From the Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of Calgary, Calgary, Alberta, Canada T2N 4N1
Received for publication, June 29, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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The activation of large conductance,
calcium-sensitive K+ (BKCa) channels by
the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be
an important cellular mechanism contributing to the relaxation of
smooth muscle. In HEK 293 cells transiently transfected with
BKCa channels, we observed that the NO donor sodium
nitroprusside and the membrane-permeable analog of cGMP, dibutyryl
cGMP, were both able to enhance BKCa channel activity 4-5-fold in cell-attached membrane patches. This enhancement
correlated with an endogenous cGMP-dependent protein kinase
activity and the presence of the The elevation of intracellular
cGMP1 in response to
endothelium-derived nitric oxide (NO) or clinically prescribed
nitrovasodilators, such as nitroglycerin and sodium nitroprusside, is
known to play an important role in the hypotensive actions of these
agents (1, 2). Similarly, elevation of cGMP by the phosphodiesterase
inhibitor sildenafil (Viagra) (3) appears to underlie the smooth
muscle-relaxing and anti-impotence effects of this drug. Although the
exact mechanism(s) by which elevated cGMP causes smooth muscle
relaxation has not been clearly defined, cGMP is known to influence a
number of cellular processes (4), such as the levels of cytosolic free
calcium, myosin light chain dephosphorylation (5), and the activity of
voltage-dependent, L-type calcium channels
(6).
In both vascular and nonvascular smooth muscle, activation of large
conductance, calcium-sensitive K+ channels (maxi-K or
BKCa channels) is reported to occur in response to
endogenous NO or exogenous NO donors (7-12). In many cases, addition
of exogenous cGMP appears to mimic the effects of NO and NO donors on
BKCa channel activation (8, 10, 12-15), suggesting that
cGMP acts downstream of NO. Physiologically, BKCa channels appear to be important cellular effectors for the vasodilatory actions
of the NO/cGMP signaling pathway because blockade of BKCa channels can interfere with the relaxation-promoting effects of NO
(16-18).
A major intracellular target for cGMP in smooth muscle is the type I
cGMP-dependent protein kinase (cGKI), a serine/threonine protein kinase that is widely expressed in mammalian tissues (19, 20).
This kinase is encoded by a single gene, which gives rise to two
alternatively spliced isoforms, To examine the role played by cGKI Rabbit polyclonal antibodies against the mouse BKCa
channel Construction and Transfection of cDNA Plasmids--
The
cDNAs encoding the mouse BKCa channel (mSlo)
Transient transfection of HEK 293 cells (50-80% confluence) was
carried out in 35-mm tissue culture dishes using the lipofection technique. Briefly, 6-8 µl of LipofectAMINE (Life Technologies, Inc.) was mixed together with ~1.5 µg of plasmid cDNA in 1 ml of serum-free culture medium (Dulbecco's modified Eagle's medium supplemented with L-glutamine and 4.5 g/liter
D-glucose) and placed on cells for 4-6 h in a humidified
incubator containing 5% CO2 at 37 °C. DNA-containing
medium was then aspirated and replaced with serum-containing medium.
The following day, cells were detached from the dish by treatment with
0.05% (w/v) trypsin/0.5 mM EDTA and replated onto sterile
glass coverslips. Electrophysiological recordings were performed on
days 3-5 after transfection (day 1). For biochemical studies, cells
detached from 35-mm dishes were replated onto 100-mm dishes to prevent
overgrowth. These cells were then harvested on days 3-4 after transfection.
Electrophysiology--
Macroscopic currents were recorded at
35 ± 0.5 °C from cell-attached membrane patches of HEK 293 cells using an Axopatch 200B patch clamp amplifier and pClamp 6.03 software. BKCa channel currents were activated by voltage
clamp pulses delivered from a holding potential of 0 mV to membrane
potentials ranging from
Transfected HEK 293 cells seeded on coverslips were placed in a
temperature-controlled recording chamber on the stage of a Nikon
Eclipse TE300 inverted microscope. Individual cells expressing BKCa channels were then identified visually by
co-expression of the marker protein green fluorescent protein under
epifluorescence using 480 nm excitation and 510 nm emission filters.
Western Blotting--
Transfected cells were detached on day 3 by a brief incubation with sterile phosphate-buffered saline containing
0.05% trypsin/0.5 mM EDTA, centrifuged in 15-ml culture
tubes at ~100 × g for 5 min, and stored at
In Vitro cGMP-dependent Protein Kinase
Assay--
This assay was performed as described previously by Wolfe
et al. (32), with minor modifications. Transiently
transfected HEK 293 cells growing on a 100-mm culture dish (50-80%
confluence) were harvested in 1 ml of lysis buffer, sonicated for 5-10
s on ice, and then centrifuged for 10 min at 4 °C at 15,000 rpm
using a microcentrifuge. The supernatants were removed and kept on ice.
The assay of cGMP-dependent protein kinase activity was
performed at 30 °C in a final reaction volume of 60 µl containing (final concentrations) 20 mM Tris-HCl, pH 7.5, 20 mM magnesium acetate, 2 mM DTT, 20 µM cGMP, 0.1 mM isobutylmethylxanthine, 160 µM synthetic peptide substrate (RKRSRAE) (33), and 200 µM Na2-ATP (radiospecific activity, 150-300
cpm/pmol). After the addition of 20-50 µg of soluble cell lysate to
start the reaction, incubation was carried out for 10 min and stopped
by spotting a 30-µl aliquot of the reaction mixture onto a P-81
phosphocellulose disc (Whatman). Discs were then placed immediately in
0.5% (v/v) phosphoric acid, washed for 3 × 5 min in 500 ml of
0.5% phosphoric acid, rinsed briefly with acetone, and dried.
Radioactivity bound to the paper discs was quantified by Cerenkov
counting using a Beckman LS6000 liquid scintillation counter.
In Vitro Phosphorylation Assay--
The BKCa
The phosphorylation reaction was carried out for 20 min at 30 °C in
a final volume of 40 µl containing (final concentrations) 20 mM Tris-HCl, pH 7.5, 20 mM magnesium acetate, 2 mM DTT, 0.1 mM isobutylmethylxanthine, 20 µM cGMP, 5,000 units of recombinant bovine type I Co-immunoprecipitation of cGKI Statistical Analysis--
A one-way analysis of variance was
used to determine statistical significance among groups of values; mean
values were considered to be significantly different at a level of
p < 0.05.
Characterization of Transfected Type I cGMP-dependent
Protein Kinase--
To examine the functional importance of cGKI
To prepare a catalytically inactive mutant of human cGKI
In the presence of cGMP, substrate phosphorylation was increased
~2-fold above background levels observed in the absence of cGMP, and
this activity was comparable to that present in mock-transfected cells.
However, cGMP-dependent substrate phosphorylation was
strongly enhanced (~8-fold) in the lysate from cells co-transfected
with BKCa channels and wild-type cGKI Co-expression of BKCa Channels and cGKI
Using an anti-cGKI antibody, we observed modest expression of an
endogenous immunoreactive protein with a molecular mass of ~75
kDa, corresponding to the Effects of the NO/cGMP Signaling Pathway on BKCaChannel Activity--
The functional importance of cGKI
In the absence of SNP, a modest level of BKCa channel
activity was observed, the magnitude of which varied from cell to cell. However, after ~4 min of exposure to SNP, we observed a large increase in the amplitude of BKCa channel macroscopic
current; this increase typically peaked during 2-6 min of exposure and was reversible over several minutes upon washout of SNP from the bath
(Fig. 3C). SNP produced an average increase of ~4-fold in current magnitude compared with control, as quantified in Fig. 3C. This observation is thus consistent with recent findings
reported by Fukao et al. (15) using whole cell voltage clamp
methodologies that SNP could augment the activity of canine colonic
BKCa channels expressed in HEK 293 cells. Using our
dominant negative strategy, we observed that in cells co-transfected
with cDNAs encoding the BKCa channel and the
catalytically dead form of cGKI
Although SNP is known to elevate intracellular cGMP in
several cell types via the NO-dependent activation of
guanylyl cyclase (39), it is also capable of generating chemical
products (i.e. peroxynitrites, S-nitrosothiols,
and ferrocyanates) that may directly influence BKCa channel
activity (40, 41). To address whether SNP may initiate other cellular
mechanisms not dependent upon activation of cGKI, we examined the
effect of dibutyryl cGMP, a membrane-permeable form of cGMP, on
BKCa channels expressed alone or in the presence of
co-transfected wild-type cGKI
Dibutyryl cGMP (1 mM) was back-filled in the recording
pipette and allowed to diffuse to the membrane patch at the pipette tip
after formation of a high resistance, gigaohm seal. Over a 10-min
recording period, we observed that dibutyryl cGMP (db-cGMP) produced an
increase (4-5-fold above control) in BKCa channel activity
that was qualitatively similar to that seen with exposure to SNP
(compare Fig. 4, A and C with Fig. 3,
A and C). In the absence of db-cGMP, no
significant change in BKCa channel activity was observed
over the same time period (Fig. 4C). These findings are thus
in agreement with those recently reported by Alioua et al.
(14) for the activation of human BKCa channels expressed in
Xenopus oocytes by db-cGMP. Most interestingly, however, in cells co-transfected with BKCa channels and dead cGKI Direct Phosphorylation of BKCa Channels by Purified
cGKI
In the lane containing immunoprecipitated BKCa channels
plus purified cGKI Interaction of cGKI
Using this co-immunoprecipitation strategy, we found a small
amount of expressed cGKI
When BKCa channel immunoprecipitates were reprobed for the
presence of channel protein, we observed that similar amounts were recovered from cells transfected under each condition (Fig.
6B). This finding indicates that unequal immunoprecipitation
of BKCa channel protein cannot account for the difference
observed in the levels of co-immunoprecipitated cGKI
This important association between BKCa channels and
cGKI
Finally, we believe that the difference in co-immunoprecipitation data
shown in Fig. 6, A and D, may reflect the
relative expression of cGKI In this study, we have examined the importance of the cGKI in the
regulation of a BKCa channel by the NO/cGMP signaling
pathway in intact cells. To do so, we created a catalytically inactive mutant of the Having established both the activity and expression of endogenous
cGMP-dependent protein kinase in our HEK 293 cells, we
examined whether stimulation of this intrinsic pathway by the NO donor SNP or membrane-permeable db-cGMP could result in altered
BKCa channel activity. Using the cell-attached recording
mode of the patch clamp technique (to keep the intracellular milieu
intact), we observed that exposure of cells to either SNP (Fig.
3A) or db-cGMP (Fig. 4A) significantly increased
the magnitude of macroscopic BKCa channel currents, in
agreement with recent observations of other investigators (14, 15).
Taken together with our biochemical data above, these
electrophysiological data would be consistent with a role for
endogenous cGMP-dependent protein kinase in the augmentation of BKCa channel activity by SNP and db-cGMP in
these cells. Given that the primary function of protein kinases is to phosphorylate selected substrates, we anticipated that BKCa
channels would undergo serine/threonine phosphorylation in the presence of cGKI If cGKI Several groups have already reported that protein kinases may
physically associate with membrane ion channels (50-54), presumably as
part of a phosphorylation-dependent regulatory mechanism.
Our findings (see Fig. 6, A and D) that cGKI In summary, the findings of our study using a dominant negative
suppression strategy implicate an important role for cGKI isoform of type I
cGMP-dependent protein kinase (cGKI). We observed that
co-transfection of cells with BKCa channels and a
catalytically inactive ("dead") mutant of human cGKI
prevented
enhancement of BKCa channel in response to either sodium
nitroprusside or dibutyryl cGMP in a dominant negative fashion. In
contrast, expression of wild-type cGKI
supported enhancement of
channel activity by these two agents. Importantly, both endogenous and
expressed forms of cGKI
were found to associate with
BKCa channel protein, as demonstrated by a reciprocal
co-immunoprecipitation strategy. In vitro, cGKI
was able
to directly phosphorylate immunoprecipitated BKCa channels,
suggesting that cGKI
-dependent phosphorylation of
BKCa channels in situ may be responsible for
the observed enhancement of channel activity. In summary, our data
demonstrate that cGKI
alone is sufficient to promote the enhancement
of BKCa channels in situ after
activation of the NO/cGMP signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
, differing only in their
N-terminal domains (36% of the first 103 amino acids of the
isoform are identical to those of the
isoform) (19, 21).
Both the
and
isoforms of the type I cGMP-dependent protein kinase functionally exist as homodimers (i.e.
/
and
/
), in which each subunit contains a catalytic
domain, 2 cGMP-binding sites, and an N-terminal dimerization region (4,
19). Smooth muscle expresses both isoforms (22), although the
biological roles of each are not well understood.
in the activation of cellular
BKCa channels by the NO/cGMP signaling cascade, we created a catalytically inactive or "dead" mutant of cGKI
that could be
co-expressed with murine BKCa channels. Utilizing a
dominant negative suppression strategy (23, 24), we observed that dead cGKI
prevented activation of BKCa channels in
cell-attached patches of HEK 293 cells in response to the
nitrovasodilator sodium nitroprusside (SNP) or dibutyryl cGMP, a
membrane-permeable analog of cGMP. Using a reciprocal
co-immunoprecipitation strategy, we found that the endogenous and
expressed forms of cGKI
were able to associate with BKCa
channel protein. We also observed that cGKI
is able to directly
phosphorylate BKCa channels in vitro, supporting
the hypothesis that a similar event may be responsible for the
enhancement of channel activity by cGMP in situ. Taken
together with previous observations, our results strongly suggest that
the
isoform of type I cGMP-dependent protein kinase
represents a major downstream effector in the activation of
BKCa channels by the NO/cGMP signaling pathway in
situ.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit and the human type I cGMP-dependent
protein kinase were obtained from Chemicon International and
Calbiochem, respectively. A horseradish peroxidase-linked, mouse
anti-rabbit IgG monoclonal antibody (clone RG-96) was purchased from
Sigma Chemical Co. The purified, recombinant cGMP-dependent
protein kinase I
enzyme was purchased from Calbiochem. The
cGMP-dependent protein kinase-selective peptide substrate
RKRSRAE was obtained from Peninsula Laboratories. The Lowry protein
assay kit (detergent compatible) was purchased from Bio-Rad Laboratories.
subunit (25), the wild-type green fluorescent protein (26), and the human cGKI
(27) were subcloned into the polylinker region of the
SV40 promoter-based mammalian expression plasmid SR
using standard
techniques. Site-directed mutagenesis was carried out using the
Transformer mutagenesis kit (CLONTECH), which is
based upon the unique restriction site elimination strategy (28). The
enzymatically dead form of cGKI
was prepared by a Lys to Met
substitution at amino acid position 393 within the kinase's catalytic domain.
90 to +150 mV; tail currents were recorded at
80 mV. Current traces were filtered at 2-5 kHz (4-pole Bessel
filter) and acquired on a Dell Pentium II-based computer at a sampling
frequency of 8-10 kHz using a Digidata 1200 analog/digital interface.
Recording micropipettes were pulled from thin-walled borosilicate glass
capillaries (1.2 mm inner diameter; 1.5 mm outer diameter; WPI,
Sarasota, FL) using a Sutter P-89 horizontal electrode puller.
Micropipettes were filled with a solution containing 5 mM
KCl, 140 mM KOH, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES (pH
adjusted to 7.3 with methanesulfonic acid) and had tip resistances of
2-3.5 megaohms. The bath solution contained 5 mM KCl, 140 mM KOH, 1 mM MgCl2, 1 mM CaCl2, and10 mM HEPES (pH
adjusted to 7.3 with methanesulfonic acid). The recording chamber
(~0.3 ml volume) was perfused by gravity flow at a constant rate of
1-1.5 ml/min, using a set of manually controlled solenoid valves to
switch between solutions. Reagents were added directly to the solution
reservoir tubes at the concentrations indicated.
80 °C as intact cell pellets. These pellets were resuspended in
0.5-1 ml of ice-cold lysis buffer containing 20 mM
Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 1%
(v/v) Triton X-100, 1 mM EGTA, 2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 5 µg/ml each of leupeptin, pepstatin A, and aprotinin and then sonicated for 5-10 s to shear the genomic DNA. After measurements of protein concentration were carried out using
a modified Lowry procedure (29), lysates were mixed with Laemmli sample
buffer containing 1% (v/v)
-mercaptoethanol and incubated for
20-30 min at 70 °C, and the proteins were then separated by
SDS-polyacrylamide gel electrophoresis (30). The resolved proteins were
electrotransferred to nitrocellulose membrane at 4 °C in a buffer
containing 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, and 20% (v/v) methanol for either ~2 h at 80-90 V or overnight at 35 V (31). Membranes were first dried in a fume hood to
fix proteins and then rinsed briefly in a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1%
(v/v) Tween 20 (TTBS). Membranes were incubated at room temperature for
20-30 min in TTBS containing 10% (w/v) skim milk powder to block
nonspecific binding of antibodies and then rinsed three times for 5 min
each time in TTBS. Incubation of membranes with primary antibodies was
carried out in TTBS containing 1% (w/v) skim milk powder for 1-2 h at
room temperature, followed by three to five 10-min washes with
TTBS alone. Membranes were then incubated for ~1 h at room temperature with a horseradish peroxidase-linked, mouse anti-rabbit secondary antibody also diluted in TTBS/1% (w/v) skim milk powder, followed by three to five 5-min washes with TTBS. After the
final wash, blots were developed immediately by applying the
SuperSignal chemiluminescence reagent (Pierce Chemical Co.) for ~2
min and then exposing the blots to x-ray film (Hyperfilm; Amersham
Pharmacia Biotech).
subunit was immunoprecipitated from transiently transfected HEK 293 cells as follows: on day 3-4 after transfection, cells growing on a
100-mm dish were harvested in 1 ml of lysis buffer, sonicated briefly,
and then centrifuged for 10 min at 4 °C at 15,000 rpm in a
microcentrifuge. The supernatant was diluted to 0.4-0.5 mg protein/ml,
and a 1.4-ml aliquot of the diluted lysate was transferred to a clean
microcentrifuge tube. Bovine serum albumin was then added to a final
concentration of 1 mg/ml. This sample was pre-cleared by the addition
of 30 µl of a 50% slurry (v/v) of rehydrated protein A-Sepharose
beads (Amersham Pharmacia Biotech), followed by rotation at 4 °C for
1 h. Samples were centrifuged for 5 min at 10,000 rpm to pellet
the beads, and the soluble material was transferred to a clean
microcentrifuge tube. Pre-cleared supernatants were then incubated for
4-16 h at 4 °C with ~1.5 µg of anti-BKCa channel
antibody, followed by further incubation for 2 h with 30 µl of
protein A-Sepharose beads (50% slurry). The beads were pelleted by
centrifugation at 4 °C for 5 min at 3,000 rpm and then washed twice
by resuspension in 1 ml of wash buffer containing 20 mM
Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mM EGTA, 1 mM benzamidine, 1 mM phenylmethylsulfonyl
fluoride, 0.1% (v/v) Triton X-100, and 5 µg/ml each of aprotinin,
leupeptin, and pepstatin A, followed by a final wash in the same buffer
minus Triton X-100. The beads were then resuspended in 20-30 µl of a
buffer containing 20 mM Tris HCl, pH 7.5, and 2 mM DTT; 15-µl aliquots were used directly in the assay.
cGMP-dependent protein kinase, and 15 µl of resuspended protein A-Sepharose beads containing immunoprecipitated
BKCa
subunit. The reaction was started by the addition
of [
-32P]ATP (20 µM; final
concentration, 20,000-30,000 cpm/pmol) and stopped by addition
of concentrated (4×) Laemmli sample buffer directly to the assay
tubes. Reaction mixtures were then resolved by
SDS-polyacrylamide gel electrophoresis. Gels were stained and destained
and then directly exposed to x-ray film in a Kodak BioMax cassette at
room temperature for 12-24 h.
and BKCa
Channels--
After transient expression of cGKIa and BKCa
channel cDNAs, either alone or together, the immunoprecipitation
protocol was carried out as described above, with minor modifications.
Upon addition of 0.5-1.0 ml of lysis buffer, cells were kept on ice for 20-30 min and then disrupted by 10-12 up and down strokes of a
disposable plastic pestle designed to fit 1.5-ml Eppendorf centrifuge
tubes. For immunoprecipitation of cGKI
, ~4 µg of rabbit polyclonal anti-cGKI antibody (Calbiochem) was added to each
pre-cleared cellular lysate. Subsequent steps in the protocol were unchanged.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in
the regulation of BKCa channels by cGMP and the
nitrovasodilator compound SNP, we hypothesized that a catalytically
inactive mutant of cGKI
could be used in a dominant negative
strategy (23, 24) to suppress or interfere with the actions of
endogenous cGKI
in an intact cell. Earlier reports have shown that
native HEK 293 cells contain an endogenous cGMP-dependent
protein kinase activity (15, 34, 35); our new data, described below,
strongly suggest that this activity can be accounted for by the
presence of the
isoform of type I cGMP-dependent
protein kinase in these cells.
(27), we
replaced a critical lysine residue (Lys393) in the
ATP-binding motif of the enzyme's catalytic domain with methionine
(36, 37). The effectiveness of this substitution was verified using two
complementary biochemical approaches. First, an in vitro
kinase assay was performed using soluble cell lysates from HEK 293 cells transiently transfected with cDNA constructs encoding a
BKCa channel
subunit together with either the wild-type or mutant form of cGKI
. Fig. 1 shows
that the detergent-solubilized lysate from cells transfected with the
BKCa channel cDNA alone displayed modest
cGMP-dependent protein kinase activity, as quantified by
measuring phosphorylation of a cGKI-selective peptide substrate (33).
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Fig. 1.
Cyclic GMP-dependent protein
kinase activities in HEK 293 cells expressing wild-type or
catalytically dead cGKI . HEK 293 cells
were transfected with BKCa channel
subunit alone
(BK alone) or together with either wild-type cGKI
(BK + cGKI
) or catalytically inactive cGKI
(BK + dead cGKI
); mock-transfected cells (Mock) were
transfected with an equal amount of empty plasmid (i.e. no
insert cDNA present). Detergent-soluble cellular lysates were
prepared from each group of cells, and cGMP-dependent
protein kinase activity was quantified using an in vitro
kinase assay (see "Materials and Methods"). Results are expressed
as the means ± S.E. of three to four separate experiments,
each carried out in duplicate.
, demonstrating
expression of intact, biochemically active protein kinase. In contrast
to this situation, cells transiently transfected with BKCa
channels and the catalytically inactive or dead form of cGKI
displayed a level of cGMP-dependent kinase activity that
was very similar to that of cells transfected with BKCa
channels alone. This finding is thus consistent with results using
other protein kinases (36, 38), in which mutation of this invariant
lysine residue within the catalytic domain is sufficient to block
expression of enzymatic activity. It is interesting that co-expression
of dead cGKI
did not lower the total observable
cGMP-dependent protein kinase activity measured in
vitro. This observation may be due to: (a) transfection efficiency of HEK 293 cells with the LipofectAMINE reagent (Life Technologies, Inc.) ranges from 20-30% in our hands; therefore, endogenous cGMP-dependent activity in the majority of cells
remains unaffected, and (b) the assay mixture for
cGMP-dependent protein kinase activity contains saturating
concentrations of Mg-ATP, cGMP, and peptide substrate, therefore the
presence of inactive kinase molecules would not be expected to
interfere with the activity of native kinase by depletion of essential
reagents in vitro.
--
Fig.
2A shows a Western blot of the
detergent-soluble cellular lysates used for the in
vitro kinase assays shown in Fig. 1.
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Fig. 2.
Protein expression levels of wild-type and
catalytically dead cGKI in transiently
transfected HEK 293 cells. A shows a Western blot of
equal amounts of total cellular lysates (~75 µg/lane) from HEK 293 cells transfected with the BKCa channel
subunit alone
(BK alone) or together with either wild-type cGKI
(BK+ cGKI
) or catalytically inactive cGKI
(BK + dead cGKI
). An antibody specifically recognizing the type I
cGMP-dependent protein kinase detected a ~75-kDa band in
each lane; however, the intensity of this band was much greater in
cells transfected with cGKI
cDNA. The lower band at ~60 kDa
most likely represents a proteolytic fragment of full-length cGKI
(19). After detection of cGKI
, the blot was stripped and reprobed
with an antibody recognizing the BKCa channel
subunit.
A single band of ~125 kDa was detected in each lysate (B).
The electrophoretic mobility of molecular mass markers (in kDa) is
indicated to the right of each panel.
isoform of cGKI (22). No
expression of a 78-80-kDa
isoform was detected in our HEK 293 cells, although both
and
isoforms were readily observed under
the same Western blotting conditions in isolated smooth muscle myocytes
from rabbit aorta (data not shown). In contrast, HEK 293 cells
co-transfected with cDNAs encoding either the wild-type or mutant
form of cGKI
showed very strong expression of a similar 75-kDa band,
consistent with the presence of exogenous cGKI
protein. The lower
immunoreactive bands with a molecular mass of ~60 kDa most likely
represent proteolytic fragments of the full-length wild-type and mutant
cGKI
(19). Taken together, the results shown in Figs. 1 and
2A demonstrate that both the wild-type and mutant forms of
recombinant human cGKI
can be strongly expressed in HEK 293 cells,
with only the wild-type enzyme displaying significant
cGMP-dependent kinase activity. We then examined whether
co-transfection of cGKI
influenced the expression pattern of
BKCa channels themselves. Fig. 2B shows a
Western blot of the same cellular extracts used in Fig. 2A, which was probed with an antibody against the BKCa channel
subunit. A single immunoreactive band of ~125 kDa was observed,
the level of which was comparable under all three transfection
conditions. Similar results were obtained in two additional
experiments. Taken together, these observations demonstrate that
co-transfection of HEK 293 cells with either wild-type or mutant
cGKI
cDNA leads to the expected expression of both protein and
kinase activity, without altering the expression pattern of
BKCa channel protein.
in the
regulation of BKCa channels was examined by using patch clamp
techniques to record BKCa channel activity in cell-attached
membrane patches of transfected HEK 293 cells in the absence and
presence of 100 µM SNP. Fig. 3A shows BKCa
channel activity before and during bath application of 100 µM SNP.
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Fig. 3.
Effect of transiently expressed wild-type and
catalytically dead cGKI on the enhancement of
BKCa channel activity by sodium nitroprusside.
A and B show cell-attached patch clamp recordings
of macroscopic BKCa channel currents from HEK 293 cells
transiently transfected with BKCa channel
subunit alone
(A) or together with catalytically inactive (dead) cGKI
(B). BKCa channel currents in cell-attached
membrane patches were evoked by voltage clamp steps from
90 to +150
mV, in 10-mV increments; the membrane patch was held at 0 mV (refer to
the inset). The top set of traces in each panel
was recorded shortly after the formation of a gigaohm seal (designated
as 0 min). Using continuous bath superfusion, cells were then exposed
to 100 µM SNP, and current families were recorded every 2 min for up to 6-7 min after the start of exposure (the bottom
set of traces in each panel). The fold change in current amplitude
at +100 mV in the absence or presence of SNP exposure versus
initial control level is plotted in C for cells transfected
with BKCa channels alone or together with either
catalytically inactive (dead) cGKI
or wild-type cGKI
. The
reversibility of stimulated current amplitude in cells expressing
BKCa channels alone after SNP wash-out (SNP W/O)
for 6-8 min is also indicated. Current amplitudes were quantified by
measuring the average steady-state current over the last 5 ms of the
depolarizing pulse. Data are presented as the means ± S.E.
(n = 4-6 cells in each group). An asterisk
indicates that these values are significantly different from the
control value in the absence of SNP, p < 0.05.
, the effect of bath-applied SNP on
BKCa channel activity was significantly blunted compared
with cells expressing BKCa channels alone (Fig. 3,
B and C). Interestingly, in cells co-transfected
with BKCa channels and wild-type cGKI
, bath application
of SNP also produced a large enhancement of BKCa channel
activity, although the effect was not significantly higher than that
observed with cells expressing BKCa channels alone (Fig.
3C). This result suggests that the bath concentration of SNP
and the amount of endogenous cGMP-dependent protein kinase
in HEK 293 cells are sufficiently high to produce maximal stimulation
of BKCa channel activity; further expression of wild-type
cGKI
via transfection does not appear to augment this already
maximal response.
or catalytically dead cGKI
. Fig.
4A shows cell-attached patch
clamp recordings of BKCa channels under control conditions
and after activation by dibutyryl cGMP.
View larger version (30K):
[in a new window]
Fig. 4.
Effect of transiently expressed wild-type and
catalytically dead cGKI on the enhancement of
BKCa channel activity by dibutyryl cGMP. A
and B show cell-attached patch clamp recordings of
macroscopic BKCa channel currents from HEK 293 cells
transiently transfected with BKCa channel
subunit alone
(A) or together with catalytically inactive (dead) cGKI
(B). To carry out stimulation of individual cells by
membrane-permeable cGMP, the extreme tip of the recording micropipette
was first filled with normal pipette solution (see "Materials and
Methods"), and then the majority of the pipette shaft was back-filled
with the same solution containing 1 mM db-cGMP. In this
strategy, dibutyryl cGMP was allowed to diffuse to the pipette tip and
then across the cell membrane over the course of several minutes.
BKCa channel currents in cell-attached membrane patches
were evoked by voltage clamp steps from
90 to +150 mV, in 10-mV
increments; steps were delivered from a holding potential of 0 mV
(refer to the inset). The top set of traces in
each panel was recorded shortly after formation of a gigaohm seal
(designated as 0 min). Cells were then continuously superfused under
constant bath flow, and current families were recorded at 2-min
intervals for up to 10 min after the initial seal formation (the
bottom set of traces in each panel). The fold change in
current amplitude at +100 mV (average steady-state current over the
last 5 ms of the pulse) in the absence or presence of db-cGMP exposure
at 10 min versus initial control level (0 min) is plotted in
C for cells transfected with BK channels alone or together
with either catalytically inactive (dead) cGKI
or wild-type cGKI
.
Data are presented as the means ± S.E. (n = 3-6
cells in each group). The asterisk indicates that these
values are significantly different from control (BK alone),
p < 0.05.
,
we observed that db-cGMP was no longer effective in enhancing channel
activity when assayed over a 10-min recording period (Fig. 4,
B and C). This result is thus similar to that
observed with exposure of co-transfected cells to SNP (see Fig.
3B) and suggests that both SNP and db-cGMP act via a type I
cGMP-dependent protein kinase to enhance BKCa
channel activity. Under the condition of BKCa channels
co-transfected with wild-type cGKI
, db-cGMP was observed to increase
current magnitude ~5-fold above control levels (see Fig.
4C). However, this increase was not significantly greater than that observed for BKCa channels expressed alone, as
was the case for stimulation by SNP. This result further supports the likelihood that under the conditions of our experiment, the level of
endogenous cGKI
is sufficient to produce maximal enhancement of
BKCa channel activity.
--
Whereas it has been generally hypothesized that the
regulation of BKCa channels by cGMP may involve a
phosphorylation event, only recently have results appeared in the
literature directly supporting such a mechanism (9, 14, 15, 42, 43). To examine whether BKCa channels expressed in HEK 293 cells
may undergo direct phosphorylation in the presence of cGKI
, we
isolated expressed BKCa channels by immunoprecipitation and
then incubated these purified channels in either the absence or
presence of purified cGKI
. Fig.
5A shows an autoradiogram of
an in vitro phosphorylation assay using immunoprecipitates
from cells transfected with BKCa channel cDNA or cells
transfected with empty vector alone (mock-transfected cells).
View larger version (28K):
[in a new window]
Fig. 5.
Direct phosphorylation of BKCa
channels by cGKI in vitro.
A, immunoprecipitates from HEK 293 cells transiently
transfected with cDNA encoding either BKCa channel
subunit (IP from BKCa transfected cells) or
empty vector (IP from mock transfected cells) were used as
substrates in an in vitro phosphorylation reaction. The
presence or absence of purified recombinant bovine cGKI
in each
reaction is indicated by + or
, respectively, above the
lanes. After termination, reaction mixtures were resolved by
SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.
B shows a Western blot of the total cellular lysates and
immunoprecipitates (IP) used in the experiment described in
A. The blot was probed with an antibody recognizing the
BKCa channel
subunit. The positions of molecular mass
standards are indicated to the right of each panel.
, a major phosphorylated band of ~125 kDa was
observed, most likely corresponding to the BKCa channel
subunit. The second major phosphoprotein observed in the same lane,
migrating at ~75 kDa, corresponds to the purified cGKI
after
autophosphorylation (4). As shown in Fig. 5A, addition of
purified cGKI
to the immunoprecipitate from mock-transfected cells
produced only a single phosphoprotein of ~75 kDa, again corresponding
to the autophosphorylated cGKI
enzyme. The absence of this 75-kDa
band from both reactions lacking addition of the purified cGKI
further supports this conclusion. To demonstrate the presence of
BKCa channel protein in our reactions, we performed a
Western blot on the total cellular lysates and immunoprecipitates from
both sets of transfected cells. Fig. 5B shows that the
BKCa channel
subunit is strongly expressed in the total
cellular lysate of positively transfected cells, with significant
recovery in the immunoprecipitate. However, a similar immunoreactive
band was not detected in either the lysate or immunoprecipitate of
mock-transfected cells, consistent with the observed lack of a
phosphoprotein of ~125 kDa in the autoradiogram of
Fig.5A.
with BKCa Channels in
Situ--
To address the potential mechanism by which expression of
the catalytically inactive form of cGKI
interfered with stimulation of BKCa channel activity by SNP and db-cGMP,
BKCa channels and cGKI
(wild-type or catalytically
inactive forms) were transiently expressed either alone or together in
HEK 293 cells. BKCa channels were then directly
immunoprecipitated, and the immunoprecipitates were probed by Western
blot for the presence of associated cGKI
.
associated with isolated BKCa
channels, and we found that brief stimulation by dibutyryl cGMP
modestly enhanced this interaction (Fig.
6A). Under these same
conditions, we further observed that the catalytically inactive form of
cGKI
associated with immunoprecipitated BKCa channels to
a much greater extent compared with the expressed wild-type kinase.
However, in cells transfected with BKCa channels alone, we
were unable to detect the presence of endogenous cGKI
in
BKCa channel immunoprecipitates. This inability to capture
such a steady-state interaction may reflect the combination of
relatively low amounts of endogenous kinase present in these cells
compared with BKCa channels and the transient nature of
interaction of cGKI
with the BKCa channel substrate.
Similarly, in their recent study, Wang et al. (44) could not
detect either endogenous cAMP-dependent protein kinase or
cSrc tyrosine kinase in immunoprecipitates of recombinant
Drosophila Slo channels transiently expressed in HEK 293 cells. However, when either kinase was co-expressed along with the
channel, channel-kinase interactions could be observed using such a
co-immunoprecipitation strategy.
View larger version (67K):
[in a new window]
Fig. 6.
Interaction of cGMP-dependent
protein kinase with BKCa channels in
situ. BKCa channels or the isoform of
human cGKI
(wild-type or catalytically inactive forms) were
transiently expressed either alone or together in HEK 293 cells;
transfection conditions for each lane in A-C and
D-F are indicated at the top of A and
D, respectively. Intact cells were stimulated for 3 min at
30 °C in either the absence (
) or presence (+) of 1 mM
db-cGMP, as indicated above each lane in A and
D. After stimulation, medium was aspirated, and cells were
lysed immediately and subjected to immunoprecipitation using either an
anti-BKCa channel
subunit antibody (A and
B) or an anti-GKI antibody (D and E).
A and E show a Western blot of the isolated
immunoprecipitates probed with an antibody versus cGKI
.
These blots were then stripped and reprobed using an
anti-BKCa channel
subunit antibody (B and
D). C and F show Western blots of the
initial cell lysates (~15 µg protein loaded/lane in C,
~45 µg protein loaded/lane in F) from each of the
transfection conditions, which were probed with an antibody
versus cGKI
(C) or the BKCa
channel
subunit (F). The prominent bands of ~55 kDa
detected in A, B, D, and E represent the heavy
chains of the rabbit polyclonal anti-BKCa channel and
anti-cGKI antibodies used for immunoprecipitation. The electrophoretic
mobility of molecular mass standards is shown to the right
of each panel.
shown in Fig.
6A. To further verify this result, we then probed equal
amounts of the starting whole cell lysates for the expression of type I
cGMP-dependent protein kinase to ensure that differences in
the level of cGKI
expression between conditions did not account for
the differential co-immunoprecipitation of cGKI
shown in Fig.
6A. Our observation that expression of the transiently
expressed, wild-type cGKI
was greater than that of the catalytically
inactive form of the kinase, which was greater than that of endogenous
cGKI
(see Fig. 6C), indicates that gross differences in
cGKI
expression can not account for the differential
co-immunoprecipitation of wild-type and dead cGKI
with
BKCa channels presented in Fig. 6A. In Fig. 6C, the amount of endogenous type I
cGMP-dependent protein kinase was below detection in cells
transfected with BKCa channels alone, likely due to the low
amount of whole cell lysate loaded per lane (i.e. ~15
µg). By restricting the amount of protein loaded per lane in this
particular experiment, we were able to achieve a better comparison of
the expression levels between the wild-type and catalytically inactive
forms of cGKI
in the whole cell lysates shown in Fig.
6A.
was further examined by performing reciprocal
co-immunoprecipitation, in which we probed anti-cGKI immunoprecipitates
for the presence of co-associated BKCa channel protein.
As expected, we observed that BKCa channels
co-immunoprecipitated with co-expressed wild-type or catalytically
inactive cGKI
(Fig. 6D), although there was not the same
marked difference as seen in Fig. 6A. Importantly, we also
observed that the endogenous form of cGKI
in HEK 293 cells is able
to co-associate with expressed BKCa channels, as demonstrated by the presence of these two proteins in the same anti-cGKI
immunoprecipitates. When these immunoprecipitates were reprobed for the presence of cGKI
protein, we observed similar levels of either expressed wild-type or inactive cGKI
, along with
modest amounts of the endogenous form of cGKI
(Fig. 6E). A Western blot of the initial whole cell lysates probed for the BKCa channel
subunit demonstrates similar expression of
BKCa channel protein in the four groups of transfected
cells (Fig. 6F), thus confirming equal starting conditions
for the immunoprecipitation results shown in Fig. 6D.
versus BKCa
channels. If we consider that cGKI
is expressed to a greater level
than BKCa channel protein under the conditions of our
transient co-transfection, then we would anticipate that there is a
greater likelihood to observe cGKI
co-associated with
BKCa channel immunoprecipitates because the kinase
molecules are present in excess quantity. However, for cGKI
immunoprecipitation, the majority of either wild-type or inactive
kinase molecules would not be associated with BKCa channel
protein, which decreases the probability that immunoprecipitated cGKI
will have a channel molecule bound to it. This situation leads
to a low recovery of kinase co-associated with the channel, which
effectively dilutes any observable differences in the detected co-associations. This idea is supported by our observation that expressed BKCa channels can be detected in
immunoprecipitates of endogenous cGKI (Fig. 6D), which is
present at much lower levels than the expressed forms of the kinase.
This low expression ratio between endogenous kinase and expressed
channel thereby increases the likelihood that an immunoprecipitated
kinase molecule will be co-associated with a channel protein.
Therefore, immunoprecipitation of the lesser of these two proteins
increases the likelihood of detecting an interaction with the more
abundant partner.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
isoform of cGKI (see "Materials and Methods") that could be transfected and expressed in mammalian cells. We anticipated that this mutant would selectively target and interfere with the function of endogenous cGKI
in a dominant negative fashion (23, 24), which is not possible with the nonselective
cGMP-dependent protein kinase inhibitor KT5823 (15) or the
disruption of the cGKI gene (45), leading to loss of both
and
isoforms. The results shown in Figs. 1 and 2 demonstrate that HEK 293 cells transiently transfected with BKCa channel cDNA
expressed a measurable level of endogenous cGMP-dependent
protein kinase activity that correlated with the presence of the
~75-kDa
isoform of cGKI, as detected by Western blotting.
Transient expression of wild-type cGKI
produced a large increase in
cGMP-dependent protein kinase activity, as measured in
total cell lysates, which correlated with a large increase in
immunoreactive cGKI
. In contrast, transient expression of the
catalytically inactive or dead mutant of cGKI
produced no change in
measurable cGMP-dependent protein kinase activity compared
with control (BKCa channel alone) but led to a similar
large increase in the expression of immunoreactive type I
cGMP-dependent protein kinase, indicating the presence of
transfected cGKI
protein.
. The results shown in Fig. 5 clearly demonstrate that purified cGKI
can readily phosphorylate immunoprecipitated
BKCa channels in vitro, in agreement with the
findings of others (14, 42). Such direct phosphorylation of
BKCa channels by cGKI
in situ would thus
serve as the basis for the enhancement of BKCa channel
activity observed electrophysiologically after the addition of cGMP,
Mg-ATP, and cGKI
to excised membrane patches (9, 15, 43, 46) and
would also explain how enhancement could be maintained after excision
of inside-out membrane patches from stimulated cells
(14).2
is indeed a critical component in the regulation of
BKCa channel activity by the NO/cGMP signaling pathway,
then we would predict that expression of the catalytically inactive cGKI
mutant described above would selectively prevent such
augmentation in response to SNP or db-cGMP. As shown in Figs.
3B and 4B, co-expression of BKCa
channels with dead cGKI
does in fact preclude augmentation of
channel activity by either SNP or db-cGMP when compared with BKCa channels expressed alone. This observation thus
suggests that the
isoform of cGKI alone is sufficient to support
the regulation of BKCa channel activity by the NO/cGMP
signaling pathway in cells expressing a type I
cGMP-dependent protein kinase. Based on these novel
results, along with the previous observations of others (9, 14, 15,
43), we conclude that cGKI acts directly on BKCa channels
in situ, resulting in enhanced channel activity. Our
conclusion thus agrees with that of a recent study by Sausbier et
al. (47), who used the cGKI-deficient mouse to demonstrate the
important role of cGKI in both the activation of BKCa
channels and nitric oxide/cGMP-dependent vasodilation.
Recent studies from Han et al. (48) and White et
al. (49) further demonstrate that cGKI is the primary protein
kinase involved in the activation of BKCa channels in
coronary smooth muscle myocytes by vasodilatory, cAMP-elevating agents
such as dopamine or forskolin.
can associate with mammalian BKCa channels are analogous to
recent results showing that endogenous cAMP-dependent
protein kinase and Src tyrosine kinase can independently associate with
native BKCa channels immunoprecipitated from
Drosophila head (44). In the context of
phosphorylation-dependent regulation, the stronger
association observed for catalytically inactive cGKI
with
BKCa channels compared with wild-type kinase may serve to explain how the dead kinase acts in a dominant negative fashion to
suppress enhancement of channel activity via the NO/cGMP signaling pathway. This stronger interaction of dead kinase with the channel could thus account for the observed suppression of channel activity in situ by (a) binding and depletion of the
channel as a phosphorylation substrate and/or (b)
displacement of active cGKI
from anchoring proteins that localize
the kinase near the BKCa channel complex (23, 24).
in the
enhancement of BKCa channel activity by the NO/cGMP
signaling pathway in intact cells. These results are further consistent with the observed phenotype of cGKI-deficient knockout mice, which display impaired endothelium and NO-dependent relaxation of
smooth muscle, resulting in vascular and intestinal dysfunction (45, 47). The question of whether NO and NO donors may also be able to
directly activate BKCa channels is not supported by our
data and remains controversial (55-58); it is possible that such a
phenomenon may depend upon the specific preparation in use, along with
the types and concentrations of agents under study.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Luisa Sy for construction of the
catalytically inactive mutant of cGKI. Dr. Leo Pallanck (University
of Wisconsin, Madison, WI) generously provided the mouse
mSlo cDNA, and Drs. J. D. Corbin (Vanderbilt
University, Nashville, TN) and M. Sandberg (Central Hospital of
Akershus, Akershus, Norway) provided the cDNA encoding the
isoform of the human type I cGMP-dependent protein kinase.
Drs. M. Walsh, W. Giles, R. Loutzenhiser, and H. Schulman provided
helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This study was supported by an Establishment Award from the Alberta Heritage Foundation for Medical Research and a Medical Research Council Operating Grant (to A. P. B.).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.
Supported by a Medical Research Council/Pharmaceutical
Manufacturers Association of Canada studentship award.
§ A Research Scholar of the Alberta Heritage Foundation for Medical Research and the Heart and Stroke Foundation of Canada.
¶ To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, The University of Calgary, 3330 Hospital Dr., N.W., Calgary, Alberta, Canada T2N 4N1. Tel.: 403-220-8861; Fax: 403-270-2211; E-mail: abraun@ucalgary.ca
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M005711200
2 A. P. Braun, unpublished observations
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: cGMP, cyclic GMP; BKCa, large conductance, calcium-sensitive potassium; cGKI, type I cGMP-dependent protein kinase; DTT, dithiothreitol; NO, nitric oxide; SNP, sodium nitroprusside; db-cGMP, dibutyryl cGMP.
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