Am J Physiol Cell Physiol
281: C1769–C1775, 2001.
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
Received 12 June 2001; accepted in final form 17 July 2001
Pérez, Guillermo J., Adrian D. Bonev, and Mark T.
Nelson. Micromolar Ca2⫹ from sparks activates Ca2⫹-sensi-
tive K⫹ channels in rat cerebral artery smooth muscle. Am J
Physiol Cell Physiol 281: C1769–C1775, 2001.—The goal of
the present study was to test the hypothesis that local Ca2⫹
release events (Ca2⫹ sparks) deliver high local Ca2⫹ concen-
tration 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 mi-
croscopy and patch-clamp techniques. BK channels had an
apparent dissociation constant for Ca2⫹ of 19 ␮M and a Hill
coefficient of 2.9 at ⫺40 mV. At near-physiological intracel-
lular 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 corre-
sponds to ⬃30 ␮M to 4 ␮M spark Ca2⫹ concentration. 1,2-
bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid ace-
toxymethyl 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
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-de-
pendent 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 differ-
ent 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⫹-sen-
Address for reprint requests and other correspondence: M. T.
Nelson, Dept. of Pharmacology, Univ. of Vermont, Burlington, VT
05405 (E-mail: mtnelson@zoo.uvm.edu).
http://www.ajpcell.org 0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
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sitive 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 ele-
ments 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 hypoth-
esis that Ca2⫹ sparks deliver high local (micromolar)
Ca2⫹ concentrations to activate nearby BK channels in
the cell membrane at the physiological membrane po-
tentials (⫺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 chan-
nels at ⫺40 mV. These measurements indicate that BK
channels experience Ca2⫹ concentrations in the range
of 4–30 ␮M during a spark. Third, the local communi-
cation of Ca2⫹ sparks to BK channels was probed by
introducing the mobile Ca2⫹ buffer 1,2-bis(2-amino-
phenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid (BAPTA)
into single smooth muscle cells. BAPTA at concentra-
tions that effectively competed with the fluorescent
Ca2⫹ indicator fluo 3 did not affect the Ca2⫹ spark-
evoked BK channel currents. Together these results
The costs of publication of this article were defrayed in part by the
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solely to indicate this fact.
C1769
C1770 LOCAL COMMUNICATION OF CA2⫹ SPARKS TO BK CHANNELS

support the concept that the Ca2⫹ signal, Ca2⫹ sparks, ries (Rahway, NJ). All other chemicals were obtained from
through proximity, are matched to the Ca2⫹ sensitivity Sigma (St. Louis, MO) and Calbiochem-Novabiochem Inter-
of a target, the BK channels, to regulate membrane national (La Jolla, CA). All experiments were conducted at
potential. room temperature (20–22°C).
Data analysis. Transient BK currents were analyzed using
METHODS
Mini analysis 5.1.1 software (Jaejin Software). Image analy-
sis was performed using custom-written analysis programs
Cell isolation. Sprague-Dawley rats (12–14 wk old) of using Interactive Data Language software (Research Sys-
either sex were euthanized by peritoneal injection of pento- tems, Boulder, CO). Baseline fluorescence (Fo) was deter-
barbital solution (150 mg/kg). Cerebral (basilar) arteries mined by averaging 30 images (of 1,200) with no activity.
were carefully dissected and then digested with papain (0.3 Ratio images were then constructed and replayed for careful
mg/ml papain and 1 mg/ml dithioerythritol for 20 min at examination to detect active areas where sudden increases in
37°C) and collagenase (1 mg/ml collagenase, type F and type fluorescence (fractional change, F/Fo) occurred. F/Fo vs. time
H in a 70%-30% mixture, respectively, incubated for 10 min traces were further analyzed in Microcal Origin (Microcal
at 37°C). The digested tissue was triturated with a fire- Software, Northampton, MA) and represent the averaged
polished glass Pasteur pipette to yield single smooth muscle F/Fo from a box region of 2.2 ⫻ 2.2 ␮m centered in the active
cells. area of interest to achieve the fastest and sharpest changes.
Simultaneous patch-clamp and fluorescence recordings.
Isolated myocytes were loaded by incubation with 10 ␮M 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 ␮M
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 ␮m (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 ␮M wort-
mannin. The pipette solution contained (in mM) 110 K as-
partate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05
EGTA (pH 7.2) with 200 ␮g/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 fluo-
rescence changes as indicated in Simultaneous patch-clamp
and fluorescence recordings.
Single-channel recordings. Single-channel currents were re- Fig. 1. Ca2⫹-activated K⫹ (BK) channels increase their activity
corded from inside-out membrane patches of isolated arterial ⬃106-fold during a (Ca2⫹ spark induced) BK transient current. A:
myocytes (9). The bathing solution contained (in mM) 140 KCl, left, representative whole cell record in symmetrical 140 mM K⫹
solutions showing that inward transient BK current (at ⫺40 mV)
10 HEPES (pH 7.2), 1 Mg2⫹, and 5 EGTA or N-(2-hydrohy- peak amplitude was ⫺148 ⫾ 4 pA and single-channel amplitude was
ethyl)ethylenediamine-N,N⬘,N⬘-triacetic acid (HEDTA) with ⫺8.5 ⫾ 0.5 pA (n ⫽ 6 cells). Right, section indicated by bar on left
different free Ca2⫹ concentrations (100 nM, 300 nM, 3 ␮M, 10 trace at an expanded time scale; *single-channel opening. B: left,
␮M, 30 ␮M, and 100 ␮M) adjusted with Ca2⫹ electrodes. The single BK channel activity recorded in whole cell configuration.
pipette solution contained 140 mM KCl, 10 mM HEPES (pH Right, single BK channel activity recorded in inside-out patches
7.2), 1 mM Mg2⫹, 5 mM HEDTA, and 10 ␮M Ca2⫹. The patches exposed to 100 nM Ca2⫹, which is close to the cytoplasmic Ca2⫹ at
were held at a steady potential of ⫺40 mV. Currents were rest. Holding potential (Vh) was ⫺40 mV, extracellular K⫹ concen-
filtered at 10 kHz and digitized at 40 kHz. The number of tration ([K⫹]o) was 140 mM. Arrows indicate closed state of the
channel. In the whole cell experiments, single BK channel activity
channels present in any given excised patch was estimated from
was measured after exposing the cell to 100 nM thapsigargin for 15
all-points histograms at depolarized voltages (greater than ⫹60 min to eliminate the contribution of Ca2⫹ release from the sarcoplas-
mV). mic reticulum (SR) to the BK channel. Open probability (Po) of BK
Chemicals. Fluo 3-AM, pluronic acid, and BAPTA-AM channels in inside-out patches was 1.16 ⫾ 0.26 ⫻ 10⫺6 (n ⫽ 16
were obtained from Molecular Probes (Eugene, OR). Dehy- patches) and activity (NPo, where N is total number of channels per
drosoyasaponin-1 was a gift from Merck Research Laborato- cell) of whole cell BK channels was ⫽ 3.1 ⫾ 0.1 ⫻ 10⫺3 (n ⫽ 5 cells).

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 LOCAL COMMUNICATION OF CA2⫹ SPARKS TO BK CHANNELS
This box size (4.8 ␮m2) was determined empirically to be the
best compromise between temporal and spatial precision of
Ca2⫹ sparks and the signal-to-noise ratio. Results are ex-
pressed as means ⫾ SE where applicable.
RESULTS
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) ob-
served in pressurized cerebral arteries. To increase the
single-channel and transient BK currents at ⫺40 mV,
the external K⫹ concentration was raised to symmet-
rical conditions (140 mM). Figure 1A illustrates the
whole cell current activity in these conditions, display-
ing the transient BK currents as downward deflections.
The mean transient BK current at ⫺40 mV in symmet-
rical 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 acti-
vates 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 chan-
nel 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 configura-
tion with single-channel activity recorded in inside-out
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C1771
excised patches. At ⫺40 mV and symmetrical K⫹, the
NPo of BK channels in the presence of 100 nM thapsi-
gargin (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 mem-
brane.
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 chan-
nels. 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
␮m2/cell surface; Ref. 20) of the cell membrane. Hence,
a Ca2⫹ spark would elevate Po of the nearby BK chan-
nels ⬃6 ⫻ 105-fold (i.e., 18 ⫼ 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 condi-
tions 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/␮m2, which is consistent with
the average number of channels observed in excised
patches of 2.3 ⫾ 0.5 channels/patch (n ⫽ 11 patches).
Fig. 2. Calcium sensitivity of BK chan-
nels at ⫺40 mV. Left, original records
of single BK channel activity of inside-
out patches exposed to 100 nM and
10 ␮M Ca2⫹. Vh ⫽ ⫺40 mV, [K⫹]o ⫽
140 mM. Arrows indicate closed level.
Right, single-channel Po vs. Ca2⫹ con-
centration plot. Experimental Po values
(F, n ⫽ 5–16 patches, total 29) were
fitted with a Hill equation Po ⫽ Po max/
[1 ⫹ (Kd/[Ca2⫹]) nH], where Po max is
maximum Po, Kd is dissociation con-
stant, [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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C1772 LOCAL COMMUNICATION OF CA2⫹ SPARKS TO BK CHANNELS

The pipette resistances ranged from 9 to 13 M⍀, which
allows an estimation of 1.6–1.2 ␮m2 of patch mem-
brane 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 ␮m2) 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 cere-
bral 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 chan-
nels was determined at physiological membrane poten-
tials (⫺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 con-
ductance of the BK channels under these conditions
was 231 pS.
Po of the BK channels at ⫺40 mV and resting intra-
cellular 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 ␮M, with a Hill coefficient of
2.9 ⫾ 0.05 and maximum Po of 0.79. This Ca2⫹ sensi-
tivity is consistent with the BK channels being assem-
bled with their ␤1-subunit, as we recently demon-
strated (3). Therefore, we decided to test further the
subunit composition of the BK channels under our
conditions. Indeed, BK channels appeared to have

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 ␮M 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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 LOCAL COMMUNICATION OF CA2⫹ SPARKS TO BK CHANNELS
functional ␤1-subunits because 100 nM DHS-1, which
activates BK channels through the ␤-subunit (17), in-
creased 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 ␮M to 4–30 ␮M. Thus the local
Ca2⫹ (4–30 ␮M) sensed by the BK channels during a
Ca2⫹ spark is far greater than registered (⬍0.5 ␮M) 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 ␮M) 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). Further-
more, 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 cur-
rents 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. How-
ever, the transient BK current characteristics were
unchanged after BAPTA, suggesting that local Ca2⫹
transients in the vicinity of BK channels are not af-
fected by this Ca2⫹ chelator. Figure 4 shows that av-
eraged transient BK currents remain almost identical
after disappearance of Ca2⫹ sparks with BAPTA appli-
cation [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.
DISCUSSION
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
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C1773
Fig. 4. BAPTA does not alter transient BK currents. Averaged tran-
sient 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 (␶) from the fit were as follows: control ⫽ 22.3 ⫾
1.7 ms; BAPTA ⫽ 21.9 ⫾ 1.1 ms.
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⫹ sen-
sitivity 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 mem-
brane potential of smooth muscle cells in pressurized
arteries, as supported by the depolarizing and con-
stricting 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 ampli-
tude 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 compari-
son 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 prepara-
tion ⬃6 ⫻ 105-fold, assuming that 1% of the BK chan-
nels are activated by a Ca2⫹ spark. Even assuming
that all BK channels in the cell membrane are centered
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C1774 LOCAL COMMUNICATION OF CA2⫹ SPARKS TO BK CHANNELS
over one spark site, BK channel Po would increase
⬃6 ⫻ 103-fold during a spark. To determine the in-
crease in Ca2⫹ required for a Ca2⫹ spark-evoked acti-
vation 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 ␮M to at least 4 ␮M 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 ␮M (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 ␮M 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 (sur-
face area ⬃1,300 ␮m2), corresponding to ⬃2–3 chan-
nels/␮m2, 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 ␮m2 (20) and homogeneous
channel distribution. These results suggest that max-
imal 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 commu-
nication of RyRs to BK channels. BK channels have a
low affinity for Ca2⫹ (Kd 19 ␮M) at physiological mem-
brane 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 ␮M) 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 ␮M
and 30 ␮M, respectively) probably reflects a dilution of
Ca2⫹ from the local subsarcolemmal volume experi-
enced by the BK channels during a transient current
into the scanning volume. Typically, our confocal scan-
ning volume is 2.2 ␮m ⫻ 2.2 ␮m ⫻ 3 ␮m, or 14.5 fl. The
difference in dilution factors can be explained by re-
ducing one of the three-dimensional micrometer di-
mensions to a range of 20–30 nm in agreement with
EM study distances for subsarcolemmal space (7, 16).
AJP-Cell Physiol • VOL
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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.
DHS-1 was kindly provided by Merck Research Laboratories
(Rahway, NJ).
This study was supported by grants from the National Institutes
of Health (HL-44455, HL-63722, and DK-53832), the National Sci-
ence 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 Labo-
ratory, 2150 Bleecker St., Utica, NY 13501
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