arrangement for specificity of Ca2+ signaling becomes apparent when one considers that the global [Ca2+] in the cells used here cannot be elevated to much more than 1 µM (1). Hence, targets with low Ca2+ sensitivity lying in the microdomain would be activated only by Ca2+ arising from sparks and would be protected from activation by global rises in Ca2+. In a different smooth muscle type, investigators at our laboratory previously showed that the same sparks activated both BK channels and Ca2+-activated Cl channels, with the latter in the form of STIC (30). No evidence for STIC was found in this preparation, hence cytosolic Ca2+ arising from sparks may have targets other than ion channels in smooth muscle. For example, investigators at our laboratory recently observed sparklike events in freshly dissociated single-nerve terminals from the posterior pituitary. We call these events syntillas (from scintilla, Latin for spark, from a nerve terminal, a synaptic structure), and our laboratory found that syntillas do not activate ion channels, although the precise role of syntillas is not yet clear (7). Moreover, because the SR network has contact with virtually every intracellular organelle, Ca2+ sparks may be capable of modulating a variety of cellular processes.
Number of spark sites.
We have found that there were many more spark sites than previously estimated in smooth muscle cells with the use of confocal imaging technology. For example, in rat cerebral arterial cells, the mean number of spark sites per cell was 2.1 (19), and in canine pulmonary arterial cells the mean number was 14/cell (2 times more in the presence of 100 µM caffeine) (12). In the present study, using a high-sensitivity, wide-field digital imaging technology, we directly observed 14 sites on average for the approximately one-third of the cell in view, leading to an estimate of 42 per cell. (Even if we defined a site as an area of 5 pixels/side, i.e., 1.67 µm2, we would have observed
13 sites per region of the cell in the field, hence
39 sites per cell.) Furthermore, our experiments with low levels of caffeine led to an even higher estimate and indicate that there are hundreds of spark sites per cell. Qualitative observations that we have made in other cell types suggest that these results are not due to a difference in the smooth muscle cell type or the animal species used. We have observed a large number of spark sites in smooth muscle cells from the guinea pig airway (30), the cat esophagus (13), and the rat cerebral artery (Supplemental Fig. 2), although we did not perform systematic quantitation of the number of sites in those cells. (Note that in Supplemental Fig. 2, 4 of the 6 images show 2 widely separated spark sites occurring at the same time.) It is possible that the difference could result from the expression of different types or different densities of RyR in different smooth muscle cells. Our findings, however, are consistent with immunocytochemical data (15) that show an extensive distribution of RyR in smooth muscle, with a meshlike pattern resembling the interconnected SR network.
Rate of spark discharge at a site and existence of bursts of sparks. We estimated that spontaneous discharge at a spark site occurred, on average, at a rate of 0.007 s1, or 1:143 s. This estimate was made simply by dividing the observed STOC frequency by the estimated number of spark sites. When we attempted to fit the frequency of spontaneous discharge at a site to a Poisson distribution, there was a small but highly significant deviation from the expected distribution. This deviation can be explained in two ways. First, different sites may discharge with different rate constants. This is similar to the conclusion reached by Gordienko et al. (10) and Bolton and Cannell (2) that there are high-frequency discharge sites in smooth muscle cells. Second, the fact that there are brief bursts of sparks could give rise to a compound Poisson process (21) that departs from a Poisson distribution. (In the latter model, all sites are identical and independent, but whenever a spark discharges, there is a certain probability that a brief burst of 24 sparks will occur.) We found no evidence that certain sites tended to burst more than others or that bursting sites had shorter interburst intervals, although we cannot rule this out. Given the present data, it is simplest to postulate that any site may discharge once or else burst each time it discharges. When such bursting behavior was eliminated from consideration, there were very few sites that discharged multiple times (<1 site/2 cells) over our observation period. Thus we favor the second explanation, i.e., the existence of spark bursts at a site, to account for the non-Poisson behavior. This, however, does not negate the existence of the high-frequency discharge sites of Gordienko et al. (10) and Bolton and Cannell (2), which are regions much larger in area than the single-pixel sites examined in the present study. These regions could simply be the result of a high density of identical single-pixel spark sites in one area of the cell. We have found evidence for such a nonuniform spatial distribution of sites in feline esophageal smooth muscle cells (13). Clearly, the kinetics of spark discharge at a site demand further examination in a quantitative fashion.
The existence of short bursts of two to four sparks within a 2-s period, although infrequent, suggests that the frequency of spontaneous spark generation with the usual 1:100 s interburst is not due to local SR Ca2+ depletion and gradual reaccumulation. If reaccumulation were required for another spark, then it certainly could occur within a second, as evidenced by the bursts, and could not explain an interspark duration of 143 s at a site. Moreover, sites where bursting is detected do not seem to be a distinct population; instead, bursting appears to be a relatively infrequent occurrence that may be a property of all sites (see above).
In summary, in the present study, we have found that sparks sites are heterogeneous in the number of RyR and BK channels and in the coupling ratio between the two, and certain sites are consistently "STOC-less." The differences in coupling ratio allow us to distinguish certain classes of models for coupling RyR and BK channels in the spark microdomain, as discussed above. By inspection alone or using the spatial extent of the region containing the BK channels activated by a spark, we found the number of spark sites per cell was to be substantially greater than previous estimates of one to four per cell, most likely many hundreds of sites per cell, a finding consistent with morphological reports of an extensive SR in smooth muscle.
The results of the present study add to, and are consistent with, the picture of the biophysics of the spark-STOC microdomain found in an earlier study (29). In that previous study, investigators at our laboratory concluded that BK channels at the spark site were exposed to a [Ca2+] of 10 µM or more during a spark. This puts the BK channels and RyR within a radial distance of 150300 nm from one another in a genuine microdomain, which we termed an "oasis." This means that the cell membrane can house many such microdomains, and we have found in the present study that indeed it does. Given a BK channel density of 1 per square micrometer in this smooth muscle cell type (23), there must be thousands of BK channels per cell. With an estimate of hundreds of spark sites, a large number of these BK channels must be located at spark sites. In the old model of fewer than five spark sites per cell, most BK channels would be inaccessible to the Ca2+ arising from sparks. In this case, most BK channels would not be activated, except at positive potentials, given 1) the voltage and Ca2+ dependence of BK channels in smooth muscle cells, and 2) that global [Ca2+] rarely reaches a value as high as 1 µM even under intense stimulation and Ca2+ influx (1). Spark sites thus are a feature spread extensively over the SR and not only in one to five privileged regions. In addition, the BK channels residing in the SR are protected from activation by changes in global [Ca2+], responding more selectively to release from the SR at the spark sites. This also suggests that Ca2+-activated proteins with low Ca2+ affinities may be located in microdomains near the ER or SR Ca2+ release sites and not necessarily near the plasma membrane. Thus Ca2+-activated proteins with low Ca2+ affinities, whether located in the cytosol or in the plasma membrane, should be considered for candidacy as residents of Ca2+ microdomains. BK channels may simply be the first examples of such target proteins.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00153.2004/DC1/.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bolton TB and Gordienko DV. Confocal imaging of calcium release events in single smooth muscle cells. Acta Physiol Scand 164: 567575, 1998.[CrossRef][ISI][Medline]
3. Bond M, Shuman H, Somlyo AP, and Somlyo AV. Total cytoplasmic calcium in relaxed and maximally contracted rabbit portal vein smooth muscle. J Physiol 357: 185201, 1984.[Abstract]
4. Brenner R, Pérez GJ, Bonev A, Eckman D, Kosek J, Wiler S, Patterson A, Nelson MT, and Aldrich RW. Vasoregulation by the 1 subunit of the calcium-activated potassium channel. Nature 407: 870876, 2000.[CrossRef][ISI][Medline]
5. Cannell MB, Cheng H, and Lederer WJ. The control of calcium release in heart muscle. Science 268: 10451049, 1995.[ISI][Medline]
6. Cheng H, Lederer WJ, and Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740744, 1993.[ISI][Medline]
7. De Crescenzo V, ZhuGe R, Velázquez-Marrero C, Lifshitz LM, Custer E, Carmichael J, Lai FA, Tuft RA, Fogarty KE, Lemos JR, and Walsh JV Jr. Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx. J Neurosci 24: 12261235, 2004.
8. Fay FS, Hoffmann R, Leclair S, Merriam P. Preparation of individual smooth muscle cells from the stomach of Bufo marinus. Methods Enzymol 85: 284292, 1982.[ISI][Medline]
9. Gomez AM, Cheng H, Lederer WJ, and Bers DM. Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat ventricular myocytes. J Physiol 496: 575581, 1996.[Abstract]
10. Gordienko DV, Bolton TB, and Cannell MB. Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells. J Physiol 507: 707720, 1998.
11. Herman B. Fluorescence microscopy. In: Fluorescence Microscopy. New York: Springer-Verlag, 1998, p. 1538.
12. Janiak R, Wilson SM, Montague S, and Hume JR. Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 280: C22C33, 2001.
13. Kirber MT, Etter EF, Bellvé KA, Lifshitz LM, Tuft RA, Fay FS, Walsh JV Jr, and Fogarty KE. Relationship of Ca2+ sparks to STOCs studied with 2D and 3D imaging in feline oesophageal smooth muscle cells. J Physiol (Lond) 531: 315327, 2001.
14. Klein MG, Cheng H, Santana LF, Jiang YH, Lederer WJ, and Schneider MF. Two mechanisms of quantized calcium release in skeletal muscle. Nature 379: 455458, 1996.[CrossRef][ISI][Medline]
15. Lesh RE, Nixon GF, Fleischer S, Airey JA, Somlyo AP, and Somlyo AV. Localization of ryanodine receptors in smooth muscle. Circ Res 82: 175185, 1998.
16. Lopez-Lopez JR, Shacklock PS, Balke CW, and Wier WG. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268: 10421045, 1995.[ISI][Medline]
17. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633637, 1995.[Abstract]
18. Parker I and Yao Y. Regenerative release of calcium from functionally discrete subcellular stores by inositol trisphosphate. Proc R Soc Lond B Biol Sci 246: 269274, 1991.[ISI][Medline]
19. Pérez GJ, Bonev AD, Patlak JB, and Nelson MT. Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries. J Gen Physiol 113: 229237, 1999.
20. Plüger S, Faulhaber J, Fürstenau M, Löhn M, Waldschütz R, Gollasch M, Haller H, Luft FC, Ehmke H, and Pongs O. Mice with disrupted BK channel 1 subunit gene feature abnormal Ca2+ sparks/STOC coupling and elevated blood pressure. Circ Res 87: e53e60, 2000.[ISI][Medline]
21. Ross SM. The exponential distribution and the poisson process. In: Introduction to Probability Models. San Diego, CA: Academic, 2000, p. 239243.
22. Somlyo AV and Franzini-Armstrong C. New views of smooth muscle structure using freezing, deep-etching and rotary shadowing. Experientia 41: 841856, 1985.[ISI][Medline]
23. Singer JJ and Walsh JV Jr. Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique. Pflügers Arch 408: 98111, 1987.[ISI][Medline]
24. Sun XP, Callamaras N, Marchant JS, and Parker I. A continuum of InsP3-mediated elementary Ca2+ signalling events in Xenopus oocytes. J Physiol 509: 6780, 1998.
25. Tsugorka A, Ríos E, and Blatter LA. Imaging elementary events of calcium release in skeletal muscle cells. Science 269: 17231726, 1995.[ISI][Medline]
26. Xu L, Lai FA, Cohn A, Etter E, Guerrero A, Fay FS, and Meissner G. Evidence for a Ca2+-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle. Proc Natl Acad Sci USA 91: 32943298, 1994.[Abstract]
27. ZhuGe R, Fogarty KE, Baker S, Tuft RA, Lifshitz LM, and Walsh JV Jr. Individual Ca2+ spark sites in smooth muscle differ in number of ryanodine receptors and BK channels (Abstract). Biophys J 80: 67a, 2001.[ISI]
28. ZhuGe R, Fogarty KE, Tuft RA, Lifshitz LM, Sayar K, and Walsh JV Jr. Dynamics of signaling between Ca2+ sparks and Ca2+- activated K+ channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca2+ current. J Gen Physiol 116: 845864, 2000.
29. ZhuGe R, Fogarty KE, Tuft RA, and Walsh JV Jr. Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca2+ concentration on the order of 10 µM during a Ca2+ spark. J Gen Physiol 120: 1527, 2002.
30. ZhuGe R, Sims SM, Tuft RA, Fogarty KE, and Walsh JV Jr. Ca2+ sparks activate K+ and Clchannels, resulting in spontaneous transient currents in guinea pig tracheal myocytes. J Physiol 513: 711718, 1998.
31. ZhuGe R, Tuft RA, Fogarty KE, Bellvé K, Fay FS, and Walsh JV Jr. The influence of sarcoplasmic reticulum Ca2+ concentration on Ca2+ sparks and spontaneous transient outward currents in single smooth muscle cells. J Gen Physiol 113: 215228, 1999.