Voltage dependence of the coupling of Ca2+ sparks to BKCa channels in urinary bladder smooth muscle

Gerald M. Herrera1, Thomas J. Heppner2, and Mark T. Nelson1,2

Departments of 1 Molecular Physiology and Biophysics and 2 Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Large-conductance Ca2+-dependent K+ (BKCa) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BKCa currents and intracellular Ca2+ revealed that BKCa currents are activated by Ca2+ release events (Ca2+ sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca2+ sparks and BKCa currents and to determine the voltage dependence of the coupling of RyRs (Ca2+ sparks) to BKCa channels in UBSM. Ca2+ sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca2+ sparks caused BKCa currents at all voltages tested, consistent with the BKCa channels sensing ~10 µM Ca2+. Membrane potential depolarization from -50 to -20 mV increased Ca2+ spark and BKCa current frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca2+ spark amplitude increasing by only 30% and BKCa current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BKCa current was quantitatively explained by the known voltage dependence of the Ca2+ sensitivity of BKCa channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca2+ sensitivity of BKCa channels, profoundly alters the coupling strength of Ca2+ sparks to BKCa channels.

guinea pig; sarcoplasmic reticulum; ryanodine receptor; iberiotoxin; thapsigargin; large-conductance Ca2+-activated K+ channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LARGE-CONDUCTANCE Ca2+-activated K+ (BKCa) channels play a key role in regulating the excitability and contractility of urinary bladder smooth muscle (UBSM). Blocking BKCa channels with iberiotoxin dramatically increases the amplitude and duration of action potentials (11) and phasic contractions (12) in UBSM.

BKCa channel activity in UBSM depends primarily on two factors: voltage and Ca2+ concentration ([Ca2+]) (21). Elevation of [Ca2+] and depolarization increase the open probability of BKCa channels from UBSM cells. The UBSM action potential is a potent activator of BKCa channels through membrane depolarization and elevation of Ca2+ entry through dihydropyridine-sensitive, voltage-dependent Ca2+ channels (VDCCs) (11). BKCa channels are also activated by Ca2+ release, in the form of Ca2+ sparks. Ca2+ sparks are local Ca2+ transients caused by the opening of ryanodine-sensitive Ca2+ release channels [referred to as ryanodine receptors (RyRs)] in the sarcoplasmic reticulum (SR) membrane (22). In smooth muscle cells from rat cerebral arteries, every Ca2+ spark activates nearby BKCa channels to cause a transient K+ current (24). In this preparation, each Ca2+ spark increases the open probability of BKCa channels 104- to 106-fold, indicating that the Ca2+ spark delivers 10-100 µM Ca2+ to the nearby BKCa channels (1, 24).

Another potentially important effect of membrane potential depolarization is to increase the apparent Ca2+ sensitivity of the BKCa channel (5, 6). This effect could theoretically increase the impact of Ca2+ sparks on BKCa channel activity, such that a given-size Ca2+ spark causes a larger BKCa current transient at depolarized potentials.

Ca2+ sparks have recently been described in UBSM. Collier and co-workers (4) observed a process whereby Ca2+ sparks in UBSM are evoked by Ca2+ entry through VDCCs. However, unlike classical Ca2+-induced Ca2+ release in cardiac muscle, which occurs on the millisecond time scale (3), this process in UBSM was relatively slow and could be attributed to the accumulation of Ca2+ through activation of VDCCs, rather than local control by Ca2+ entry through a single VDCC (4).

Although membrane potential is thought to regulate the coupling of Ca2+ sparks to BKCa channels in smooth muscle, little is known about this important relationship. Membrane potential depolarization could act on the BKCa channels by increasing the driving force for K+, increasing channel open probability, and by increasing channel Ca2+ sensitivity. Membrane potential could indirectly affect BKCa channel activity by altering Ca2+ spark frequency and amplitude.

The primary goal of the present study is to understand the effect of voltage on the coupling of Ca2+ sparks to BKCa channels. To accomplish this, we characterize Ca2+ sparks in UBSM and provide the first characterization of the effect of membrane potential on the coupling of Ca2+ sparks to BKCa channels by simultaneously measuring Ca2+ sparks and K+ currents in isolated UBSM cells. We provide the first evidence that voltage modulates the strength of coupling of Ca2+ sparks to BKCa channels, such that Ca2+ sparks cause larger BKCa currents as the membrane potential is depolarized. The results can be explained by the voltage dependence of the Ca2+ sensitivity of BKCa channels and provide a mechanism for enhanced negative-feedback regulation of UBSM membrane potential by BKCa channels.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. All procedures were reviewed and approved by the Office of Animal Care Management at the University of Vermont. Guinea pigs (250-350 g) were euthanized by halothane overdose and then exsanguinated. The urinary bladder was removed and placed in a Sylgard-coated petri dish containing cold dissection solution (DS) made up of (in mM) 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 HEPES, and 2 MgCl2, with pH adjusted to 7.3 with NaOH. After adipose and connective tissue were removed, the bladder was cut open and remaining traces of urine were washed away. The urinary bladder was cut into small strips (0.5 mm wide, 1 mm long) and transferred to a water-jacketed vial (2 ml volume, 37°C) containing DS supplemented with 1 mg/ml BSA, 1 mg/ml papain (Worthington), and 1 mg/ml dithioerythritol for 35 min. The papain-containing solution was then replaced with fresh DS containing 1 mg/ml BSA, 1 mg/ml collagenase (Fluka), and 100 µM CaCl2. The tissue was left in this solution for 5 min (37°C), and then the solution was replaced with cold BSA-containing DS and kept on ice. After two subsequent washes with cold BSA-containing DS, the tissue pieces were passed through the tip of a fire-polished glass Pasteur pipette to free individual cells. Cells were kept on ice until use, generally within 6 h. All experiments were performed at room temperature (22°C).

Confocal fluorescence microscopy. Isolated myocytes were loaded with the Ca2+-sensitive fluorophore fluo 3-AM (Molecular Probes) by mixing 250 µl of cell suspension with 250 µl of bath solution containing (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH, and supplemented with 5 µM fluo 3-AM. One hundred microliters of this fluo 3-containing cell suspension were placed on the bottom surface (glass coverslip) of a recording chamber (~1 ml volume). Cells were allowed 20 min to adhere to the coverslip, at which time dye loading was accomplished. Cells were subsequently washed with fresh bath solution to remove extraneous fluo 3 from the extracellular fluid. This wash progressed for 20-45 min, allowing sufficient time for fluo 3 deesterification. Ca2+ imaging was conducted on a laser-scanning confocal microscope (Oz, Noran Instruments) with a ×60 water immersion objective (NA 1.2; Nikon), with the 488-nm line of a krypton-argon laser used for illumination. Images were acquired at 240 Hz (image field 56.3 × 26.4 µm, 4.17 ms/image), 120 Hz (48.4 × 50.6 µm, 8.33 ms/image), or 60 Hz (56.3 × 52.8 µm, 16.7 ms/image). The relatively large fields acquired with this imaging system allow the majority of the cell to be scanned in a single image. Scan durations were 10-20 s. Pharmacological agents were applied to the test chamber at stated concentrations via a gravity-fed reservoir at a flow rate of ~2.5 ml/min.

Imaging experiments were analyzed with custom software written by Dr. Adrian Bonev using IDL 5.0.2 (Research Systems). Ca2+ sparks were defined as local increases in fluorescence of 1.2 F/FO (where F is the instantaneous fluorescence at a given time point and FO is the baseline fluorescence) that persisted for at least two images. FO was obtained by averaging 10 images containing no discernable Ca2+ transients. For quantitation of Ca2+ sparks, a square-box region (2.2 × 2.2 µm or 10 × 10 pixels) was placed over an area of the cell in which Ca2+ sparks were observed, and F/FO traces were generated for this region of the cell. Event frequency, amplitude, and kinetics were determined from entire experimental recordings lasting for 10-20 s.

Electrophysiology. A single drop of cell suspension was placed on a glass coverslip in the bottom of a recording chamber (~1 ml volume). Cells were allowed up to 20 min to adhere to the coverslip, and then fresh bath solution (see above for composition) was applied. Whole cell currents were measured using the perforated-patch technique (14). The pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA, with pH adjusted to 7.2 with NaOH. Amphotericin B (200 µg/ml) was also added to the pipette solution. Pharmacological agents were applied to the recording chamber via a gravity-fed reservoir at a flow rate of ~2.5 ml/min. For the cells used in the present study, resting membrane potential was -36 ± 2 mV, cell capacitance was 35 ± 1 pF, and series resistance was 38 ± 3 MOmega (n = 44 cells). Currents were recorded using an Axopatch 200A amplifier (Axon Instruments) filtered at 0.5-1 kHz and digitized at 1-4 kHz. For pharmacological characterization of transient outward currents in UBSM cells, experiments were conducted at a holding potential of -20 mV. These currents were characterized pharmacologically in a subset of 24 cells for a 5-min period. For control data, the 5-min period immediately before drug application was used for analysis, and to determine the effect of a given drug, a 5-min period was used during which activity was at a steady state. Transient outward currents were analyzed using Mini Analysis (Synaptosoft) with an amplitude threshold of three times the unitary BKCa channel current for guinea pig UBSM cells at the given holding potential (11, 22).

Simultaneous current and Ca2+ measurements. To examine the temporal relationship between Ca2+ sparks and BKCa channel activation, Ca2+ sparks and whole cell currents at holding potentials of -50 to -20 mV were measured simultaneously using the methods described above. All simultaneous current and fluorescence recordings were conducted in a standing bath. A trigger source output on the confocal microscope was used to align the fluorescence and electrical records. Entire experimental records lasting for 10-20 s were used for analysis of electrical and fluorescence events (frequency, amplitude, and kinetics). Simultaneous electrical events and Ca2+ sparks were analyzed using custom software written by Dr. Adrian Bonev using IDL 5.0.2 (Research Systems).

Chemicals. Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical. Ryanodine was obtained from L. C. Laboratories.

Calculations and statistics. Values are means ± SE. Data were compared using one-way ANOVA or one- or two-tailed t-test, where appropriate. Student-Newman-Keuls method was used for all pairwise multiple comparisons. P < 0.05 was considered statistically significant.

In experiments where Ca2+ sparks were recorded in cells voltage clamped to different membrane potentials, the [Ca2+] associated with a fluo 3 transient (Ca2+ spark) was estimated using the following equation to normalize for changes in background fluorescence, FO (global [Ca2+])
[Ca<SUP><IT>2+</IT></SUP>]<IT>=</IT><FR><NU><IT>K</IT>R</NU><DE>(<IT>K/</IT>[Ca<SUP><IT>2+</IT></SUP>]<SUB>rest</SUB><IT>+1</IT>)<IT>−</IT>R</DE></FR> (1)
where K is the apparent affinity of fluo 3 for Ca2+ (~400 nM; see 3), R is the fractional fluorescence increase (F/Fo), and [Ca2+]rest is the cytosolic [Ca2+] at Fo.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacological characterization of transient outward currents in UBSM cells. Transient outward currents have been previously observed in UBSM cells (9, 15). These currents were proposed to be conducted by BKCa channels, since tetraethylammonium (1 mM) or iberiotoxin (30 nM) attenuated these currents by 80% (15). Furthermore, transient outward currents in UBSM are thought to be activated by Ca2+ released from the SR, since the RyR agonist caffeine has an initial stimulatory effect on these currents (15). However, a detailed analysis of transient outward currents in UBSM has not been performed, so our first objective was to characterize transient outward currents in this tissue and determine the source of Ca2+ initiating these electrical events.

Single UBSM cells were held at a potential of -20 mV using the perforated-patch configuration of whole cell voltage clamp (see MATERIALS AND METHODS). The frequency of transient BKCa currents was 1.23 ± 0.24 Hz (n = 24), and the average amplitude of these currents was 43.2 ± 9.9 pA. Iberiotoxin (100 nM), a selective blocker of BKCa channels (8, 23), inhibited the currents by 97% (n = 10; Fig. 1, A and B). To examine the possibility that the transient currents remaining in the presence of iberiotoxin were conducted by K+ channels other than BKCa channels, the potent and selective small-conductance Ca2+-dependent K+ (SKCa) channel blocker apamin (100 nM) was applied in the presence of 100 nM iberiotoxin. Apamin did not affect these currents (n = 3), suggesting that they are not conducted by SKCa channels (average amplitude of transient currents in the presence of iberiotoxin = 15.6 ± 2.3 pA, average amplitude in the presence of iberiotoxin and apamin = 14.2 ± 1.5 pA, P > 0.05). Furthermore, increasing the concentration of iberiotoxin to 600 nM completely abolished all transient outward current activity in UBSM cells, with an apparent EC50 of 27 nM at -20 mV (n = 3). Thus the transient currents in UBSM can be attributed to activation of BKCa channels.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 1.   Large-conductance Ca2+-dependent K+ (BKCa) currents depend on ryanodine receptor (RyR)-mediated Ca2+ release from the sarcoplasmic reticulum (SR) in urinary bladder smooth muscle (UBSM). A: original recording of whole cell currents in a UBSM cell before and after BKCa channels were blocked with iberiotoxin (100 nM). B: blocking BKCa channels decreased transient outward current frequency. C: original recording of currents in a UBSM cell before and after application of the SR Ca2+-ATPase inhibitor thapsigargin (100 nM). D: thapsigargin decreased BKCa current frequency. E: original recording of currents in a UBSM cell before and after inhibition of RyRs with ryanodine (10 µM). F: blocking RyRs decreased BKCa current frequency. All experiments were performed at a holding potential of -20 mV. *P < 0.05 vs. control.

Transient BKCa currents in other preparations are caused by local Ca2+ release (Ca2+ sparks) through RyRs in the SR (17, 22). To determine the importance of SR Ca2+ release in initiating BKCa currents in UBSM, UBSM cells were treated with thapsigargin (100 nM), which blocks the SR Ca2+-ATPase and thereby leads to depletion of SR Ca2+ stores (n = 5). Figure 1C shows an original record of BKCa currents measured from a cell before and after thapsigargin. Thapsigargin reduced the BKCa current frequency by 96% (from 1.05 ± 0.26 to 0.04 ± 0.04 Hz, P < 0.05, n = 5; Fig. 1D). This observation suggests that BKCa channels are activated by SR Ca2+ release. Next, the role of RyRs in causing transient BKCa currents was examined by treating UBSM cells (n = 9) with ryanodine (10 µM) at a concentration that inhibits RyRs (18, 22, 26). Figure 1E shows an original recording of BKCa currents in a single UBSM cell before and after ryanodine (10 µM) treatment. Within 15 min of application, ryanodine caused a marked reduction in transient BKCa current activity. At steady state, ryanodine reduced BKCa current frequency by 94%, from 1.32 ± 0.57 to 0.09 ± 0.03 Hz (P < 0.05, n = 9 cells; Fig. 1F). These observations suggest that BKCa currents are caused by brief Ca2+ release events through RyRs in the SR.

Identification of local Ca2+ transients ("Ca2+ sparks") in UBSM cells. To determine whether localized transient increases in intracellular [Ca2+] ([Ca2+]i) could be observed that may serve as a stimulus for BKCa channel activity in UBSM cells, myocytes were loaded with the Ca2+-sensitive indicator fluo 3 and scanned with a laser-scanning confocal microscope (Fig. 2). Localized transient increases in fluorescence were observed in UBSM cells. The spatial and temporal characteristics of these events suggest that they correspond to Ca2+ sparks, which have been described in other types of smooth muscle (17, 22), as well as skeletal (19) and cardiac (3) muscle. An average of 2.9 ± 0.3 spark sites per cell were observed (n = 18). Spark frequency was 0.62 ± 0.08 Hz. The fractional fluorescence increase (F/Fo, see MATERIALS AND METHODS) associated with Ca2+ sparks in UBSM cells was 2.12 ± 0.03 (n = 344 sparks from 18 cells). Figure 2A is a pseudocolor image of a single UBSM cell loaded with the fluorescent Ca2+ indicator fluo 3. In this cell, transient increases in [Ca2+]i, corresponding to Ca2+ sparks, were found in three distinct regions (red, orange, and green boxes).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   Identification of Ca2+ sparks in UBSM cells. A: pseudocolor image of a single fluo 3-loaded UBSM cell. Colored boxes show regions of the cell containing Ca2+ spark sites. Each box measures 2.2 × 2.2 µm. Dashed white box highlights a region of the cell that is shown in time lapse in B. Color bar below the cell image shows the F/FO scale (where F is instantaneous fluorescence at a given time point and F0 is baseline fluorescence). B: time-lapse series of images highlighting 3 phases of a Ca2+ spark: 1) baseline, 2) peak, and 3) decay to baseline. Image scale is the same as in A. C: F/FO vs. time plots for the 3 spark sites in A. *Spark shown in B.

Pharmacological characterization of Ca2+ sparks in UBSM cells. In other preparations, Ca2+ sparks are attributed to Ca2+ release via RyRs in the SR (3, 17, 19, 22, 27). To examine the nature of the Ca2+ sparks in UBSM, cells were treated with thapsigargin (100 nM) to inhibit the SR Ca2+-ATPase (n = 6). Figure 3A shows an image of a single fluo 3-loaded UBSM cell before (control) and after application of thapsigargin. Under basal conditions, Ca2+ sparks were observed at five sites in this cell (regions a-e, Fig. 3A, left). Fluorescence transients in each region are shown below the image of the cell. The same cell is shown ~8 min after thapsigargin (100 nM). Thapsigargin abolished spark activity in this cell. In the same regions where Ca2+ sparks were observed under control conditions, no Ca2+ transients were seen after thapsigargin treatment (Fig. 3A, right). Thapsigargin decreased Ca2+ spark frequency from 0.62 ± 0.09 to 0.06 ± 0.03 Hz (n = 6 cells, P < 0.05; Fig. 3C).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Ca2+ sparks reflect Ca2+ release through RyRs in the SR membrane. A: thapsigargin inhibits Ca2+ spark activity. Left: image of a cell (constructed by averaging 10 frames of 2,400 containing no Ca2+ transients) under resting conditions. There were 5 spark sites in this cell (a-e). Below the image are the F/FO traces for each spark site (a-e). Right: cell at left ~8 min after application of thapsigargin (100 nM). The same regions of the cell with active spark sites under control conditions were silent after inhibition of the SR Ca2+-ATPase. B: ryanodine inhibits Ca2+ spark activity. Left: Ca2+ sparks from 2 sites (a and b) recorded in a UBSM cell under resting conditions. Right: fluorescence traces from the same regions after ~10 min of exposure to ryanodine (10 µM). C: summary of effects of thapsigargin and ryanodine on Ca2+ spark frequency. *P < 0.05 vs. control.

To address the role of RyRs in UBSM Ca2+ sparks, a separate group of cells was treated with the RyR inhibitor ryanodine (10 µM, n = 5). Figure 3B shows fluorescence recordings from two Ca2+ spark sites (a and b) recorded in another cell before and 10 min after ryanodine. In the presence of ryanodine, no Ca2+ sparks were observed at the same sites that were active under resting conditions (or in any other regions of this cell). Ryanodine decreased Ca2+ spark frequency from 0.65 ± 0.22 to 0.11 ± 0.07 Hz (n = 5, P < 0.05; Fig. 3C).

Simultaneous measurements of Ca2+ sparks and BKCa currents in UBSM cells. To examine directly the hypothesis that SR Ca2+ release from RyRs activates BKCa channels in the cell surface, Ca2+ spark activity and whole cell membrane currents were recorded simultaneously. Figure 4A shows the simultaneous electrical and fluorescence recordings from a 20-s scan in a UBSM cell held at -40 mV. Each Ca2+ spark is associated with a nearly synchronous transient BKCa current. Larger Ca2+ sparks are associated with larger BKCa currents (see below). The peak of the current preceded the peak of the Ca2+ spark by 21.5 ± 4.4 ms (n = 27). The onset time for BKCa currents and sparks can be determined using the rise times (~16 ms for BKCa currents, ~35 ms for Ca2+ sparks; Table 1), and it is evident that these events start within the imaging resolution (8.33 ms/image). The close temporal relationship (Fig. 4B) between Ca2+ sparks and BKCa currents suggests that SR Ca2+ release from RyRs (detected as a change in fluorescence) activates BKCa channels.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Ca2+ sparks cause BKCa currents in UBSM cells. A: original recordings of whole cell membrane currents and Ca2+ sparks from a UBSM cell held at -40 mV. Every Ca2+ spark is associated with a simultaneous BKCa current. B: expanded sweeps showing regions from A (*) to illustrate the simultaneous nature of a Ca2+ spark and its associated BKCa current. HP, holding potential.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Summary of BKCa current and Ca2+ spark kinetics from simultaneous measurements

Membrane potential depolarization increases frequency and amplitude of Ca2+ sparks and transient BKCa currents. The relationship between Ca2+ sparks and BKCa currents was assessed at membrane potentials ranging from -50 to -20 mV. Figure 5A shows Ca2+ sparks from two sites and the associated membrane currents in a cell held at -50 mV. At this membrane potential, some Ca2+ sparks (11 of 55) were observed that were not associated with resolvable BKCa currents. Figure 5B shows Ca2+ sparks from the same cell shown in Fig. 5A and membrane currents at a holding potential of -20 mV. At -20 mV, all Ca2+ sparks (n = 127) were associated with BKCa currents, but many currents (61 of 188) were observed with no detectable fluorescence transients. This result is not unexpected, since only about one-third of the cell volume is scanned with the confocal microscope, whereas current is recorded across the entire cell membrane (24). Thus, although we can detect electrical events associated with nearly every Ca2+ spark (94%), we only detect Ca2+ sparks that occur in the volume of the cell that is scanned.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   Voltage dependence of Ca2+ sparks and BKCa currents in UBSM cells. A: original recordings of whole cell currents (top trace) and Ca2+ sparks (middle and bottom traces) in a UBSM cell held at -50 mV. There were 2 active spark sites in this cell. B: original recording of BKCa currents (top trace) and Ca2+ sparks (middle and bottom traces) in a UBSM cell held at -20 mV. The same 2 spark sites were active at -20 mV. Note the large amplitude and high frequency of the currents at this potential compared with -50 mV. At -20 mV, several of the smaller currents occur with no detectable Ca2+ sparks, but every spark is associated with an electrical event.

Figure 6A illustrates the relationship between holding potential and Ca2+ spark and associated BKCa current frequency. Fewer sparks than transient currents were detected at all potentials positive to -50 mV. A summary of kinetic data for Ca2+ sparks and BKCa currents recorded at various membrane potentials is given in Table 1.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Ca2+ spark and BKCa current amplitude and frequency increase with depolarization. A: voltage dependence of Ca2+ spark and BKCa current frequency. Em, membrane equilibrium potential. B: voltage dependence of Ca2+ spark amplitude {peak spark Ca2+ concentration ([Ca2+]) minus resting intracellular [Ca2+]}. C: BKCa channel activity (IBK/i, where IBK is BKCa current and i is unitary BKCa current) increases with membrane potential depolarization from -50 to -20 mV (n = 5-8 cells at each membrane potential).

Global [Ca2+]i increased with depolarization. Baseline whole cell fluorescence at -50 mV was taken as Fo, and when expressed relative to -50 mV, whole cell fluorescence (F/Fo) increased with depolarization 1.04 ± 0.17-fold at -40 mV (n = 8), 1.12 ± 0.19-fold at -30 mV (n = 8), and 1.76 ± 0.49-fold at -20 mV (n = 5); these values are consistent with previous measurements of steady-state [Ca2+]i in UBSM (9). Ca2+ spark amplitude also increased with depolarization. The fractional increase in fluorescence, F/Fo, of the Ca2+ sparks was used to calculate [Ca2+] during a spark (see Eq. 1). Values for [Ca2+]rest were taken from measurements of [Ca2+]i in voltage-clamped UBSM cells and were 115 nM at -50 mV, 150 nM at -40 mV, 180 nM at -30 mV, and 250 nM at -20 mV (9). Figure 6B shows the increase in Ca2+ spark amplitude at depolarized potentials. The increase in spark amplitude and increased global [Ca2+]i suggest that SR Ca2+ content increases at depolarized potentials.

Membrane potential depolarization increases the coupling strength of RyRs to BKCa channels. A striking observation is that BKCa current amplitude increases dramatically at depolarized potentials (cf. Fig. 5, A and B). BKCa current amplitude increased from 8.6 ± 0.8 pA at -50 mV to 144.1 ± 14.3 pA at -20 mV (~16-fold). To account for the effects of the K+ driving force on measured BKCa currents, the peak transient BKCa currents (IBK) were divided by the unitary BKCa current (i) (11) at each potential (Fig. 6C). BKCa channel activity, measured as IBK/i, increased sixfold over the range of -50 to -20 mV (Fig. 6C). The elevation in BKCa current at depolarized potentials could reflect an increase in the Ca2+ released during a Ca2+ spark (Fig. 6B) or an increase in the sensitivity of BKCa channels to Ca2+ sparks, such that a Ca2+ spark of a given amplitude causes a larger transient BKCa channel current at more depolarized voltages.

To explore the issue of the elevation of transient BKCa currents with membrane depolarization, the relationship between peak BKCa channel activity (IBK/i) and peak [Ca2+]i during a spark was determined at each membrane potential. Peak transient BKCa channel activity, corrected for driving force (IBK/i), increased with peak [Ca2+] during a spark at all voltages (Fig. 7). Membrane potential depolarization increased the steepness of the relationship between peak [Ca2+] during a spark and IBK/i, indicating that a given-size Ca2+ spark is more effective at activating BKCa channels at more positive voltages. Thus the coupling strength of a Ca2+ spark to BKCa channels increased with depolarization.


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 7.   Membrane potential depolarization increases the coupling strength of RyRs to BKCa channels. BKCa channel activity (IBK/i) is plotted as a function of Ca2+ spark amplitude (peak spark [Ca2+]). Each color group is a scatter plot of Ca2+ spark amplitude vs. IBK/i at a given membrane potential, fit by least-squares regression (n = 5-8 cells at each potential). Peak Ca2+ spark amplitudes were obtained using Eq. 1. Correlation coefficients were 0.75, 0.80, 0.81, and 0.83 at -20, -30, -40, and -50 mV, respectively. Dashed lines, 95% confidence intervals for each fit.

It is possible that the increase in Ca2+ spark-induced BKCa channel activity with depolarization reflects a greater spatial spread of Ca2+ sparks, such that a spark would recruit more BKCa channels on a greater surface of the cell at more positive voltages. Despite the slight increase in Ca2+ spark amplitude with depolarization (Fig. 6B), Ca2+ spark spatial spread (area of spark at half-maximum amplitude) did not change with membrane potential depolarization (Fig. 8). This observation is in contrast to reports in striated muscle, where larger-amplitude sparks also have a larger spatial profile (10), and suggests that the increased BKCa channel activity (IBK/i) at depolarized potentials cannot be explained by an increased spread of the Ca2+ sparks but, instead, is due to a higher activity of BKCa channels activated by a given-size Ca2+ spark.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 8.   Spatial spread of Ca2+ sparks does not change with membrane potential. Ca2+ spark spatial spread was measured at half-maximum amplitude at each membrane potential. Scatter plot shows individual measurements obtained in 5-8 cells at each membrane potential. Horizontal lines, group means.

BKCa channel activity in the absence of Ca2+ sparks. To estimate the increase in BKCa channel activity caused by a Ca2+ spark, it is necessary to determine BKCa channel activity in the absence of sparks. To do this, whole cell membrane currents were recorded (perforated patch) in UBSM cells that were pretreated with thapsigargin (100 nM) for 10 min to eliminate Ca2+ sparks (see Ref. 25 for a similar procedure in vascular smooth muscle). UBSM cells were held at 0 mV, and single BKCa channel activity was recorded. BKCa channel activity was 0.073 ± 0.039 (n = 4; not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BKCa channels as targets for Ca2+ sparks in UBSM. BKCa channels are key elements in the regulation of UBSM function. BKCa channels are involved in the repolarization of the action potential (11, 20). The specific BKCa channel inhibitor iberiotoxin causes a membrane potential depolarization and increases the action potential amplitude, duration, and frequency (11), and this causes a dramatic increase in the amplitude and duration of phasic UBSM contractions (12).

Step depolarization of UBSM cells leads to transient SR Ca2+ release, which is associated with activation of Ca2+-dependent K+ channels (16). These Ca2+ transients (referred to as "hot spots") coalesced into a global Ca2+ transient over the course of ~100 ms and were rarely observed in cells at rest (16). This situation is different from the present study, where Ca2+ sparks occurred in unstimulated cells, and had very little effect on global [Ca2+]i. However, the observation that SR Ca2+ release can activate BKCa channels provides a possible mechanism to account for the involvement of BKCa channels in the regulation of the membrane potential.

Ca2+ sparks and SKCa channels. Apamin-sensitive SKCa channels play an important role in regulating UBSM contractility (12). Because these channels are activated by Ca2+ in the submicromolar range (13), Ca2+ sparks should activate these channels. We found that transient outward currents in UBSM were not sensitive to the SKCa channel blocker apamin. This finding suggests that apamin-sensitive SKCa channels are not present in sufficient density near a spark site to generate measurable currents.

Ca2+ sparks dramatically elevate BKCa channel activity. The results of the present study have significant implications for communication between RyRs and BKCa channels. On the basis of our measurements, the increase in BKCa channel activity during a Ca2+ spark can be estimated (24). Dividing the transient BKCa current by the unitary current gives an estimate for the minimum number of BKCa channels activated by a Ca2+ spark. At -20 mV, a Ca2+ spark activates >= 35 BKCa channels in ~0.3% of the cell membrane (Figs. 6C and 8). The whole cell activity (open channel probability) of BKCa channels in the absence of sparks is ~10-2 at -20 mV on the basis of our single BKCa channel measurements in whole cells. Therefore, a Ca2+ spark increases the mean activity of BKCa channels above the spark site 106-fold at -20 mV. For such a substantial increase in BKCa channel activity to occur, [Ca2+] in the vicinity of BKCa channels would have to increase ~30-fold, with the assumption that channel activity increases with the fourth power of [Ca2+] (2). This means that a Ca2+ spark increases [Ca2+]i in its local domain from ~250 nM at rest to ~7.5 µM at -20 mV (see Ref. 9 for resting [Ca2+]i in UBSM). Therefore, the [Ca2+]i sensed by the BKCa channel is far greater than that reported by the fluorescent indicator fluo 3 (24). To communicate high [Ca2+]i (~10 µM) to the BKCa channel, the RyRs in a spark site must be close to the BKCa channels in the surface membrane (7, 17).

Voltage dependence of coupling strength of RyRs to BKCa channels. In the present study, we characterized the relationship between Ca2+ sparks and their associated BKCa currents over a range of membrane potentials (-50 to -20 mV). Ca2+ spark frequency and amplitude increased with depolarization (Fig. 6, A and B). Membrane depolarization from -50 to -20 mV elevated global [Ca2+] from 115 to 260 nM on the basis of an increase in whole cell fractional fluorescence (resting [Ca2+]i at -50 mV from Ref. 9). This would lead to an elevation of SR Ca2+ content. The elevation of cytoplasmic and SR Ca2+ should elevate Ca2+ spark frequency (28). An elevation of SR Ca2+ would also increase spark amplitude (28).

Membrane potential dramatically impacted the amplitude of the BKCa channel currents activated by a spark (Fig. 6C). Because the Ca2+ sensitivity of the BKCa channel increases with depolarization (5, 6, 21), we propose that changes in the apparent Ca2+ sensitivity of the BKCa channel could underlie the depolarization-induced increase in transient BKCa current amplitude activated by a given-size Ca2+ spark. At each membrane potential, BKCa current amplitude correlated with spark [Ca2+] (Fig. 7). Depolarization caused a steepening of the relationship between BKCa currents and peak [Ca2+] during a spark, such that a given Ca2+ spark caused a larger increase in BKCa channel activity. In fact, the voltage dependence of BKCa channel activity (IBK/i) for a given-size Ca2+ spark mirrors the voltage dependence of the Ca2+ sensitivity of the BKCa channel (6-fold increase in BKCa channel activity at 10 µM Ca2+ from -50 to -20 mV). Cui and co-workers (6) found that the apparent dissociation constant of the BKCa channel decreased approximately sixfold over the voltage range -50 to -20 mV, and their results suggest that the coupling strength of Ca2+ sparks to BKCa channels should increase approximately e-fold per 20 mV of depolarization, until BKCa channels saturate with [Ca2+]i.

In addition to the enhanced Ca2+ sensitivity, two other factors contribute to the elevation of transient BKCa current with membrane potential depolarization. The amplitude of Ca2+ sparks increased by 30% with membrane depolarization from -50 to -20 mV (Fig. 6B). The K+ driving force also increased with depolarization. These three factors lead to a ~16-fold increase in BKCa current amplitude over the range of -50 to -20 mV. In contrast, the frequency of Ca2+ sparks and transient BKCa currents increased nearly fivefold over this same range of membrane potentials (Fig. 6A). Therefore, amplitude modulation of BKCa currents at depolarized potentials reflects three main factors: 1) an increase in the driving force for K+, 2) an increase in the amount of Ca2+ released during a spark, and 3) augmented coupling strength of RyRs to BKCa channels. The coupling strength increased about sixfold for 30 mV (Fig. 6C) and, as such, was the most significant contributor to the amplitude modulation of the transient BKCa currents by voltage.

In conclusion, the present study supports the idea of local communication between RyRs in the SR and BKCa channels in the sarcolemma of UBSM cells. This communication involves Ca2+ release events (Ca2+ sparks), which serve as the stimulus to activate BKCa channels, thus comprising a negative-feedback system to regulate Ca2+ entry via voltage-dependent Ca2+ channels (22). Surprisingly, the coupling strength between Ca2+ sparks and BKCa channels increases with membrane potential depolarization, resulting in much larger BKCa currents for a given-size Ca2+ spark at depolarized potentials. On the basis of previous studies (24), we expected that BKCa channels would be saturated with Ca2+ during a spark at all membrane potentials, and the coupling strength would not increase with membrane potential depolarization but would simply reflect the electrochemical gradient for K+. Our data suggest that BKCa channels are not saturated by Ca2+ during a spark and that the BKCa channel Ca2+ sensitivity is under dynamic regulation, depending on the membrane potential. Thus the gain on this feedback system is effectively increased at depolarized potentials, owing to the voltage-dependent nature of the BKCa channel Ca2+ sensitivity (5, 6). It is likely that this local signaling scheme plays an important role in controlling UBSM contractility by affecting membrane excitability, especially at depolarized membrane potentials when the coupling of RyRs to BKCa channels is enhanced. These results suggest that other factors (e.g., the beta -subunit or protein kinase A or G) that regulate the Ca2+ sensitivity of BKCa channels should also modulate the coupling of Ca2+ sparks to BKCa channel activation.


    ACKNOWLEDGEMENTS

The authors thank Drs. Adrian Bonev, David Hill-Eubanks, and Guillermo Pérez for comments and discussions regarding the manuscript.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-53832 and HL-44455 (to M. T. Nelson). G. M. Herrera is a National Science Foundation Graduate Research Fellow.

Address for reprint requests and other correspondence: M. T. Nelson, Dept. of Pharmacology, University of Vermont College of Medicine, Burlington, VT 05405 (E-mail: nelson{at}salus.med.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 11 August 2000; accepted in final form 2 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bonev, AD, Pérez GJ, and Nelson MT. Communication of ryanodine receptors and Ca2+-sensitive K (KCa) channels in smooth muscle from rat cerebral arteries requires close proximity (Abstract). Biophys J 78: 438A, 2000.

2.   Carl, A, and Sanders KM. Regulation of ion channels in smooth muscle by calcium. Am J Physiol Cell Physiol 271: C9-C34, 1996[Abstract/Free Full Text].

3.   Cheng, H, Lederer WJ, and Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740-744, 1993[ISI][Medline].

4.   Collier, ML, Ji G, Wang Y-X, and Kotlikoff MI. Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. J Gen Physiol 115: 653-662, 2000[Abstract/Free Full Text].

5.   Cox, DH, Cui J, and Aldrich RW. Allosteric gating of a large-conductance Ca-activated K+ channel. J Gen Physiol 110: 257-281, 1997[Abstract/Free Full Text].

6.   Cui, J, Cox DH, and Aldrich RW. Intrinsic voltage dependence and Ca2+ regulation of large-conductance Ca-activated K+ channels. J Gen Physiol 109: 647-673, 1997[Abstract/Free Full Text].

7.   Devine, CE, Somlyo AV, and Somlyo AP. Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J Cell Biol 52: 690-718, 1972[Abstract/Free Full Text].

8.   Galvez, A, Gimenez-Gallego G, Reuben JP, Roy-Contacin L, Feigenbaum P, Kaczorowski GJ, and Garcia ML. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem 265: 11083-11090, 1990[Abstract/Free Full Text].

9.   Ganitkevich, VY, and Isenberg G. Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder. J Physiol (Lond) 435: 187-205, 1991[Abstract].

10.   González, A, Kirsch WG, Shirokova N, Pizarro G, Stern MD, and Ríos E. The spark and its ember: separately gated local components of Ca2+ release in skeletal muscle. J Gen Physiol 115: 139-157, 2000[Abstract/Free Full Text].

11.   Heppner, TJ, Bonev AD, and Nelson MT. Ca2+-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle. Am J Physiol Cell Physiol 273: C110-C117, 1997[Abstract/Free Full Text].

12.   Herrera, GM, Heppner TJ, and Nelson MT. Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am J Physiol Regulatory Integrative Comp Physiol 279: R60-R68, 2000[Abstract/Free Full Text].

13.   Hirschberg, B, Maylie J, Adelman JP, and Marrion NV. Gating of recombinant small-conductance Ca-activated K+ channels by calcium. J Gen Physiol 111: 565-581, 1998[Abstract/Free Full Text].

14.   Horn, R, and Marty A. Muscarinic activation of ionic currents measured by a new whole cell recording method. J Gen Physiol 92: 145-159, 1988[Abstract].

15.   Imaizumi, Y, Henmi S, Uyama Y, Atsuki K, Torii Y, Ohizumi Y, and Watanabe M. Characteristics of Ca2+ release for activation of K+ current and contractile system in some smooth muscles. Am J Physiol Cell Physiol 271: C772-C782, 1996[Abstract/Free Full Text].

16.   Imaizumi, Y, Torii Y, Ohi Y, Nagano N, Atsuki K, Yamamura H, Muraki K, Watanabe M, and Bolton TB. Ca2+ images and K+ current during depolarization in smooth muscle cells of the guinea-pig vas deferens and urinary bladder. J Physiol (Lond) 510: 705-719, 1998[Abstract/Free Full Text].

17.   Jaggar, JH, Porter VA, Lederer WJ, and Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278: C235-C256, 2000[Abstract/Free Full Text].

18.   Jaggar, JH, Stevenson AS, and Nelson MT. Voltage dependence of Ca2+ sparks in intact cerebral arteries. Am J Physiol Cell Physiol 274: C1755-C1761, 1998[Abstract/Free Full Text].

19.   Klein, MG, Cheng H, Santana LF, Jiang Y-H, Lederer WJ, and Schneider MF. Two mechanisms of quantized calcium release in skeletal muscle. Nature 379: 455-458, 1996[ISI][Medline].

20.   Klöckner, U, and Isenberg G. Action potentials and net membrane current of isolated smooth muscle cells (urinary bladder of the guinea-pig). Pflügers Arch 405: 329-339, 1985[ISI][Medline].

21.   Markwardt, F, and Isenberg G. Gating of maxi-K+ channels studied by Ca2+ concentration jumps in excised inside-out multichannel patches (myocytes from guinea pig urinary bladder). J Gen Physiol 99: 841-862, 1992[Abstract].

22.   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: 633-637, 1995[Abstract].

23.   Nelson, MT, and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799-C822, 1995[Abstract/Free Full Text].

24.   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: 229-237, 1999[Abstract/Free Full Text].

25.   Porter, VA, Bonev AD, Knot HJ, Heppner TJ, Stevenson AS, Kleppisch T, Lederer WJ, and Nelson MT. Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides. Am J Physiol Cell Physiol 274: C1346-C1355, 1998[Abstract/Free Full Text].

26.   Rousseau, E, Smith JS, and Meissner G. Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am J Physiol Cell Physiol 253: C364-C368, 1987[Abstract/Free Full Text].

27.   ZhuGe, R, Sims SM, Tuft RA, Fogarty KE, and Walsh Jr JV. Ca2+ sparks activate K+ and Cl- channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J Physiol (Lond) 513: 711-718, 1998[Abstract/Free Full Text].

28.   ZhuGe, R, Tuft RA, Fogarty KE, Bellve K, Fay FS, and Walsh Jr JV. The influence of sarcoplasmic reticulum Ca2+ concentration on Ca2+ sparks and spontaneous transient outward currents in single smooth muscle cells. J Gen Physiol 113: 215-228, 1999[Abstract/Free Full Text].


Am J Physiol Cell Physiol 280(3):C481-C490
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society