Departments of 1 Molecular Physiology and Biophysics and 2 Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 M
(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+])
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(1) |
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RESULTS |
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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
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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).
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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).
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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.
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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.
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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.
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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).
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DISCUSSION |
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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).
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Adrian Bonev, David Hill-Eubanks, and Guillermo Pérez for comments and discussions regarding the manuscript.
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FOOTNOTES |
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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.
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