Department of Pharmacology, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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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RESULTS |
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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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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.
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 (
) 22.3 ± 1.7 ms vs. BAPTA
21.9 ± 1.1 ms]. These results are also consistent with local
communication of Ca2+ sparks to BK channels.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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DHS-1 was kindly provided by Merck Research Laboratories (Rahway, NJ).
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FOOTNOTES |
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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.
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