Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

Guillermo J. Pérez, Adrian D. Bonev, and Mark T. Nelson

Department of Pharmacology, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+ concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 µM and a Hill coefficient of 2.9 at -40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (-40 mV), the open probability of a single BK channel was low (1.2 × 10-6). A Ca2+ spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ~30 µM to 4 µM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+ sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.

ryanodine receptor; local calcium release; BK potassium channel


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTRACELLULAR CALCIUM IONS (Ca2+) regulate a variety of cellular processes, ranging from cellular contraction to gene expression (for review see Ref. 6). Ca2+ enters the cytoplasm of smooth muscle cells through voltage-dependent Ca2+ channels in the plasma membrane and through inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ release channels and ryanodine-sensitive Ca2+ release ("ryanodine receptor," RyR) channels in the sarcoplasmic reticulum (SR) membrane. "Local" Ca2+ transients or "Ca2+ sparks," which occur in <1% of the cell volume, have been observed in a number of different types of smooth muscle (Ref. 19; for review, see Ref. 12) and were originally described in cardiac muscle (5). Ca2+ sparks are caused by the opening of a cluster of RyR channels. Ca2+ sparks may communicate with a number of cytoplasmic targets including the Ca2+-sensitive K+ (BK) channel (20, 23, 24) and Ca2+-sensitive Cl- channels (23). The coding of Ca2+ sparks to cellular processes depends on a number of factors, including the amount of Ca2+ released during a spark, the proximity of the Ca2+ spark site to a target protein, and the Ca2+ sensitivity of the target.

Transient BK currents in isolated smooth muscle cells were first described by Benham and Bolton (1) and were thought to be caused by sudden discharges of Ca2+ stores near the membrane. Indeed, transient BK currents have been shown to be caused by Ca2+ sparks in arterial smooth muscle (19, 20). Virtually every Ca2+ spark caused a transient BK current, suggesting a close proximity of Ca2+ spark sites to the plasma membrane of arterial smooth muscle cells. SR elements can be observed in close apposition, within 20 nm, to the inside of the plasma membrane (7, 16), and RyRs appear to be near the cell membrane (8, 16). Unfortunately, the relative distances (<100 nm) for this local communication of Ca2+ are far below the resolution of confocal microscopy (>700 nm).

The goal of the present study was to test the hypothesis that Ca2+ sparks deliver high local (micromolar) Ca2+ concentrations to activate nearby BK channels in the cell membrane at the physiological membrane potentials (-40 mV) for pressurized cerebral arteries (2, 14). First, the effect of a Ca2+ spark on the activity of BK channels at -40 mV was determined. A single Ca2+ spark evoked an ~105- to 106-fold increase in the open probability (Po) of nearby BK channels, if it is assumed that ~1% of the BK channels are activated by one calcium spark, which is consistent with previous estimates (20). Second, the increase in Ca2+ required to elevate BK channel activity 106-fold was determined by measuring the Ca2+ sensitivity of single BK channels at -40 mV. These measurements indicate that BK channels experience Ca2+ concentrations in the range of 4-30 µM during a spark. Third, the local communication of Ca2+ sparks to BK channels was probed by introducing the mobile Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into single smooth muscle cells. BAPTA at concentrations that effectively competed with the fluorescent Ca2+ indicator fluo 3 did not affect the Ca2+ spark-evoked BK channel currents. Together these results support the concept that the Ca2+ signal, Ca2+ sparks, through proximity, are matched to the Ca2+ sensitivity of a target, the BK channels, to regulate membrane potential.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Cell isolation. Sprague-Dawley rats (12-14 wk old) of either sex were euthanized by peritoneal injection of pentobarbital solution (150 mg/kg). Cerebral (basilar) arteries were carefully dissected and then digested with papain (0.3 mg/ml papain and 1 mg/ml dithioerythritol for 20 min at 37°C) and collagenase (1 mg/ml collagenase, type F and type H in a 70%-30% mixture, respectively, incubated for 10 min at 37°C). The digested tissue was triturated with a fire-polished glass Pasteur pipette to yield single smooth muscle cells.

Simultaneous patch-clamp and fluorescence recordings. Isolated myocytes were loaded by incubation with 10 µM fluo 3-acetoxymethyl ester (AM) for 30 min followed by a 30-min wash period. In experiments with BAPTA, it was likewise introduced into the cells with a bath concentration of 1 µM BAPTA-AM. Fluo 3-loaded cells (20) were scanned with a Noran OZ (Middleton, WI) laser scanning confocal system hosted by an Indy workstation (Silicon Graphics, Mountain View, CA) and the Intervision software package. The confocal system is mounted in an inverted Nikon Diaphot microscope with a ×60 water immersion lens (NA = 1.2). Images were typically 48.4 × 50.6 µm (or 220 × 230 pixels) and were acquired every 8.33 ms (120 images/s) over 10 s. Cells were simultaneously voltage-clamped as indicated in Whole cell current recordings.

Whole cell current recordings. K+ currents were measured in the whole cell perforated-patch configuration of the patch-clamp technique (9, 10) with an Axopatch 200A amplifier (Axon Instruments, Foster City CA). The bathing solution contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4). To minimize contraction, in some cases the bathing solution also contained 5 µM wortmannin. The pipette solution contained (in mM) 110 K aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (pH 7.2) with 200 µg/ml amphotericin B. Membrane currents were recorded while the cells were held at a steady membrane potential of -40 mV. Currents were filtered at 500 Hz and digitized at 2.5 kHz. For measuring whole cell single-channel activity, external K+ was isosmotically raised to 140 mM to enhance single-channel currents at -40 mV. In simultaneous experiments, cells were also scanned for fluorescence changes as indicated in Simultaneous patch-clamp and fluorescence recordings.

Single-channel recordings. Single-channel currents were recorded from inside-out membrane patches of isolated arterial myocytes (9). The bathing solution contained (in mM) 140 KCl, 10 HEPES (pH 7.2), 1 Mg2+, and 5 EGTA or N-(2-hydrohyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA) with different free Ca2+ concentrations (100 nM, 300 nM, 3 µM, 10 µM, 30 µM, and 100 µM) adjusted with Ca2+ electrodes. The pipette solution contained 140 mM KCl, 10 mM HEPES (pH 7.2), 1 mM Mg2+, 5 mM HEDTA, and 10 µM Ca2+. The patches were held at a steady potential of -40 mV. Currents were filtered at 10 kHz and digitized at 40 kHz. The number of channels present in any given excised patch was estimated from all-points histograms at depolarized voltages (greater than +60 mV).

Chemicals. Fluo 3-AM, pluronic acid, and BAPTA-AM were obtained from Molecular Probes (Eugene, OR). Dehydrosoyasaponin-1 was a gift from Merck Research Laboratories (Rahway, NJ). All other chemicals were obtained from Sigma (St. Louis, MO) and Calbiochem-Novabiochem International (La Jolla, CA). All experiments were conducted at room temperature (20-22°C).

Data analysis. Transient BK currents were analyzed using Mini analysis 5.1.1 software (Jaejin Software). Image analysis was performed using custom-written analysis programs using Interactive Data Language software (Research Systems, Boulder, CO). Baseline fluorescence (Fo) was determined by averaging 30 images (of 1,200) with no activity. Ratio images were then constructed and replayed for careful examination to detect active areas where sudden increases in fluorescence (fractional change, F/Fo) occurred. F/Fo vs. time traces were further analyzed in Microcal Origin (Microcal Software, Northampton, MA) and represent the averaged F/Fo from a box region of 2.2 × 2.2 µm centered in the active area of interest to achieve the fastest and sharpest changes. This box size (4.8 µm2) was determined empirically to be the best compromise between temporal and spatial precision of Ca2+ sparks and the signal-to-noise ratio. Results are expressed as means ± SE where applicable.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+ spark causes 105- to 106-fold increase in activity of BK channels during transient BK current. A Ca2+ spark causes a transient macroscopic BK current in smooth muscle cells isolated from cerebral arteries (19, 20). To estimate the increase in Po of the BK channels caused by a Ca2+ spark, the activities of BK channels at the peak transient BK current and in the absence of any contribution from Ca2+ sparks were determined at the physiological membrane potential (-40 mV) observed in pressurized cerebral arteries. To increase the single-channel and transient BK currents at -40 mV, the external K+ concentration was raised to symmetrical conditions (140 mM). Figure 1A illustrates the whole cell current activity in these conditions, displaying the transient BK currents as downward deflections. The mean transient BK current at -40 mV in symmetrical K+ solution was -148 ± 4 pA (n = 787 events from 6 different cells). The activity (NPo, where N is total number of channels per cell) of the BK channels reaches a value of 18 during the peak of a transient current event. This value was determined by dividing the mean transient BK current (148 pA) by the mean BK unitary current under these conditions (-8.5 ± 0.5 pA; n = 6 at -40 mV). Therefore, a Ca2+ spark activates at least 18 BK channels in the nearby plasma membrane. To determine the activity of BK channels in the absence of Ca2+ sparks, single-channel BK channel activity was measured in the same cells across the entire cell membrane with the whole cell perforated-patch configuration (10). Figure 1B compares single-channel activity recorded in the whole cell configuration with single-channel activity recorded in inside-out excised patches. At -40 mV and symmetrical K+, the NPo of BK channels in the presence of 100 nM thapsigargin (to abolish the contribution of Ca2+ sparks) was 3.1 ± 0.1 × 10-3 (n = 5). This activity presumably reflects BK channel function over the entire cell membrane.


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Fig. 1.   Ca2+-activated K+ (BK) channels increase their activity ~106-fold during a (Ca2+ spark induced) BK transient current. A: left, representative whole cell record in symmetrical 140 mM K+ solutions showing that inward transient BK current (at -40 mV) peak amplitude was -148 ± 4 pA and single-channel amplitude was -8.5 ± 0.5 pA (n = 6 cells). Right, section indicated by bar on left trace at an expanded time scale; *single-channel opening. B: left, single BK channel activity recorded in whole cell configuration. Right, single BK channel activity recorded in inside-out patches exposed to 100 nM Ca2+, which is close to the cytoplasmic Ca2+ at rest. Holding potential (Vh) was -40 mV, extracellular K+ concentration ([K+]o) was 140 mM. Arrows indicate closed state of the channel. In the whole cell experiments, single BK channel activity was measured after exposing the cell to 100 nM thapsigargin for 15 min to eliminate the contribution of Ca2+ release from the sarcoplasmic reticulum (SR) to the BK channel. Open probability (Po) of BK channels in inside-out patches was 1.16 ± 0.26 × 10-6 (n = 16 patches) and activity (NPo, where N is total number of channels per cell) of whole cell BK channels was = 3.1 ± 0.1 × 10-3 (n = 5 cells).

To calculate Po of BK channels above a spark site, two boundary conditions were chosen: 1) homogeneous distribution of BK channels across the cell membrane and 2) all BK channels clustered above one spark site. The latter condition is highly unlikely, because most cells have more than one spark site that activates BK currents and every excised patch contains BK channels. It is likely, therefore, that the true distribution resides between these two boundary conditions. In the case of homogenous distribution, baseline activity of BK channels above a Ca2+ spark site should be ~1% of 3.1 × 10-3 because a Ca2+ spark affects ~1% (13 µm2/cell surface; Ref. 20) of the cell membrane. Hence, a Ca2+ spark would elevate Po of the nearby BK channels ~6 × 105-fold (i.e., 18 divide  3.1 × 10-5). In the case of all BK channels positioned above one spark, a Ca2+ spark would elevate Po of BK channels from 3.1 × 10-3 to 18, or 6 × 103-fold. Thus, with the boundary conditions of homogenous BK channel distribution and all channels clustered over one spark site, a Ca2+ spark causes a very significant increase in BK channel Po (range 6 × 103- to 6 × 105-fold).

To estimate the number of BK channels in a single cell, the whole cell activity (NPo) of BK channels was divided by Po (1.2 × 10-6) of BK channels in excised patches, which was determined at the same voltage (-40 mV) and similar intracellular Ca2+ (100 nM; Fig. 1B). This approach yielded an estimate of 3,000 BK channels or ~2 channels/µm2, which is consistent with the average number of channels observed in excised patches of 2.3 ± 0.5 channels/patch (n = 11 patches). The pipette resistances ranged from 9 to 13 MOmega , which allows an estimation of 1.6-1.2 µm2 of patch membrane area (21). Therefore, a Ca2+ spark would affect an area of ~30 BK channels in the nearby plasma membrane, based on the spatial spread of a Ca2+ spark (13 µm2) and assuming a homogeneous distribution of BK channels. Thus a Ca2+ spark increases average Po of BK channels to <0.6 at -40 mV, based on a peak activity of BK channels of 18 and 30 BK channels in the surface membrane above a Ca2+ spark. These results indicate that a Ca2+ spark caused a large increase in mean channel Po of ~30 BK channels but still to a level well below 1. If all BK channels are centered over one spark site, then a Ca2+ spark increases average Po of the BK channels to <0.006, clearly well below 1.

Calcium sensitivity of single BK channels from cerebral artery myocytes. Our results indicate that a Ca2+ spark causes an ~106-fold increase in Po of nearby BK channels. To translate this change in Po to a change in local activator Ca2+, the Ca2+ sensitivity of BK channels was determined at physiological membrane potentials (-40 mV) in inside-out patches. To determine channel Po at -40 mV, the concentration of external K+ was elevated to 140 mM. The single-channel conductance of the BK channels under these conditions was 231 pS.

Po of the BK channels at -40 mV and resting intracellular Ca2+ (100 nM), as mentioned above, was 1.2 × 10-6. The apparent dissociation constant (Kd) for Ca2+ at -40 mV was 19 ± 0.6 µM, with a Hill coefficient of 2.9 ± 0.05 and maximum Po of 0.79. This Ca2+ sensitivity is consistent with the BK channels being assembled with their beta 1-subunit, as we recently demonstrated (3). Therefore, we decided to test further the subunit composition of the BK channels under our conditions. Indeed, BK channels appeared to have functional beta 1-subunits because 100 nM DHS-1, which activates BK channels through the beta -subunit (17), increased channel Po 11.2 ± 4.9-fold (n = 4). To increase Po of the BK channels from 6 × 103-fold to 6 × 105-fold, as is caused by Ca2+ spark, intracellular Ca2+ would have to rise from ~0.1 µM to 4-30 µM. Thus the local Ca2+ (4-30 µM) sensed by the BK channels during a Ca2+ spark is far greater than registered (<0.5 µM) by the Ca2+ indicator fluo 3 (19, 20), even under the extreme condition of all BK channels being centered over one spark site.

BAPTA does not affect activation of BK channels by Ca2+ sparks. Our previous results (20) showing that every Ca2+ spark activates a detectable BK channel current, the close apposition (20 nm) of SR elements to surface membrane (7, 16), and the results in Figs. 1 and 2 support the idea that Ca2+ spark sites are very close to the BK channels. To explore this proposition further, the effects of the fast Ca2+ chelator BAPTA on Ca2+ sparks, measured optically, and on evoked BK currents were examined. To maintain cell integrity, Ca2+ sparks and BK currents were measured using the perforated-patch configuration of the whole cell patch-clamp technique. The AM form of BAPTA was applied extracellularly at a concentration (1 µM) that would effectively compete with the fluorescent Ca2+ indicator fluo 3 but is not so high as to disrupt SR and cellular Ca2+ handling. BAPTA effectively eliminates all local fluorescent changes caused by Ca2+ sparks, but it does not abolish transient BK currents (n = 7). Furthermore, after causing the disappearance of Ca2+ sparks, BAPTA-AM did not change mean transient BK current amplitude compared with independent control cells (control 40.4 ± 1.6 pA, 308 events from 5 cells; BAPTA 41.9 ± 4 pA, 282 events from 5 cells; P > 0.7; -40 mV). Figure 3 illustrates the changes before and after the application of BAPTA-AM in both whole cell current and fluorescence. The frequency of transient BK currents in the presence of BAPTA decreased ~40% (from 0.7 to 0.4 Hz), probably because of steady-state changes in global Ca2+ concentration or SR load. However, the transient BK current characteristics were unchanged after BAPTA, suggesting that local Ca2+ transients in the vicinity of BK channels are not affected by this Ca2+ chelator. Figure 4 shows that averaged transient BK currents remain almost identical after disappearance of Ca2+ sparks with BAPTA application [control peak amplitude 54.1 ± 1.6 pA vs. BAPTA peak amplitude 55.3 ± 1.7 pA; control rise time 17 ± 0.6 ms vs. BAPTA rise time 16 ± 1 ms; control decay constant (tau ) 22.3 ± 1.7 ms vs. BAPTA tau  21.9 ± 1.1 ms]. These results are also consistent with local communication of Ca2+ sparks to BK channels.


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Fig. 2.   Calcium sensitivity of BK channels at -40 mV. Left, original records of single BK channel activity of inside-out patches exposed to 100 nM and 10 µM Ca2+. Vh = -40 mV, [K+]o = 140 mM. Arrows indicate closed level. Right, single-channel Po vs. Ca2+ concentration plot. Experimental Po values (, n = 5-16 patches, total 29) were fitted with a Hill equation Po = Po max/[1 + (Kd/[Ca2+])<SUP><IT>n</IT><SUB>H</SUB></SUP>], where Po max is maximum Po, Kd is dissociation constant, [Ca2+] is Ca2+ concentration, and nH is Hill coefficient; solid line. The slope conductance of single BK channels was 231 ± 3 pS (n = 10 patches; inset).



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Fig. 3.   1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) abolishes Ca2+ sparks but not the transient BK currents. Top, original continuous record of BK currents before and after application of 1 µM BAPTA-AM. Cell was held at -40 mV. Red bars below the trace indicate simultaneous imaging. Middle, 2-dimensional confocal images (8.33 ms apart) of the smooth muscle cell. Center image in each panel corresponds to the time indicated by a green star in the traces below. F/Fo, fractional change in fluorescence. Bottom, time course of F/Fo derived from the location of a spark site (red traces) and the corresponding changes in the current (blue traces). The lag in the BAPTA effect (~20-30 min) can be explained by the time that is required for BAPTA-AM hydrolysis by intracellular esterases to free the Ca2+ chelator BAPTA.



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Fig. 4.   BAPTA does not alter transient BK currents. Averaged transient BK currents obtained from the experiment in Fig. 3. Black trace represents the average of 89 events in control conditions (peak amplitude = 54.1 ± 1.6 pA). Gray trace represents the average of 130 events after BAPTA effect was established (peak amplitude = 55.3 ± 1.7 pA). Traces were automatically selected on the basis of their rise time (<40 ms), to avoid overlapping events, and their amplitude (>35 pA), to minimize the contribution of sparkless events (20). Traces were aligned at the rise time, averaged, and fitted to a single-exponential decay function from 10% to 90% of the peak. Decay time constants (tau ) from the fit were as follows: control = 22.3 ± 1.7 ms; BAPTA = 21.9 ± 1.1 ms.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we provide quantitative evidence that the local Ca2+ release events, Ca2+ sparks, from the SR of cerebral artery smooth muscle cells occur very close to BK channels in the cell membrane, such that the BK channels experience a micromolar Ca2+ concentration from a Ca2+ spark.

A central issue in signaling is to match amplitude and frequency of a signal with the response elements of the target. With respect to arterial smooth muscle, a Ca2+-sensitive target, the BK channel, has very low activity at membrane potentials (about -40 mV) and average intracellular Ca2+ (100-200 nM) that occur in pressurized cerebral arteries with tone (see Ref. 14). We provide the first direct measurements of Ca2+ sensitivity of BK channels at -40 mV (Fig. 2). Given the number (~3,000) of BK channels in the membrane of cerebral arterial myocytes, BK channels would not contribute significantly to the membrane conductance. However, these channels clearly regulate the membrane potential of smooth muscle cells in pressurized arteries, as supported by the depolarizing and constricting effects of blockers of BK channels such as iberiotoxin (2, 13, 15, 19). The discovery of Ca2+ sparks in smooth muscle (19) provided a mechanism by which BK channel Po could be elevated so as to contribute to the regulation of membrane potential. However, there appeared to be a mismatch between the level of Ca2+ released during a Ca2+ spark, as measured by the Ca2+-sensitive fluorescent indicator fluo 3, and amplitude of the evoked BK current (20).

To explore the issue of concentration of Ca2+ sensed by the BK channel, we first determined the elevation in Po of BK channels during a Ca2+ spark at physiological membrane potentials (-40 mV; Fig. 1). The comparison of BK channel activity in the absence of Ca2+ sparks and at the peak of a Ca2+ spark indicated that a Ca2+ spark elevates BK channel Po in this preparation ~6 × 105-fold, assuming that 1% of the BK channels are activated by a Ca2+ spark. Even assuming that all BK channels in the cell membrane are centered over one spark site, BK channel Po would increase ~6 × 103-fold during a spark. To determine the increase in Ca2+ required for a Ca2+ spark-evoked activation of BK channels, the Ca2+ sensitivity of BK channels was measured at the same voltage from the same preparation. Our measurements indicate that intracellular Ca2+ rises from 0.1 µM to at least 4 µM to activate BK channels during a Ca2+ spark, even with the condition that all BK channels are above one spark site. In contrast, the peak of Ca2+ spark, as measured with fluo 3, was ~0.2-0.3 µM (19, 20). These results strongly support the idea that Ca2+ release sites are very close to the BK channels, such that BK channels experience 4-30 µM Ca2+ concentrations.

We provided an additional test for close proximity of Ca2+ spark sites to BK channels. Mobile Ca2+ buffers should have little effect on Ca2+ within 20 nm of the Ca2+ release site (18), which is a likely distance from the release site to BK channels on the basis of electron microscopy (EM) studies (7, 16). Indeed, the fast Ca2+ chelator BAPTA effectively competed with fluo 3 for Ca2+, but it had no effect on transient BK currents (Figs. 3 and 4). These results lend additional support to the idea that Ca2+ spark sites in the SR are very close to the BK channels.

Our results also indicate that cerebral artery smooth muscle cells have at least 3,000 channels per cell (surface area ~1,300 µm2), corresponding to ~2-3 channels/µm2, similar to estimates of channel density from other preparations (1, 4, 11, 22). These results indicate that ~30 BK channels experience a Ca2+ spark, based on a spatial spread of 13 µm2 (20) and homogeneous channel distribution. These results suggest that maximal average Po of BK channels at -40 mV is still significantly less than 0.6 and that there is no need to invoke BK channel clustering to explain the presence of transient BK currents.

Our results support the concept that Ca2+ spark sites in smooth muscle are positioned close to their target, BK channels, so as to precisely match communication of RyRs to BK channels. BK channels have a low affinity for Ca2+ (Kd 19 µM) at physiological membrane potentials found in pressurized cerebral arteries (-40 mV). Our results are consistent with EM studies indicating that SR elements are within 20 nm of the cell membrane (7, 16). The close proximity of RyRs that cause sparks and BK channels enables a Ca2+ spark event to deliver a high local Ca2+ concentration (~10-30 µM) to the BK channels. Furthermore, the 100-fold mismatch in the estimated Ca2+ concentration for Ca2+ sparks and transient BK currents (0.2-0.3 µM and 30 µM, respectively) probably reflects a dilution of Ca2+ from the local subsarcolemmal volume experienced by the BK channels during a transient current into the scanning volume. Typically, our confocal scanning volume is 2.2 µm × 2.2 µm × 3 µm, or 14.5 fl. The difference in dilution factors can be explained by reducing one of the three-dimensional micrometer dimensions to a range of 20-30 nm in agreement with EM study distances for subsarcolemmal space (7, 16). Alternatively, cytoplasmic Ca2+ buffers may effectively compete with fluo 3, attenuating Ca2+ spark amplitude as BAPTA does in our study. In either case, a close proximity of RyRs and BK channels is required for activation of transient BK currents. The very local nature of the release events minimizes direct effects on other Ca2+-sensitive processes, which are spread through the cell's cytoplasm.


    ACKNOWLEDGEMENTS

DHS-1 was kindly provided by Merck Research Laboratories (Rahway, NJ).


    FOOTNOTES

This study was supported by grants from the National Institutes of Health (HL-44455, HL-63722, and DK-53832), the National Science Foundation (IBN-9631416 and BIR-9601682), and the Totman Medical Research Trust Fund and by a fellowship from the American Heart Association (G. J. Pérez).

Present address of G. J. Pérez: Masonic Medical Research Laboratory, 2150 Bleecker St., Utica, NY 13501

Address for reprint requests and other correspondence: M. T. Nelson, Dept. of Pharmacology, Univ. of Vermont, Burlington, VT 05405 (E-mail: mtnelson{at}zoo.uvm.edu).

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.

Received 12 June 2001; accepted in final form 17 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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