Address correspondence to John V. Walsh, Jr, Department of Physiology, University of Massachusetts Medical Center, Worcester, MA 01655. Fax: (508) 856-5997; E-mail: john.walsh{at}umassmed.edu
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ABSTRACT |
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Key Words: calcium signaling ion channel STOC sarcoplasmic reticulum smooth muscle
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INTRODUCTION |
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The precise relationship between a Ca2+ spark and the STOC which it generates has attracted considerable attention for two reasons. First, STOCs provide a mechanism for vascular smooth muscle relaxation by driving the membrane potential more negative, thereby closing L-type Ca2+ channels which appear to supply the Ca2+ to initiate contraction. This mechanism of spark-induced relaxation, proposed originally for vascular smooth muscle by Nelson et al. (1995), has recently been confirmed in striking fashion using knock-out mice lacking the ß1 subunit of the BK channels (Brenner et al., 2000
; Pluger et al., 2000
; see also Meera et al., 1996
; Tanaka et al., 1997
). Without the ß1 subunit, BK channels do not respond adequately to the Ca2+ released in a spark, and consequently the ß1 knock-out mice suffer from hypertension and cardiomegaly. Second, spark-STOC coupling in smooth muscle has emerged as an ideal system for the study of signaling within Ca2+ microdomains. The reason, in part, is that both the Ca2+ signal, i.e., the spark, and the activity of the Ca2+ target, the BK channels, can be monitored at high temporal resolution using imaging and patch-clamp technology, respectively. The mechanism of BK channel activation studied at this resolution may provide insight into the action of Ca2+ on other targets within a Ca2+ signaling microdomain.
A critical factor in the generation of STOCs is the level of Ca2+ concentration ([Ca2+]) sensed by the BK channels during a Ca2+ spark, but an experimental determination of this [Ca2+] is fraught with difficulties. An image-based determination of the [Ca2+] in the spark microdomain using a fluorescent Ca2+ indicator is problematical since the local Ca2+ and indicator do not necessarily come into equilibrium during the spark (Stern, 1992; Naraghi and Neher, 1997
). Moreover, the relevant microdomain might encompass only a fraction of the smallest region which can be monitored optically, and hence the estimated [Ca2+], which will be an average for the entire region monitored, will be too low. Attempts to make estimates of the [Ca2+] based on a number of assumptions, for example the area of membrane occupied by the activated BK channels, have led to [Ca2+] values ranging from 300 nM to 100 µM (Nelson et al., 1995
; Perez et al., 1999
, 2001
; ZhuGe et al., 1999
).
We report here a new approach for determining the [Ca2+] that activates BK channels at the spark site. Our approach relies on a "signal mass" methodology for measuring the total amount of Ca2+ released during a spark from which the current flowing through RyRs (ICa(spark)) can be determined, a methodology which we developed previously (ZhuGe et al., 2000). We combine this with an analysis of the voltage dependence of the conductance for K+ that is activated during the STOC, a quantity which we designate g(STOC). This conductance is then compared with the voltage dependence of BK channels at various [Ca2+]s previously obtained in excised membrane patches (Singer and Walsh, 1987
). We show that, during a Ca2+ spark, the BK channels causing an STOC are exposed to a mean [Ca2+] on the order of 10 µM. Such concentrations are at least an order of magnitude higher than the highest concentration of global Ca2+ that can be reached, which is
1 µM for the cells employed here (Becker et al., 1989
). The area of membrane over which BK channels are activated during a spark is estimated to be well under 1 µm2. We also conclude that the BK channels are not distributed uniformly over the membrane of the entire cell but are found at high concentration at the spark site, with a lower density elsewhere. The method described here can readily be employed in other preparations which have STOCs.
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MATERIALS AND METHODS |
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Two measures of Ca2+ sparks were employed: the conventional fluorescence ratio, F/F0, within a restricted volume; and the change in total fluorescence, FT - FT0, over a larger volume, designated as the Ca2+ signal mass. These measurements have been described previously in detail (ZhuGe et al., 2000
); a brief description follows. For the fluorescence ratio measure, the fluo-3 images, with pixel size 333 by 333 nm, were first smoothed by convolution with a 3 x 3 pixel approximation to a two-dimensional Gaussian (
= 1). Fluorescence ratios were then calculated and expressed as a percentage on a pixel to pixel basis. The
F/F0 traces in the figures follow the time course of the single pixel that had the highest fluorescence ratio, that is, the brightest pixel, which we call the epicenter pixel of a given spark. The value of the second measure, the Ca2+ signal mass, for each spark was computed from the two-dimensional, wide-field fluorescence images of fluo-3 according to the equations:
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The signal mass (sm[t]) is the product of the detector gain (G) times the change in total fluorescence (FT) summed over a 13.7-µm square region (41 pixels on a side in the x-y plane), surrounding the spark epicenter pixel (x,y) as determined from the F/F0 images. The number of moles of Ca2+ bound to fluo-3 was calculated by the equation:
, where 2.44 is a calibration factor determined previously (ZhuGe et al., 2000
). We have shown previously that the signal mass at each point in time until the peak is reached is directly proportional to the total amount of Ca2+ released into the cytosol from the onset of the spark up to that point in time. Thus, the rate of rise of the signal mass is proportional to the Ca2+ current through RyRs during a Ca2+ spark, ICa(spark).
Modeling of Spark and BK Channel Microdomain
To model the voltage- and [Ca2+]-dependent activation of BK channels in an STOC, we first performed a series of spark simulations. Finite difference approximations were used to solve a set of partial differential equations for the reaction-diffusion kinetics in a cylindrical coordinate system and implemented using custom software as described previously (ZhuGe et al., 2000). Briefly, the cell was modeled as a circular cylinder with a radius of 3 µm, containing 50 µM fluo-3 (Kd = 1.1 µM, kon = 8 x 10-7 M-1s-1, koff = 90 s-1, D = 22 µm2s-1) and either 27 or 230 µM "fixed buffer" (Kd = 1 µM, kon = 10-8 M-1s-1, koff = 100 s-1), and with resting [Ca2+] = 100 nM. The spark event was modeled as a constant release of Ca2+ (D = 250 µm2s-1) for a duration of 15 ms, the average spark duration for the cells in this study (ZhuGe et al., 2000
). The spark current was modeled as 1 pA for the 27 µM fixed buffer simulation, and 3 pA for the 230 µM fixed buffer simulation. These values for the current were determined from the mean signal mass that we measured for a Ca2+ spark in an earlier study on the same cells employed here (ZhuGe et al., 2000
). That is, we measured a mean Ca2+ signal mass whose rate of rise corresponded to a mean ICa(spark) of 0.6 pA. Since the measured signal mass and hence ICa(spark) will depend on the amount of fixed buffer, then the assumption of a greater amount of fixed buffer for a simulation demands that the underlying Ca2+ current be greater. Thus, the measurement of the Ca2+ signal mass places constraints on our choice of the values of ICa(spark) and fixed buffer. For one set of simulations, 230 µM fixed buffer with the rate constants given was chosen, simply because that is the estimate published for smooth muscle (Bond et al., 1984
). We also did another set of simulations using a relatively low concentration of fixed buffer (27 µM), because results of our earlier study suggested that the amount of fixed buffer is low (ZhuGe et al., 2000
) and because this small amount of buffer made the simulation and modeling well behaved in the absence of fluo-3. The specific value of 27 µM also means that the rate of rise of the smallest measured mean signal mass corresponds to an ICa(spark) of 0.35 pA, a value equal to a careful estimate of the single RyR current in physiological conditions (Mejia-Alvarez et al., 1999
). The spark "site" was modeled as a small, cylindrically shaped volume, placed along the center axis of the cell at a distance of 25 nm from the flat end of the cylinder into which the Ca2+ was released. This spark site was itself 25 nm in height and had a diameter of either 60 nm for the 1 pA release or 104 nm for the 3 pA release. In this way the Ca2+ current density for the different simulations was held constant.
These spark simulations provided an estimate of the spatial distribution of the [Ca2+] immediately under the plasma membrane in the area surrounding the spark. We next modeled the voltage and [Ca2+] relationship of a single BK channel. The relationship between Po and voltage was characterized previously for single BK channels from these cells (Singer and Walsh, 1987) and was well fit by the Boltzmann relationship:
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Statistics
Data were presented as means ± SEM and were analyzed statistically with Student's t test.
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RESULTS |
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As noted, is a constant at the K+ concentrations and over the range of voltages employed here, and N is also a constant at different voltages. Hence, g(STOC) reflects the Po of the BK channels under these conditions. As shown below, the sparks do not change in magnitude as a function of voltage, and hence the [Ca2+] at the spark site remains constant at various potentials. Therefore, comparison of the g(STOC)-Vm relationship to the family of Po-Vm curves at different [Ca2+]'s for the BK channels should provide information about the [Ca2+] at the cytosolic surface of the BK channels during a spark as well as insight into the spatial structure of the spark-STOC microdomain.
g(STOC) Is Smaller at More Negative Membrane Potentials
Fig. 1 A shows typical STOC traces obtained at the potentials indicated, with EK at -28 mV as it was in all the experiments described here. The conductance for each STOC was determined as follows:
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In Fig. 1 B, the distribution of values for g(STOC) at each of the six potentials is shown for all cells studied. It can be seen that the values decrease at more negative potentials with a sharp decline between -40 and -60 mV. The entire distribution appears to shift to the right at more positive potentials, the significance of which is considered below. Fig. 1 C shows the mean g(STOC) over a range of potentials for all the cells studied. As in Fig. 1 B, a sharp decrease in g(STOC) magnitude can be seen between -40 and -60 mV. It is also noteworthy that the magnitude of g(STOC) does not increase as the potential is increased from -40 to 20 mV. Fig. 1 D shows the frequency of the corresponding STOCs at each potential. Fig. 1 E shows the time constants for the rate of rise and decay of the STOCs over this range of potentials. The decay time constant does not change significantly over the range of potentials shown and corresponds approximately to the mean open time of the BK channels as measured in excised inside-out patches in the same cell type (Singer and Walsh, 1987).
The Magnitude of Ca2+ Sparks Does not Change as a Function of Potential
A possible reason for the decrease in g(STOC) at potentials more negative than -40 mV is that the amount of Ca2+ released from the SR during a spark decreases at negative potentials. We therefore examined Ca2+ sparks at 0 and -80 mV. The results are shown in Fig. 2. Fig. 2 A shows the temporal evolution of a typical Ca2+ spark, with images above and the traces indicating the fluorescence ratio and the signal mass below. Fig. 2 B, a, provides a histogram of the distribution of F/Fo at 0 and -80 mV, and Fig. 2 B, c, does the same for the Ca2+ signal mass. Fig. 2 B, b, d, and e, compare the mean
F/F0, signal mass, and frequency at 0 and -80 mV. While spark frequency at 0 mV was greater, there was no difference in spark amplitude at 0 and -80 mV by either of the two measures of spark magnitude,
F/Fo or Ca2+ signal mass.
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The g(STOC)-Vm Relationship Corresponds Most Closely to the Po-Vm Relationship for BK Channels in the Presence of 10 µM Ca2+
Since the magnitude of the Ca2+ sparks does not change with membrane potential, the smaller magnitude of g(STOC) at negative potentials must be due to a decrease in Po of the BK channels as a function of voltage at a given [Ca2+]. For each [Ca2+], there is a characteristic Po-Vm curve for the BK channels for smooth muscle cells. The Po-Vm relationship for BK channels in the cell type used here has been studied previously in excised, inside-out patches (Singer and Walsh, 1987) and is in agreement with those found in other smooth muscle preparations (Carl et al., 1996
). This Ca2+ sensitivity reflects the presence of the ß1 subunit, which appears to be characteristic of BK channels in smooth muscle cells (Tanaka et al., 1997
). Three such curves for BK channels in the same cell type at 100 µM, 10 µM, and 1 µM Ca2+ (Singer and Walsh, 1987
) are shown in Fig. 3 A along with the g(STOC)-Vm curve from Fig. 1. The shaded area shows the variation that was observed at 10 µM Ca2+. This variation resulted only in a shift of the curve along the x-axis without a change in the slope factor (Singer and Walsh, 1987
). The mean V0.5 at 10 µM is the same as that found for the cloned
and ß1 subunits of the BK channel from human smooth muscle coexpressed at a 1:1 ratio in Xenopus oocytes (Meera et al., 1996
).
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The data in Fig. 3 A lead us to conclude that the BK channels contributing to an STOC are exposed to a [Ca2+] on the order of 10 µM. (An upper limit is placed on this concentration by the fact that the free [Ca2+] in the SR is 150 µM [ZhuGe et al., 1999].)
The Spatial Profile of [Ca2+] in the Spark Microdomain Derived from Measures of ICa(spark)
The signal mass (Fig. 2 A) provides a measure of the total amount of Ca2+ or the total charge carried by Ca2+ that is released during a spark. A trace of the signal mass, like that shown in Fig. 2 A, provides a time-integrated index of the Ca2+ or charge released; hence the derivative of its rising phase provides a measure of the Ca2+ current through the RyR's during a spark or ICa(spark) (ZhuGe et al., 2000). The mean duration of ICa(spark) was found to be
15 ms. Using this value and the value for ICa(spark), the spatial profile of [Ca2+] can be calculated at the end of the 15-ms period, a time when the STOC is at its peak amplitude. Here we are referring to the lateral extent of the [Ca2+] profile along the plasma membrane as we proceed outward from the Ca2+ release site. For these calculations we place the Ca2+ release site 25 nm from the plasma membrane, in accord with measures of the distance from the SR membrane to the plasma membrane in smooth muscle cells (Somlyo and Franzini-Armstrong, 1985
). However, both the actual magnitude of ICa(spark), as determined by the signal mass method, and its spatial extent depend on the Ca2+ buffers in the cell. That is, with more buffering the slope of the signal mass must be multiplied by a greater proportionality constant to obtain the actual magnitude of the ICa(spark) (ZhuGe et al., 2000
). Fig. 3 B shows the spatial profile of [Ca2+] for two conditions: 50 µM fluo-3 as the sole mobile buffer with a uniform concentration, either 27 or 230 µM, of fixed buffer with a Kd of 1 µM. The former condition (27 µM) is one where the fluo-3 dominates and the latter (230 µM) is the one estimated for smooth muscle (Bond et al., 1984
). (For reasons discussed below, it appears that this estimate of fixed buffer constitutes a maximum.) Fig. 3 B indicates that the radius of the region where [Ca2+] is
10 µM is
150 nm (area = 0.07 µm2) or 300 nm (area = 0.28 µm2) for the two buffer conditions. (For comparison, at a [Ca2+] equal to 2 µM, the radius is equal to 380 and 540 nm for the low and high buffer conditions, respectively.)
The Areas Containing the BK Channels Activated by a Spark Are Homogenous in their Spatial Extent
The following considerations suggest that the areas over which BK channels are activated during a spark make up a single population. Fig. 1 B indicates that there are not several qualitatively different types of microdomains containing the activated BK channels, those large in area and those quite restricted in area. The latter would have RyRs and BK channels necessarily closer since they lie in a more restricted space. In this case one might imagine that the small conductances observed at potentials of -80 and -100 mV in Fig. 1 B are due to a class of STOC domains that are of lesser spatial extent. If this were the case, then upon raising the potential to more positive levels, these small conductances would not disappear. Rather, they would remain as one of two or more peaks in the distributions of Fig. 1 B at potentials positive to -80 mV. However, such a multimodal distribution is not seen, and so heterogeneity of this sort does not appear to exist. To a first approximation, the areas containing the activated BK channels appear to be normally distributed. The small conductances that remain at the very negative potentials (Figs. 1 B and 3 A) appear to be due to the BK channels closest to the RyRs at the spark site. (Such homogeneity does not, however, mean that the spark microdomain is a highly stereotyped arrangement. We have demonstrated in earlier work that the ratio of RyRs to BK channels varies among sparks [ZhuGe et al., 2000] and among spark sites [unpublished data]. Hence, at any spark site the ratio of RyRs to BK channels can vary considerably.)
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DISCUSSION |
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Simulations of Ca2+ Sparks and STOCs Are Consistent with an "Oasis" Model of BK Channels Concentrated in the Spark Microdomain, but not with a "Handshake" Model or a Uniform Distribution of BK Channels
Using spatial profiles of [Ca2+] shown in Fig. 3 B and curves relating Po(BK) to [Ca2+] and Vm of the sort shown in Fig. 3 A, the relationship between g(STOC) and Vm can be predicted by simulation using different models of BK channel distribution at the spark site. The results of such simulations are shown in Fig. 4 for three different models of spatial distribution of BK channels. We emphasize that these simulations are meant only to predict the general shape of the g(STOC)-Vm relationship as shown by the open circles of Fig. 3 A which are also shown in Fig. 4.
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Uniform BK Channel Density throughout the Cell Membrane
The curves marked "b" in Fig. 4 indicate the results to be expected if there is a uniform density of BK channels everywhere in the plasma membrane. The channel density is critical in determining these curves, with higher densities, bringing the curves of "b" closer to the actual data given by the open circles. We have estimated the density of BK channels in the smooth muscle cell type employed here to be 1/µm2 (Singer and Walsh, 1987
), and we used this estimate to generate the curves of "b." Clearly the data of Fig. 3 A do not fit these curves.
In other smooth muscle types, determination of the density has yielded values of 2/µm2 for rabbit jejunum (Benham and Bolton, 1986) and rat cerebral artery (Perez et al., 2001
) and 4/µm2 for human coronary artery (Tanaka et al., 1997
). These differences appear to result from differences among cell types, with cells having higher mean density also generating larger STOCs. For example, in rat cerebral artery (Perez et al., 2001
), the mean BK channel density is twice that observed here, but the STOCs comprise current from
18 BK channels,
3 times the number in the preparation employed here. This would seem to require an even higher degree of concentration of BK channels at a spark site. However, even if the highest estimate of BK channel density (4/µm2) is employed in the simulations, a "best case" for the uniform distribution model, the curves do not match the g(STOC) data. In this case, the black curve plateaus at a value of
447 pS at 0 mV, as compared with the actual value of 760 pS. And the red curve does not plateau at all, but is concave upward with no plateau. Hence, a model of uniform distribution does not match the g(STOC) data.
The Oasis Model: High Density of BK Channels in the Spark Microdomain
The curves marked "c" belong to a set of simulations where the BK channels exist at a uniform density within a certain lateral distance from the [Ca2+] release site along the membrane. Beyond this region the BK channel density falls to a negligible level. This is termed an "oasis" model. Given the dual constraints of the influence of [Ca2+] and Vm on the BK channel Po, the simulation is designed to converge on an area for the oasis and a density for the BK channels which best fit the g(STOC) data. The plots resulting from this simulation match the general shape of the plot for the actual data shown in circles, i.e., flat at the more positive potentials, with a steep decline in the region of -40 to -60 mV and a failure to reach zero even at a potential of -100 mV. The curves "c" limit the extent of BK channels to an area of 250-nm radius for the low ICa(spark) and buffering condition (red), and 450 nm for the high ICa(spark) and buffering condition (black). For the 250-nm radius (area = 0.20 µm2), the mean [Ca2+] in the oasis is 12.3 µM and the [Ca2+] at the oasis periphery is 5.5 µM; the mean number of BK channels is 5.3 and the BK channel density in the oasis is 26.5 channels/µm2. For the 450-nm oasis (area = 0.64 µm2), the mean [Ca2+] is 16.4 µM and the [Ca2+] at the oasis periphery is 4 µM; the mean number of BK channels is 5.4 and the BK channel density in the oasis is 8.4 channels/µm2. Since we do not know the precise disposition of the BK channels or the RyRs within the oasis or the exact nature and spatial disposition of the fixed buffers, we can expect these simulations to correspond to the actual data only in an approximate way. Finally, we note that for the oasis model the fit to the data does not depend on the nonlinear relationship between V0.5 and log [Ca2+] used for these simulations (Meera et al., 1996). If instead a linear relationship is employed (Carl et al., 1996
), the fit to the actual data is somewhat better and the oasis shrinks to a radius of 150 and 350 nm for the low and high ICa(spark) conditions, respectively.
An alternative to the oasis model is a "fortress" model in which there is a uniform density of BK channels everywhere in the plasma membrane but a physical barrier to Ca2+ movement at the same location as the periphery of the oasis. One such barrier could simply be a membrane or other wall-like structure. However, an effective barrier to ion diffusion must act as a barrier to K+ and hence should appear as a series resistance for the K+ current of the STOCs. In this case the reversal potential for STOCs should deviate from EK, but in fact the reversal potential follows EK quite closely (Fig. 1; see also Benham and Bolton, 1986). Moreover, for such a model to match the g(STOC) data, the mean channel density has to be almost an order of magnitude greater than what is estimated. Hence, it does not appear that the fortress model can be correct.
An Extended Oasis of BK Channels Larger than the Zone of BK Channel Activation
A second version of the oasis model will also yield a fit to the g(STOC) data. Like the oasis model described in the previous section, a high local density of BK channels is required. But unlike the above model, the oasis is larger than the zone of channel activation. Thus, an oasis of BK channels at higher than average density remains, but within it there is a zone of activated BK channels. The results of simulations using this formulation yield the results given by the inset to Fig. 4. In this case the simulation matches the g(STOC) data only for high level of ICa(spark) and buffer (black curve, inset) and not for the low level (red curve, inset), which unlike the g(STOC) data exhibits no plateau at potentials positive to -60 mV. The simulation yields a density of 8 BK channels/µm2 within the oasis. With this model it is not necessary to revise upward the estimate of total number of BK channels per cell (or the mean density over the cell) since a high density of BK channels is only required up to the limit of the zone of activation. The size of the oasis is unspecified in this model, but the zone of activation is defined and sets a lower limit on the size of the oasis.
What then is the size of the zone of activated BK channels within such an extended oasis? The zone of activation will depend on the membrane potential, expanding at more positive potentials and constricting at more negative potentials. Hence, for example, at -40 mV, often used as an estimate of resting potential, 95% of the conductance in an STOC will come from a zone with a radius of 470 nM (area = 0.7 µm2) over which the mean [Ca2+] is 15 µM, and the [Ca2+] at the periphery is 3.6 µM. At 0 mV, 95% of the conductance in an STOC will come from a zone of 490 nm (area = 0.75 µm2) with a mean [Ca2+] of 14 µM and a peripheral [Ca2+] of 3.4 µM. This set of values is not much different at the two potentials, 0 and -40 mV, as might be expected from the fact that the value for g(STOC) is unchanged at the two potentials. At -80 mV, the values are: radius, 390 nm (area = 0.73 µm2) from which 95% of the STOC conductance derives; mean [Ca2+], 21 µM; peripheral [Ca2+], 4 µM. Fig. 5 provides a depiction of the oasis and extended oasis models.
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Spatial Separation of Spark Sites Prevents Sparks from Becoming Global Increases in [Ca2+]
The location of BK channels in oases separate from one another suggests that the RyRs causing sparks sites might be similarly arranged. At least one previous study suggests clustering of RyRs in adult vascular smooth muscle cells (Gollasch et al., 1998). Our results indicate that the Ca2+ from a spark arising at one STOC oasis cannot activate BK channels at a nearby oasis. Like the BK channels at potentials close to 0 mV, the RyRs have a Kd of
1 µM Ca2+ (Bezprozvanny et al., 1991
). Hence, we would expect that Ca2+ sparks would not trigger Ca2+ waves. And indeed we do not see the generation of Ca2+ waves by sparks in these smooth muscle cells. In general, if sparks are to cause relaxation in smooth muscle cells, then they must not cause Ca2+ waves since the waves would elevate global [Ca2+] and initiate contraction. Spatially isolated oases of RyRs and BK channels appear to be a way of ensuring that the Ca2+ sparks will cause relaxation without triggering contraction.
The Spark Microdomain: a Summary
In all of the simulations which fit the g(STOC) data, the mean [Ca2+] sensed by the BK channels in the spark microdomain is on the order of 10 µM (range: 1221 µM). The mean radius over which BK channel activation occurs may range from 250 nm (area = 0.2 µm2) to 490 nm (area = 0.75 µm2), depending on the fixed buffers, the magnitude of ICa(spark), and in the case of the extended oasis model, the membrane potential. The values for mean [Ca2+] and spatial extent of BK channel activation provided by the simulations above are in excellent agreement with estimates from a simple qualitative examination of Fig. 3, A and B. Whereas the values for the mean area containing activated BK channels are only a fraction of one square micron, the mean spatial extent at full-width, half max (FWHM) in images like those in Fig. 2 is 2.7 µm. Hence, the FWHM does not seem to be a meaningful measure of the extent of the spark-STOC microdomain, and its use leads to an overestimate of the area of BK channel activation. In summary, the spark-STOC microdomain is a genuine microdomain with an area which is a fraction of one square micron, allowing the mean [Ca2+] to rise to 10 µM.
The simulations above also demand that the BK channels exist at higher than average density at the spark sites. There have been previous indications that the BK channels are not uniformly distributed. Xiong et al. (1992), using portal vein smooth muscle and, more recently, Ohi et al. (2001)
, using vas deferens and bladder smooth muscle, have found evidence for clustering of BK channels based on activity of a high density of BK channels recorded in some patches of smooth muscle membrane excised with some SR apparently attached.
Meera et al. (1996) have called attention to the fact that the BK channel behaves as a "switch" operated by Ca2+. At [Ca2+] below 1 µM, the channels are in the closed state at all physiological membrane potentials. At [Ca2+] of
10 µM the channels are in the open state over the range of physiological potentials. Since global [Ca2+] generally remains at levels <1 µM, the channels must be located within a microdomain where [Ca2+] reaches levels >1 µM for the switch to function reliably. As we have shown here, the spark microdomain satisfies this demand and provides a highly localized region where Ca2+ rises to a sufficient concentration to operate the BK channel switch.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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This study was supported by a grant from the National Institutes of Health (HL61297) and by grants from the National Science Foundation (DBI-9724611 and DIR9200027).
Submitted: 31 January 2002
Revised: 12 April 2002
Accepted: 6 May 2002
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REFERENCES |
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