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INTRODUCTION |
Large conductance Ca2+-activated K+
(BKCa)1 channels
are ubiquitously distributed among tissues and are particularly
abundant in smooth muscle (1, 2). The activity of BKCa
channels is regulated by membrane potential, intracellular
Ca2+, and phosphorylation (3, 4). Although BKCa
channels are usually not involved in setting resting potential, they
play a key role as a negative feedback mechanism to limit
depolarization and contraction (5-7). Activation of BKCa
channels is increased by nitric oxide (NO) and atrial natriuretic
peptide, which hyperpolarize the membrane and increase the sensitivity
of BKCa channels to Ca2+ (8-11). Membrane
hyperpolarization closes voltage-dependent Ca2+
channels, reduces Ca2+ influx, and leads to a reduction in
intracellular Ca2+ concentration and relaxation (1). NO has
been reported to stimulate BKCa channels directly as well
as through stimulation of guanylate cyclase and the subsequent increase
in cGMP (12-15). In addition, activation of BKCa channels
plays an important role in NO-induced relaxation of smooth muscle
(16-20). cGMP activates cGMP-dependent protein kinase
(PKG), which phosphorylates various cytosolic and membrane proteins
that regulate smooth muscle tone either directly or indirectly (21,
22). Recent studies in native cells suggest that PKG activates
BKCa channels through phosphorylation of the channel (23).
These results are supported by biochemical studies of cloned
BKCa channels, which demonstrate PKG-induced
phosphorylation of the channel (24).
The primary sequence of BKCa has been determined using
molecular cloning techniques in Drosophila (25) and mammals
(26-28). These studies indicate that BKCa isoforms belong
to the voltage-gated K+ (KV) channel
superfamily. The primary sequence of the S1-S6 segment of
BKCa channels is homologous to the corresponding regions in KV channels. The long carboxyl terminus is the region of
Ca2+-sensing (29, 30), and cslo-
contains a
single high affinity phosphorylation site for PKG at Ser-1072 (3).
However additional putative PKG phosphorylation sites have been
identified in other splice variants (31). Expression of the
slo channel in Xenopus oocytes or mammalian cells
gives rise to voltage-gated, Ca2+-sensitive currents with
electrophysiological and pharmacological features similar to those of
native BKCa (32-34). However, although many studies of
native cells suggest that BKCa channel activity is also
modulated by various protein kinases (35-38), this property has been
difficult to reproduce in cloned channels. Two studies in which
slo channels have been expressed in either oocytes (27) or
Chinese hamster ovary cells (39) have reported that PKG was without
effect on slo channel activity. In contrast, Perez et al. (33) reported that an endogenous cAMP-dependent
protein kinase-like activity activated dslo-
channels
expressed in Xenopus oocytes. A recent study showed that
PKG-I
phosphorylated hslo channels reconstituted into
lipid bilayers but had no effect on channel activity in inside-out
patches expressed in Xenopus oocytes (24).
The purpose of this study was to examine PKG-induced modulation of
cloned BKCa channels and determine whether direct
phosphorylation of the channel was involved. The
-subunit of
cslo, a BKCa channel
-subunit cloned from
canine colon, was expressed in HEK293 cells, and currents were measured
using both the whole cell mode as well as cell-attached and detached
patches. Evidence was obtained suggesting that the activity of cloned
BKCa channels is enhanced by the NO/PKG pathway and that
stimulation is mediated by direct phosphorylation of Ser-1072 of
cslo-
by PKG.
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EXPERIMENTAL PROCEDURES |
Expression of cslo-
Channels--
The cDNA encoding the
-subunit of the BKCa channel (cslo-
) was
previously cloned from canine colonic smooth muscle using reverse
transcription and a polymerase chain reaction (GenBankTM accession
number U-41001). Northern blot analysis showed that the
cslo-
transcripts are expressed in the muscles of the
canine gastrointestinal tract and blood vessels (27). The
cslo-
construct was subcloned into the mammalian
expression vector pZeoSV (Invitrogen, CA).
The S1072A mutation of cslo-
was created by recombinant
mutagenesis (40). Briefly, linearized cslo-
plasmid was
modified and amplified simultaneously by PCR in two separate reactions. Two primer pairs were used for PCR. In the first amplification reaction, one-half of the plasmid was amplified using a forward primer
containing the S1072A mutation, spanning nucleotides 3194 to 3233 (5'-AGTCCTCCAGCAAGAAGAGCGCCTCCGTGCACTCCATCCC-3') and a reverse primer
complementary to the plasmid sequence
(5'-GAACGGCACTGGTCAACTTGGCCATGGTGGCCCTC-3'). The second half of the
reaction amplified the remaining half of the plasmid using the
reverse-mutating primer
(5'-AGTCCTCCAGCAAGAAGAGCGCCTCCGTGCACTCCATCCC-3') and the forward
plasmid-specific primer (5'-ATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCAC-3'). Both the mutating and plasmid primers were designed to contain a
24-base pair homology at their 5' ends, which generated an overlap
between the ends of the two PCR products. The homologous ends of the
PCR products undergo recombination in vivo following transformation of RecAEscherichia coli cells. PCR
amplification was performed in 50-µl reactions containing 1× PCR
buffer (50 mM KCl, 10 mM Tris-Cl, 1.5 mM MgCl2 (pH 8.3); Invitrogen), 200 µM each dNTP (Invitrogen), 25 pmol of primer, 2 ng of
plasmid template, 10% Me2SO, and 2. 5 units of
Taq polymerase (Promega, Madison, WI). Reactants underwent
an initial denaturation (94 °C × 1 min), 30 amplification
cycles (94 °C × 30 s, 50 °C × 30 s, and
72 °C × 3 min) and a final extension of 72 °C × 10 min. PCR products were gel-purified, and 2.5 µl of each PCR reaction were mixed and transformed directly into 50 µl of Max
CompetentTM DH5
E. coli (Life Technologies,
Inc) and selected on low salt LB zeocin plates (25 µg/ml). Plasmid
DNA was prepared from overnight cultures using the QIAprep Miniprep kit
(Qiagen, CA). Plasmid DNA of the correct size was sequenced using the
ABI Prism cycle sequencing kit (Perkin-Elmer, CA) and analyzed on a
Perkin-Elmer 310 Genetic Analyzer.
HEK293 cells were obtained from ATCC (cell line number CRL-1573,
Manassas, VA) and maintained in modified RPMI medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated horse serum
(Summit Biotechnology, Fort Collins, CO) and 1% glutamine (Life
Technologies, Inc.) in a humidified 5% CO2 incubator at 37 °C. Cells were subcultured twice a week by treatment with
trypsin-EDTA (Life Technologies, Inc.). The cslo-
DNA was
transfected into HEK cells by electroporation. Electroporation was
performed as follows. After harvesting the HEK cells by trypsin-EDTA,
the cells were washed twice with phosphate buffer solution and
resuspended in ice-cold phosphate buffer solution at a density of
5 × 106 cells/ml in the cuvette for electroporation.
Each cuvette was supplemented with appropriate combinations of CD8 (a
lymphocyte cell-surface antigen) in the
H3-CD8 plasmid construct as
a marker for transfection (4 µg), cslo-
, or S1072A
cslo-
in the pZeoSV vector (20 µg). After a 10-min
incubation on ice, electroporation was done by applying a 330-V pulse
using a pulse generator (Electroporator II, invitrogen, CA). HEK cells
expressing cslo-
were subcultured on glass coverslips for
electrophysiological recording. Current recording was performed 1 to 4 days after the electroporation procedure. Transfected cells were
identified by their binding to CD8-coated beads (Dyna-beads M-450 CD8;
Great Neck, NY) (41).
Electrophysiological Recording--
The patch-clamp technique
was used to measure membrane currents in whole cell and single cell
configuration. Patch pipettes were made from borosilicate grass
capillaries pulled with a three-stage micropipette puller (P.80/PC,
Sutter, CA) and heat-polished with a microforge (MF-83, Narishige,
Japan). The pipettes had tip resistances of 2 to 5 megaohm for whole
cell recordings and 8 to 10 megaohm for single-channel recordings.
Coverslips containing HEK cells were placed in a recording chamber
(volume 1.0 ml) mounted on the stage of an Olympus inverted microscope
and superfused with bath solution at a rate of 1.0 ml/min. Standard
gigaohm seal patch-clamp recording techniques were used to measure the
currents of whole cell, cell-attached, and excised inside-out
configurations. An Axopatch 200A patch-clamp amplifier (Axon
Instruments, CA) was used to measure whole cell and single-channel
recordings. Capacitance and series resistance compensation were
performed. The output signals were filtered at 1 kHz with an 8-pole
Bessel filter, digitized at a sampling rate of 3 kHz, and stored on the
hard disk of a computer for off-line analysis. Data acquisition and
analysis were performed with pClamp software (version 6.0.4., Axon
Instruments). Channel open probability (NPo) in patches was determined
from recordings of more than 3 min by fitting the sum of Gaussian
functions to an all-points histogram plot at each potential. Single
channel conductance was determined from all-point amplitude histograms using Fechan and Pstat programs (Axon Instruments).
Solutions and Drugs--
For whole cell recordings of HEK cells,
the bath solution contained 135 mM NaCl, 5 mM
KCl, 1.8 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, 10 mM glucose
(pH 7.4), and the pipette solution contained 50 mM KCl, 70 mM L-aspartic acid monopotassium, 8 mM NaCl,
0.826 mM CaCl2, 1 mM
MgCl2, 2 mM MgATP, 0.3 mM NaGTP, 10 mM HEPES, 1 mM
N-(2-hydroxyethyl)ethylenediaminetriacetic acid (pH
7.2). For single channel recordings in the inside-out mode, the bath
solution contained 140 mM KCl, 1 mM
MgCl2, 10 mM HEPES, 1 mM
N-(2-hydroxyethyl)ethylenediaminetriacetic acid (pH
7.2). The concentration of free Ca2+ in the bath solution
was changed from 10
8 M to 10
4
M to determine the Ca2+ sensitivity of
BKCa channels. Ca2+ concentration was estimated
by a computer program (42), and the appropriate amounts of
CaCl2 were added. The ionized Ca2+
concentration was confirmed using a Ca2+-sensitive
electrode. The pipette solution contained 140 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2,
10 mM HEPES (pH 7.4). For single channel recordings in the
cell-attached mode, the bath solution contained 140 mM KCl,
1.8 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, 10 mM glucose (pH 7.4), and the pipette solution contained 140 mM KCl,
1.8 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES (pH 7.4). All patch-clamp
experiments were performed at room temperature (22 °C). PKG-I
and
KT5823 were purchased from Calbiochem. Diethylenetriamine/nitric oxide (DETA/NO) was from RBI (Natick, MA) and other drugs were from Sigma.
Statistics--
Data are expressed as mean ±S.E. Statistical
significance was determined using Student's t test for
paired observations.
 |
RESULTS |
Characterization of cslo-
Currents Expressed in HEK
Cells--
Membrane currents of nontransfected native HEK cells were
measured in whole cell voltage clamp configuration. Depolarizing steps
triggered an outward current in native HEK cells. The current showed
little or no inactivation during the pulse. The steady-state current at
+50 mV was 0.29 ± 0.04 nA (n = 10). This current
was suppressed by the K+ channel blocker 4-aminopyridine
(0.09 ± 0.03 nA at 1 mM; n = 4) but
was unaffected by the specific BKCa channel inhibitor
iberiotoxin (IBTX 100 nM; n = 3).
Expression of CD8 DNA (marker plasmid) in HEK cells had no effect on
the current (n = 6).
The amplitude of outward current in HEK cells expressing
cslo-
was considerably larger than in native HEK cells.
Mean current amplitude obtained under steady-state conditions at +50 mV
in cells expressing cslo-
was 4.56 ± 0.42 nA
(n = 10). Representative whole cell currents obtained
in transfected and native cells are shown in Fig.
1A. The current-voltage
relationships of transfected and native cells are shown in Fig.
1B. Membrane conductance plotted as a function of voltage in
HEK cells expressing cslo-
is shown in Fig.
1C. Discernible conductance was apparent at potentials positive to
40 mV, and maximum conductance was reached at
approximately +60 mV. The V0.5 was +20.3 mV, and
the slope was 15.1.

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Fig. 1.
Characterization of the
cslo- current expressed in
HEK293 cells. A, representative currents
cslo- transfected and nontransfected HEK cells. Currents
were recorded in whole cell voltage mode. The membrane potential of the
cell was held at 70 mV and depolarized with a series of 200-ms step
pulses from 50 mV to +80 mV with 10-mV increments at 15-s intervals.
B, summary of the current-voltage relationship of
cslo- ( , n = 10) and native ( ,
n = 10) currents. Values are means ±S.E. C,
average relative membrane conductance plotted as a function of voltage
in HEK cells expressing cslo- .
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Effect of IBTX on Whole Cell cslo-
Currents--
BKCa channels are specifically blocked by
IBTX purified from venom of the scorpion (43). Experiments were
therefore undertaken to determine whether outward currents recorded in
cslo-
cells were blocked by IBTX. The addition of IBTX
(100 nM) to the bathing solution produced a marked
reduction in outward current at all voltages tested as seen in Fig.
2, A and B. In 5 cells expressing cslo-
, IBTX significantly
(p < 0.01) reduced current amplitude by greater than
90% (Fig. 2C). The current remaining in the presence of
IBTX was not different from that of native currents recorded in HEK
cells (p > 0.05).

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Fig. 2.
Effect of IBTX on
cslo- current in HEK293
cells. A, representative traces of
cslo- current expressed in HEK cells before and after
treatment with IBTX (100 nM). Currents were recorded in
whole cell voltage mode. The membrane potential of the cell was held at
70 mV and depolarized with a series of 200-ms step pulses from 50
mV to +80 mV with 10-mV increments at 15-s intervals. B,
current-voltage relationship of cslo- currents before
( ) and after ( ) treatment witn IBTX. C, average
cslo- currents before and after treatment with IBTX
(n = 5). Data shows average peak currents at voltage
clamp steps from 70 to +50 mV at 30-s intervals (n = 5). Values are means ± S.E. *, p < 0.01 compared
with control.
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Single Channel Recordings of cslo-
Current--
To further
examine the properties of outward currents in cells transfected with
cslo-
, single channel activity was recorded in inside-out
patches in a symmetrical KCl solution (140 mM KCl). Channel
openings could be detected at membrane potentials from
60 mV to +60
mV (Fig. 3A). The activity of
these channels was voltage-dependent, i.e. NPo
increased from 0.064 ± 0.030 at
60 mV to 0.942 ± 0.192 at
+60 mV (n = 5). The current voltage relationship of
channels was linear between
60 mV to +60 mV (Fig. 3B) with a mean slope conductance of 253 ± 9.7 pS (n = 8)
and a reversal potential of
0 mV. When the Ca2+
concentration on the cytosolic side of the membrane patch was increased, ranging from 10
8 to 10
4
M, channel activity increased dramatically (holding
potential = +40 mV, see Fig. 3C). The relationship between
Ca2+ concentration and Po of the cslo-
channel at +40 mV is shown in Fig. 3D.

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Fig. 3.
Single channel recordings of
cslo- current in inside-out
patches of HEK cells. A, representative recording of
cslo- channel recorded from an inside-out excised
membrane patch at membrane potentials ranging from 60 to +60 mV.
Cytosolic Ca2+ concentration was maintained at
10 5 M. C indicates closed.
B, current-voltage relationship of cslo-
channel current from inside-out patches of HEK cells using symmetrical
140 mM KCl solution. C, a representative
recording of cslo- channel recorded from an inside-out
excised membrane patch at cytosolic Ca2+ concentration
ranging from 10 8 to 10 5 M. The
membrane potential was clamped at +40 mV. D, open-state
probability versus cytosolic Ca2+ concentration
at membrane potential +40 mV. The line is the Boltzmann fit
to the data.
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Effect of SNP on Whole Cell cslo-
Currents--
The NO donor
sodium nitroprusside (SNP) increased the activity of native
BKCa channels in smooth muscle (13-18). Experiments were
therefore undertaken to determine the action of SNP on cells expressing
cslo-
using the whole cell patch-clamp mode. The addition of SNP (10
4 M) to the bathing solution led to
a significant increase in whole cell outward current (Fig.
4, A and B). In 8 cells tested, SNP significantly (p < 0.01) increased
outward current amplitude 2.3-fold at + 50 mV (Fig. 4C).
When SNP was removed from the bathing solution, current amplitude
returned to the prestimulus amplitude. Cytosolic Ca2+
concentration was buffered at 10
5 M with
N-(2-hydroxyethyl)ethylenediaminetriacetic acid in these experiments.

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Fig. 4.
Effect of SNP on
cslo- current expressed in
HEK cells. A, representative traces of
cslo- current expressed in HEK cells before and after
application of SNP (10 4 M). Currents were
recorded in whole cell voltage mode. The membrane potential of the cell
was held at 70 mV and depolarized with a series of 200-ms step pulses
from 50 mV to +80 mV with 10-mV increments at 15-s intervals.
B, current-voltage relationship of cslo-
currents before ( ) and after ( ) application of SNP. C,
average cslo- currents of control, SNP and after washout
(WO) of SNP. Data shows average peak currents at voltage
clamp steps from 70 to +50 mV at 30-s intervals. Values are means
±S.E. (n = 8). *, p < 0.01 compared
with control.
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Effect of NO Donors on cslo-
Channel Activity--
Because SNP
significantly enhances whole cell cslo-
current
amplitude, additional experiments were performed to determine whether
changes also occur in single channel activity recorded in cell-attached
patches. The addition of SNP (10
4 M) to the
bathing solution led to a marked increase in cslo-
channel activity, which returned to near control levels 10-15 min
after wash-out (Fig. 5, A and
C). In Fig. 5B, the time course of changes in
cslo-
activity after the addition of SNP is shown. SNP
increased NPo from 0.092 to 0.656 in this cell. In 8 cells, SNP
significantly (p < 0.01) increased NPo 3.3-fold
(holding potential = +40 mV; see Fig. 5C) but had no effect
on unitary current amplitude (control 253 ± 11.3, SNP 262 ± 13.1, wash-out 258 ± 8.8 pS, n = 8, p > 0.05). SNP had no significant effect on
cslo-
activity in the presence of the PKG-specific
inhibitor KT5823 (10
6 M) in cell-attached
patches (Fig. 5D). SNP had no effect on cslo-
channels in inside-out patches (NPo; control 0.318 ± 0.123, SNP 0.370 ± 0.184, n = 8, p > 0.05).
Additional experiments were performed with the NO donor, DETA/NO (300 µM), because there is evidence that different donors may
have differing effects upon potassium channels (44). In contrast to
SNP, DETA/NO increased cslo-
channel activity in the
absence (NPo: control, 0.036 ± 0.023; DETA/NO, 0.072 ± 0.029, n = 6, p < 0.05) and in the
presence (NPo: control, 0.152 ± 0.121, DETA/NO, 0.249 ± 0.117, n = 8, p < 0.05) of KT5823 in
cell-attached patches.

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Fig. 5.
Effect of SNP on
cslo- channel activity in
cell-attached patches. A, representative recording of
cslo- channel under control conditions, after application
of SNP to the bath solution and after wash-out in cell-attached patch
at a membrane potential of +40 mV. C indicates closed.
B, time course of the activity of cslo-
channel (NPo). SNP (10 4 M) was
added to the bath as indicated in the figure. NPo of
cslo- channel activity was calculated every 1 min.
C, summary of the effect of SNP on NPo of the
cslo- currents (n = 8). Values are means
±S.E. *, p < 0.01 compared with control.
D, summary of the effect of SNP on NPo of the
cslo- currents in the presence of PKG inhibitor KT5823
(10 6 M). Values are means ±S.E.
WO, wash-out.
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Effect of PKG-I
on cslo-
Channel Activity--
Our
experiments with SNP suggest that cslo-
channels are
activated by the cGMP/PKG pathway. To provide more direct evidence for
PKG-induced modulation of cslo-
, we examined the effects of PKG-I
on cslo-
channel activity in inside-out
patches. Application of PKG-I
to the cytosolic side of the membrane
did not modify cslo-
currents (Fig.
6, B and C). The
effect of ATP (1 mM) on cslo-
channel was
variable (11 of 21 increased, 7 of 21 decreased, 3 of 21 showed no
change), so that overall, no significant change was observed in the
pooled data (Fig. 6C). ATP (1 mM) plus cGMP (0.1 mM) also had no effect on cslo-
current (Fig.
6, A and C). However, when PKG-I
was added to
the bath solution in the presence of ATP (1 mM) plus cGMP
(0.1 mM), cslo-
channel activity
significantly (p < 0.05) increased (Fig. 6,
A and C). Wash out of PKG led to a return of
channel activity to the control level (Fig. 6C). PKG had no
effect on the unitary conductance of cslo-
channels
(control 253 ± 9.7; PKG + ATP + cGMP, 252 ± 8.3; wash-out,
248 ± 8.6 pS, n = 10, p > 0.05).

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Fig. 6.
Effect of PKG-I on
cslo- channel activity in
inside-out patches. A, representative recording of
cslo- channel recorded from an inside-out excised
membrane patch at membrane potential of +40 mV. PKG increased the
cslo- channel activity in the presence of ATP (1 mM) and cGMP (0.1 mM). Cytosolic
Ca2+ concentration was maintained at 10 6
M. C indicates closed. B,
representative recording of cslo- channel recorded from
an inside-out excised membrane patch at membrane potential of +40 mV.
PKG alone did not change the cslo- channel activity.
C, summary of the effect of ATP (n = 21),
ATP + cGMP (n = 10), PKG (5 kilounits/ml,
n = 4), and ATP + cGMP + PKG (n = 10)
on cslo- current. The effect was compared with control
conditions (normalized to 1). Cytosolic Ca2+ concentration
was 10 6 M, and membrane potential was clamped
at +40 mV. Values are means ± S.E. *, p < 0.05 compared with control. WO, wash-out.
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Effect of PKG-I
on Mutated cslo-
Channel
Activity--
Activation of PKG may indirectly activate
BKCa channels through other kinases or related proteins. To
clarify this point, we made a point mutation on the cslo-
channel. The amino acid sequence of cslo-
has only one
optimal consensus sequence for PKG phosphorylation at Ser-1072. Thus a
point mutation of cslo-
was created in which Ser-1072 was
replaced by Ala. The general characteristics of mutated
cslo-
channels is shown in Fig.
7, A-C. The
mutated cslo-
channel was activated by membrane
depolarization, and its conductance (247.2 ± 13 pS,
n = 10) was not different (p > 0.05)
from that of wild-type cslo-
channels. Increases in Ca2+ concentration at the cytosolic surface activated the
channel in a concentration-dependent manner. These
characteristics of the mutated cslo-
channel were
comparable with wild-type cslo-
channels. However,
application of PKG-I
in the presence of ATP (1 mM) plus
cGMP (0.1 mM) was without effect on mutated
cslo-
channel activity (n = 10; Fig. 7,
D and E). The single channel conductance of the
cslo-
channel was also unchanged by PKG
(n = 10; Fig. 7F). SNP also had no effect on
mutated cslo-
channel activity in cell-attached patches
(NPo: control, 0.049 ± 0.042; SNP, 0.054 ± 0.051, n = 6, p > 0.05).

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Fig. 7.
Effect of PKG-I on
mutated cslo- channel in
inside-out patches. A, representative recording of
cslo- channel recorded from an inside-out excised
membrane patch at membrane potentials ranging from 60 to +60 mV.
Cytosolic Ca2+ concentration was maintained at
10 5 M. C indicates closed.
B, current-voltage relationship of cslo-
channel current from inside-out patches of using symmetrical 140 mM KCl solution. C, representative recording of
cslo- channel recorded from an inside-out excised
membrane patch at cytosolic Ca2+ concentration ranging from
10 8 to 10 5 M. Membrane
potential was clamped at +40 mV. D, effect of PKG on mutated
cslo- current expressed in HEK cells in inside-out
patches recorded at cytosolic Ca2+ concentration
10 6 M, and membrane potential +40 mV.
E, summary of the effect of PKG on mutated
cslo- channel. Values are means ±S.E. (n = 10). F, effect of PKG on conductance of the mutated
cslo- current. Values are means ±S.E. WO,
wash-out.
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|
 |
DISCUSSION |
This study provides direct evidence that cloned BKCa
channels expressed in HEK cells can be activated by
cGMP-dependent protein kinase. Activation required ATP and
cGMP, suggesting that PKG stimulates BKCa channels through
phosphorylation. Mutation of cslo-
at Ser-1072, the only
optimal consensus phosphorylation site for PKG on cslo-
,
abolished the stimulatory effect of PKG on cslo-
channels. These results indicate that PKG activates cslo-
channels through direct phosphorylation at Ser-1072.
Native outward currents in HEK cells were small and inhibited by
4-aminopyridine but not by IBTX, suggesting that these currents were
largely because of delayed rectifier-type K+ channels with
little contribution from BKCa channels. This result agrees
with another recent study of these cells (45). Several kinds of
Cl
channels also contribute to native HEK cell currents
(46). However, under the conditions of our experiments, both delayed rectifier and chloride currents were minimal compared with currents recorded in cells transfected with cslo-
. Whole cell
outward currents recorded in transfected cells were blocked by IBTX and exhibited a voltage dependence comparable with native BKCa
channel currents. cslo-
channel activity recorded in
single channel mode was enhanced by membrane depolarization and by
increases in Ca2+ concentration at the cytosolic surface.
Half-maximal activation of cslo-
for Ca2+ at
+40 mV was 10
5 M. This Ca2+
sensitivity is comparable with the sensitivity observed by others when
only slo-
is expressed (3, 27, 39) but is 10 to 20 times
less than the Ca2+ sensitivity observed when
slo-
is co-expressed with the
-subunit (27) or when
native BKCa channel currents are recorded (47). In
addition, the single channel conductance of cslo-
(253 pS) was similar to that of native BKCa channels. These
results indicate that cslo-
current expressed in HEK
cells exhibits functional features of native BKCa channels
in smooth muscle cells.
In this study, SNP activated whole cell BKCa channel
currents and increased NPo of cslo-
channels in
cell-attached patches without a change in single channel conductance.
There are three possible mechanisms by which SNP could activate the
cslo-
channel. First, NO derived from SNP may directly
activate the cslo-
channel (12, 39). Second, NO may
activate PKG, which then leads to direct phosphorylation of the
cslo-
channel. Third, activation of PKG by NO may lead to
stimulation of a phosphatase (possibly phosphoprotein phosphatase 2A),
which dephosphorylates the channel (39, 48). Our results suggest that
the mechanism involved in activation of cslo is dependent
upon the NO donor used. In the case of SNP, activation of
cslo appears to be because of a PKG-dependent
mechanism without an appreciable contribution from the direct
activation of channels by NO. This conclusion was reached because 1)
SNP was without affect when applied to the cytosolic surface of the
membrane in inside-out patches, 2) the stimulatory effect of SNP was
blocked by the PKG inhibitor KT5823 in cell-attached patch recordings,
and 3) SNP was without effect on mutated cslo-
channel activity.
Further support for this conclusion comes from studies by other
laboratories suggesting that the cGMP-PKG pathway is functional in HEK
cells (49-52). In contrast to SNP, the effect of DETA/NO was not
blocked by the PKG inhibitor KT 5823, suggesting that this NO-donor may
have direct effects upon the cslo-
channel. This
conclusion is in good agreement with studies by Zhou et al. (39) who recently reported that hslo-
channels expressed
in Chinese hamster ovary cells are directly activated by NO derived from diethylamine/NO via S-nitrosylation. The differences
between SNP and DETA/NO may be because of the differences in the redox state of NO generated by these NO-donors as suggested by others (44).
Additional experiments were undertaken to distinguish between direct
PKG-mediated phosphorylation of the channel versus more indirect effects of PKG. Exposure of the cytosolic surface of inside-out patches to PKG-I
in the presence of ATP plus cGMP increased NPo of cslo-
channels as previously reported
for native BK channels (10, 23). This suggests that the action of PKG involves a phosphorylation event. In a previous study by our laboratory using cslo-
(27) and a study using hslo-
channels (24) expressed in oocytes, PKG did not activate slo
channels, although, interestingly, PKG-induced regulation was observed
when the hslo-channels were reconstituted in a lipid bilayer
(24).
In our studies, neither ATP alone nor ATP plus cGMP increased the
activity of channels, suggesting that significant quantities of PKG are
not bound to the cytoplasmic surface of the isolated patch. This result
differs from a study by Fujino et al. (14), who reported
that cGMP plus ATP increased BKCa channel activity in
isolated patches of porcine coronary artery myocytes. However, it is in
agreement with studies of vascular (7, 10) and tracheal (36) smooth
muscles in which cGMP plus ATP were without affect in isolated patches.
These disparate results suggest that mammalian expression systems and
different smooth muscle preparations may contain differing amounts of
bound PKG.
PKG has been reported to phosphorylate many proteins that regulate
smooth muscle tone (21, 22), and it is possible that PKG could regulate
BKCa channel activity indirectly by phosphorylating a
protein that then regulates channel activity. A particularly intriguing
target in this regard are phosphatases that could regulate channel
activity through dephosphorylation (39, 48, 53). To investigate whether
PKG directly acts on the channel or, alternatively, requires some
intermediary protein, we mutated the single optimal consensus sequence
for PKG phosphorylation in the carboxyl-terminal region of the
cslo-
channel (i.e. KKSS at 1069-1072).
Mutation of Ser-1072 abolished PKG-induced modulation of channel
activity but did not change the electrophysiological characteristics of the channel. The mutated cslo-
channels exhibited all of
the features described for wild-type channels, i.e. they
were activated by membrane depolarization and by elevation of
Ca2+ on the cytosolic side of the membrane and had the same
single channel conductance as the wild-type cslo-
channel. Thus, the lack of effect of PKG on mutated channels could not
be attributed to general channel dysfunction. These mutation
experiments suggest that PKG enhances channel activity through direct
phosphorylation of the channel rather than requiring the actions of a
phosphatase. In studies of reconstituted hslo-
channels,
it was also concluded that activation of channels by PKG involved
phosphorylation rather than dephosphorylation (24). Furthermore, the
cloned human slo channel hslo-
has the same
optimal consensus phosphorylation site as cslo-
, and this
channel has been reported to be directly phosphorylated by PKG-I
(24).
In summary, we have found that the cslo-
channel activity
recorded in whole cell and single channel configuration is increased by
the NO donor SNP, presumably through activation of PKG. Direct application of PKG-I
also activated cslo-
channels but
only in the presence of ATP and cGMP. A point mutation at the only optimal consensus phosphorylation site for PKG on cslo-
abolished the stimulatory effects of PKG. From these results we
conclude that PKG activates cslo-
channel by direct
phosphorylation at serine 1072.