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(Received for publication, November 7, 1996, and in revised form, December 15, 1996)
From the Department of Physiology, McGill University, 3655 Drummond
Street, Montréal, Québec H3G 1Y6, Canada
ABSTRACT Protein kinase A (PKA) stimulates Cl secretion by
activating the cystic fibrosis transmembrane conductance regulator
(CFTR), a tightly regulated Cl INTRODUCTION The regulatory domain of cystic fibrosis transmembrane conductance
regulator (CFTR)1 has nine dibasic
consensus sequences for phosphorylation by protein kinase A (PKA) and
at least seven potential sites for phosphorylation by protein kinase C
(PKC). There is direct evidence for phosphorylation of serines 660, 700, 712, 737, 753, 768, 795, and 813 by PKA (1-3) and serines 660, 686, 700, and 790 by PKC (2). In freshly excised membrane patches, CFTR
Cl The role of PKC in regulating the CFTR channel is less certain.
Exogenous PKC causes a small stimulation of channel activity when added
during the first few minutes after patches are excised (4, 13). PKC
enhances both the rate and magnitude of subsequent PKA stimulation of
open probability (po) by approximately 2-fold and 50%, respectively (4). Potentiation of cAMP responses has also
been observed in phorbol-stimulated Xenopus oocytes
expressing CFTR (14), HT-29 cells (15), and pancreatic duct cells, in which the PKC agonist Studying CFTR function after phorbol treatment is problematic when both
its expression (19) and degradation (20) are strongly influenced by PKC
activity. Another problem has been the difficulty of distinguishing
acute PKC-induced changes in po from changes in
the number of functional channels (N). Finally, the
responsiveness of CFTR to PKA stimulation varies widely among different
cell types even when channels are studied in excised patches. The
reasons for this variation are not known.
Here we present evidence that PKC-mediated phosphorylation not only
potentiates CFTR responses but is, in fact, essential for acute PKA
activation of CFTR channels. This requirement for PKC only became
apparent when the PKA response was studied after prolonged rundown in
excised patches or after short-term pretreatment with PKC inhibitors,
presumably because constitutive PKC phosphorylation was slowly removed
by phosphatase activity under these conditions (4, 21, 22).
EXPERIMENTAL PROCEDURES Chinese hamster ovary (CHO) cells and baby
hamster kidney (BHK) cells that had been stably transfected with
plasmids directing the expression of wild-type CFTR were provided by
Drs. X.-B. Chang and J. R. Riordan (Mayo Clinic, Scottsdale, AZ; Ref.
6) and F. Siebert (University of Toronto, Toronto, Ontario, Canada;
Ref. 7). CHO and BHK cells were cultured as described previously (4,
42), subcultured on glass coverslips in 35-mm Petri dishes, and used in
patch clamp experiments 2-5 days after plating.
Single channels were recorded using
the cell-attached and inside-out patch clamp configurations (23; also
see Ref. 4). The bath and pipette solutions contained 150 mM NaCl, 10 mM TES, and 2 mM
MgCl2 (pH 7.2). Fresh MgATP (1 mM final
concentration) and forskolin (5 µM) were added to the
bath before each experiment. Experiments were carried out at room
temperature (~22 °C). Single channel currents were amplified
(Axopatch 200; Axon Instruments Inc., Foster City, CA), recorded on
video tape (PCM 418; Medical Systems Corp, Greenvale, NY), and analyzed
as described previously (4). The pipette voltage was held at In whole cell experiments, the bath
(extracellular) solution contained 150 mM NaCl, 10 mM TES, and 2 mM MgCl2 (pH 7.40)
and was supplemented with 50 mM sucrose to minimize
swelling-activated Cl currents. The pipette (intracellular) solution
contained 110 mM
N-methyl-D-glucamine-aspartate, 30 mM N-methyl-D-glucamine-Cl, 1 mM MgCl2, 10 mM TES, and 0.1 mM
1,2-bis-(o-aminophenoxy)ethane-N,N,N Whole cell currents were recorded using pipettes pulled from
thick-walled borosilicate glass (1.5-3 M The number of opening transitions during each
segment of >3 min was counted and used to estimate the mean burst
( RESULTS Exposing transfected CHO cells to forskolin activated
CFTR Cl
Fig. 2 summarizes the dependence of the PKA response on
time after excision and the effect of exogenous PKC on channel
responsiveness to PKA. All channels were locked open at the end of each
experiment using AMP-PNP to determine N. This number was
then used to calculate po, as described under
"Experimental Procedures." CFTR channels were consistently
reactivated if PKA was added within 2 min after excision; however, this
stimulation of po was progressively reduced with
time after excision (Fig. 2A). The mean
po values during the PKA responses are shown in
Fig. 2B. The interval between excision and PKA addition was
inversely related to the po during PKA
stimulation, which eventually declined to 0. By contrast,
responsiveness to PKA did not decline when patches were exposed to PKC,
DiC8, and MgATP throughout the 10-min period after
excision. Single-channel po in the presence of
PKA and PKC was 0.46 ± 0.07 (n = 3), similar to
that observed for the same patches during forskolin stimulation prior
to excision (po = 0.42 ± 0.02;
p > 0.05; Fig. 2C). This po is higher than that calculated for patches
excised from unstimulated cells into the bath solution containing PKA
and ATP (0.22 ± 0.04; Ref. 24), when the number of channels in
each patch was corrected using AMP-PNP. Thus, in addition to having a
permissive effect on po (Fig. 1B),
PKC also potentiated the PKA response under these conditions by
increasing the maximum po elicited by PKA
approximately 2-fold, as reported previously (4).
To further investigate whether
PKC regulates the number of channels opened by PKA or the
po of individual channels, patches containing a
small number of CFTR channels were excised from CHO cells and exposed
to PKA after a delay of 5 min. Adding PKC and DiC8 to the
same patches 5 min after PKA addition did not increase the maximum
number of channels open simultaneously, which was identical to the
value of N determined using AMP-PNP at the end of the
experiment (Fig. 3A). By contrast,
po of single channels calculated from the mean
number of channels open and N was approximately doubled
(p < 0.005; Fig. 3B). When BHK cells, which
express more than a 10-fold higher density of CFTR channels, were
pretreated with the PKC inhibitor chelerythrine (1 µM)
for 5 min, adding PKA to the excised patches caused only a small
increase in channel activity, although the number of channels locked
open by AMP-PNP was high, indicating the channels were active when
exposed to PKA alone but simply had low po. For
example, the patch shown in Fig. 3D contained at least 40 active channels, each with po < 0.05. Addition
of PKC did not increase the number of channels locked open by AMP-PNP
(Fig. 3D), further evidence that channels were already
functional after PKA alone. Thus, PKC acts by enhancing PKA stimulation
of po rather than by increasing N.
PKC shortened the mean closed time (p < 0.01) but had
no effect on mean burst duration (open time; p > 0.05)
during PKA stimulation (Fig. 3C). The lower
po in Fig. 3D (<0.05) compared with
Fig. 3B (~0.2) is explained by pretreatment of the former
with chelerythrine to reduce constitutive PKC phosphorylation. These
results suggest there is some constitutive PKC activity in unstimulated
cells, which permits submaximal responses to PKA stimulation. This
possibility is examined below using cell-attached and whole cell
patches.
To investigate whether endogenous PKC activity
is required for cAMP activation of CFTR in intact cells, we tested the
effect of preincubating cells with chelerythrine, a specific PKC
inhibitor (26, 27). Pretreatment with chelerythrine abolished forskolin activation of CFTR channels in 13 of 16 cell-attached patches (Fig.
4). In parallel control experiments forskolin activated CFTR Cl
DISCUSSION The CFTR chloride channel is regulated by PKA- and PKC-mediated
phosphorylation; however, the relationship between these kinases has
not been studied in detail. Previous work suggested that PKC increases
the stimulation of po by PKA approximately
2-fold (4); however, no precautions were taken in those early
experiments to minimize PKC phosphorylation prior to testing kinase
effects. A major finding of the present study is that PKC regulation of CFTR is mainly constitutive, at least in the two mammalian expression systems studied. Using somewhat different protocols we found that PKC
not only potentiates responses but is required for acute stimulation of
CFTR by PKA. This dependence on PKC was observed in excised and whole
cell patches and in intact cells, but only when protocols were designed
to allow dephosphorylation of PKC sites, which may explain why a strict
dependence on PKC has not been observed previously. Although PKC is
permissive and modulates the PKA responses over a wide range in excised
patches (po = 0-0.7), basal PKC activity in
resting CHO cells appears sufficient to maintain PKA responsiveness near its maximal level.
The present results confirm that forskolin stimulates CFTR channels in
CHO and BHK cells, and that CFTR channel activity declines rapidly when
patches are excised in the absence of PKA, consistent with
dephosphorylation of PKA sites by membrane-associated phosphatases (4).
Moreover, PKA reactivates CFTR chloride channels when added soon after
patches are excised but not when more than 10 min have elapsed. We
attribute this decline in responsiveness to PKA to slow
dephosphorylation of PKC sites, because it did not occur in the
presence of PKC and was reversed by the addition of PKC to the bath.
Although PKC alone caused an apparent, partial (10%) activation in
this and previous studies, the small PKC response may be a
manifestation of the permissive effect of PKC. The early experiments
suggesting activation by PKC alone were not preceded by a lengthy
rundown period such as the one shown in Fig. 1C. Thus the
stimulation by PKC may have been due to the reactivation of channels
that had become dephosphorylated at a permissive PKC site but retained
some residual PKA phosphorylation. Consistent with this explanation,
some CFTR activity was observed in the present work when patches were
excised directly into PKC-containing buffer lacking PKA, comparable
with that reported previously when PKC was added immediately after
channel rundown (po = 0.04, (4) and 0.034 ± 0.0125, (31)). Regulation of Cl PKA reactivated CFTR channels when added 2 min after excision but not
when added 10 min after excision; therefore, the permissive PKC site(s)
may be dephosphorylated more slowly than the PKA sites mediating
initial rundown of channel activity. The rundown caused by
dephosphorylation of PKA sites occurs within 100 s when patches are excised from these cells at room temperature (22). Biochemical studies will be needed to assess whether constitutive PKC
phosphorylation is required for phosphorylation of CFTR by PKA as in
hierarchical phosphorylation (37) or whether PKA phosphorylation of
CFTR is normal in the absence of PKC and only the functional response of the channel to PKA phosphorylation is attenuated. Prestimulating CHO
cells with phorbol ester did not noticeably increase phosphorylation of
CFTR protein during forskolin stimulation (6); however, the functional
responses may depend on trace phosphorylation at cryptic sites, which
could escape detection. Interdependent PKC and PKA regulation may allow
more precise control of CFTR activity, as would regulation by multiple
phosphatases acting at PKA or PKC sites.
Permissive control of cAMP stimulation by PKC was also observed in
intact cells; PKC inhibitors greatly reduced forskolin-stimulated CFTR
activity in cell-attached patches and abolished acute stimulation of
whole cell CFTR currents by cpt-cAMP. Thus constitutive, endogenous PKC
activity is sufficient for activation of CFTR by forskolin or cAMP. The
fact that exogenous PKC needs to be added to excised patches implies
that the endogenous PKC activity responsible for priming CFTR is not
membrane-associated, although it remains a formal possibility that PKC
activity in the patches is membrane-associated but requires an
additional cytoplasmic factor for activity. A membrane-associated PKC
isoform has been shown previously to stimulate CFTR channels when added
to patches excised from NIH-3T3 cells (13).
CFTR channels were weakly responsive to PKA when cells were briefly
pretreated with chelerythrine (5 instead of >30 min) or when patches
were excised from cells and allowed to run down for only 5 min so that
some PKC phosphorylation would remain. To assess whether the permissive
effect of PKC involved an increase in po or the
number of functional channels under these conditions, we took the
advantage of the previous findings that the poorly hydrolyzable analog
AMP-PNP causes CFTR to become locked in an open burst state in which
po To ensure that increases in N were not missed by studying
cells with low CFTR channel density, we also examined BHK cells that
contained between 40 and several hundred CFTR channels per patch. When
BHK cells were pretreated with chelerythrine for 5 min, subsequent
exposure of excised patches to PKA caused only a small stimulation,
although the number of functional channels as defined by their
susceptibility to locking by AMP-PNP was high and was not increased
further by PKC. If exocytotic insertion of CFTR channels is stimulated
by PKC, it was not apparent in the patches we studied and did not
contribute to the potentiation reported in this article. Kinetic
analysis of patches from CHO cells revealed that PKC decreased the mean
closed time during PKA stimulation and had no effect on the mean open
time. These results are consistent with an increased rate of channel
opening, such as the one that accompanies phosphorylation-induced
increases in ATP affinity (24, 31). In a CFTR mutant with alterations at all 10 dibasic consensus PKA sequences (10SA; Ref. 6), the reduction
in po was associated with a lower bursting rate
(i.e. a longer closed time) rather than a shortening of the
bursts (24). Strong potentiation by PKC was still observed in the
10SA mutant, which lacks serines at positions 660, 686, and 700;
therefore, these dual PKA and PKC sites are not essential for
PKC-dependent regulation of PKA responsiveness.
PKC isozymes have been classified into several distinct groups (40).
The "Ca-dependent" group ( Although the initial experiments in this study suggested that PKA
responsiveness is completely dependent on PKC phosphorylation, we were
surprised to find that whole Cl In summary, these results indicate that PKA-mediated phosphorylation
alone is not a sufficient stimulus to activate the CFTR chloride
channel. We propose that under normal conditions, constitutive phosphorylation of CFTR by PKC has a permissive role in priming the
channel for acute activation by PKA. Thus maneuvers that lower this
constitutive phosphorylation reveal PKC effects that are much more
profound than previously suspected. These results have implications for
functional studies of CFTR phosphoforms; PKA-stimulated channel
activity in membrane patches and bilayers will be dictated by residual
PKC phosphorylation. Variations in constitutive phosphorylation by PKC
may also help explain the variable responsiveness of CFTR channels to
stimulation by PKA in different expression systems (7, 41).
FOOTNOTES Acknowledgment We thank Jie Liao for culturing the cells.
REFERENCES
Volume 272, Number 8,
Issue of February 21, 1997
pp. 4978-4984
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
channel in the apical
membrane of many secretory epithelia. The CFTR channel is also
modulated by protein kinase C (PKC), but the regulatory mechanisms are
poorly understood. Here we present evidence that PKA-mediated
phosphorylation alone is not a sufficient stimulus to open the CFTR
chloride channel in the presence of MgATP; constitutive PKC
phosphorylation is essential for acute activation of CFTR by PKA. When
patches were excised from transfected Chinese hamster ovary cells, CFTR
responses to PKA became progressively smaller with time and eventually
disappeared. This decline in PKA responsiveness did not occur in the
presence of exogenous PKC and was reversed by the addition of PKC to
channels that had become refractory to PKA. PKC enhanced PKA
stimulation of open probability without increasing the number of
functional channels. Short-term pretreatment of cells with the PKC
inhibitor chelerythrine (1 µM) reduced the channel
activity that could be elicited by forskolin in cell-attached patches.
Moreover, in whole cell patches, acute stimulation of CFTR currents by
chlorophenylthio-cAMP was abolished by two chemically unrelated PKC
inhibitors, although an abrupt, partial activation was observed after a
delay of >15 min. Modulation by PKC was most pronounced when basal PKC
phosphorylation was reduced by briefly preincubating cells with
chelerythrine. Constitutive PKC phosphorylation in unstimulated cells
permits the maximum elevation of open probability by PKA to reach a
level that is ~60% of that attained during in vitro
exposure to both kinases. Differences in basal PKC activity may
contribute to the variable cAMP responsiveness of CFTR channels in
different cell types.
channels are activated by exposure to the PKA
catalytic subunit and MgATP (4, 5). Altering the four major sites of
in vivo phosphorylation reduces forskolin-stimulated
Cl permeability (1, 6), whereas removal of all 10 dibasic sites and the additional monobasic site Ser753 further
reduces, but does not eliminate, PKA stimulation of CFTR in both intact
cells and excised patches (6, 7). There is little doubt that
PKA-mediated phosphorylation is the primary mechanism controlling CFTR
Cl channel activity, consistent with early evidence
that cAMP stimulates transepithelial secretion, in part by increasing
apical membrane Cl conductance (8-12).
-phorbol 12,13-dibutyrate increased
cAMP-induced whole cell CFTR Cl current by 31% although
having no effect on basal currents (16). Potentiation has received
several different interpretations. The effects of PKC on excised
patches (4) and phorbols on T84 and C127 were suggested to involve PKC
regulation of the CFTR channel itself (17, 18). However, to explain the
apparent increase in Cl current by PKC in pancreatic duct
cells (expressed as pA/pF), it was proposed that PKC reduces cell
membrane area rather than increasing channel activity (16). By
contrast, exocytotic addition of new channels to the plasma membrane
was suggested to explain the potentiation in HT-29 cells (15).
30 mV
during cell-attached recordings and then switched to +30 mV after
excision so that the potential across the membrane patch would be
similar in both configurations (approximately 30 mV). Channel
activity was allowed to run down for 10 min after excision to allow
dephosphorylation by membrane-associated phosphatase activity prior to
the addition of protein kinases. To estimate the N in the
patch, AMP-PNP (1 mM) was added to the bath at the end of
each experiment to lock CFTR channels in the open state so they could
be counted (24). po was calculated as the mean
number of channels open during segments of the record
(Npo) divided by the value of N
determined using AMP-PNP. The catalytic subunit of PKA was obtained
from Promega (Madison, WI). Rat brain PKC II, predominantly
Ca2+- and phospholipid-dependent PKC isoforms,
was prepared in the laboratory of M. P. Walsh (University of Calgary,
Alberta, Canada; see Ref. 4). MgATP and the lipid activator
1,2-dioctanoylglycerol (8:0) (DiC8) were purchased from
Sigma. ATP was added from a 100 mM stock
solution in buffer. DiC8 was dissolved in chloroform at 5 mM and stored under nitrogen at 20 °C before use.
Chelerythrine chloride (Biomol, Plymouth Meeting, PA) was prepared as a
10 mM stock in ME2SO, stored at 20 °C, and
diluted in bath solution immediately before use (1 µM
final concentration).
,N-tetraacetic acid tetrasodium salt; (Biomol) (pH 7.20). MgATP (1 mM) was
added to the pipette solution from a 100 mM stock in TES
buffer. Stock cpt-cAMP and 8-bromo-cAMP solutions (50 mM)
were prepared in distilled water using their sodium salts and used at a
final concentration of 100 µM. The PKC inhibitor
Gö6976 (Calbiochem) was prepared in ME2SO and diluted
in bath solution immediately before use (final concentration, 500 nM). It was also included in the pipette solution, and
experiments were begun 4 min after obtaining the whole cell configuration to allow its diffusion into the cell (25). When chelerythrine was used, it was prepared as described above and added at
a final concentration of 1 µM for 30 min prior to
recording whole cell currents.
). After obtaining seals, pipette capacitance was canceled using the internal circuitry of the
patch clamp (Axopatch 1C; Axon). The whole cell recording configuration
was obtained by applying excess suction, and cell capacitance was
measured and then canceled. In most experiments, Vm
was held at 0 mV for 1 s between alternating pulses to ± 60 mV (1-s duration). Current-voltage relationships were generated by
stepping from 100 to +100 mV in 10-mV increments, returning to the
holding potential of 0 mV between pulses. Currents are expressed per
unit cell capacitance (pA/pF) to allow comparison of data from
different cells. Outward (positive) current corresponds to
Cl ions entering the cell. The Nernst prediction for a
perfectly Cl-selective membrane was 39 mV under the
conditions used. The leak current at +60 mV (+63.4 ± 14.7 pA/pF)
was small compared with the mean cAMP-stimulated CFTR current (8.6 ± 1.4%), and therefore was not subtracted. Mean pipette capacitance
was 7.87 ± 0.08 pF (n = 46), mean whole cell
capacitance was 13.1 ± 0.6 pF (n = 51), and the
average access resistance of the pipettes was 15.6 ± 1.0 M (n = 47).
open) and interburst (closed) durations
according to: open = (N × po) (time)/(number of openings) and
closed = (N N × po) (time)/(number of openings 1), where
N is the number of channels estimated by locking channels
open with AMP-PNP, and po is the single-channel open probability (31).2 Data are expressed
as means ± S.E. Paired t tests and Mann-Whitney U tests were performed using GraphPad InStat software
(Biosoft, Cambridge, United Kingdom).
channels in 15 of 15 cell-attached patches (Fig.
1). The po under cell-attached
conditions was 0.32 ± 0.038 (n = 4). As in
previous studies, po declined spontaneously when
patches were excised into a bath solution containing 1 mM
MgATP. When exogenous PKA (200 units/ml) was added to the bath, the
magnitude of the CFTR channel response depended on the time that had
elapsed since excision. Robust PKA stimulation was observed ~2 min
after patches were excised, whereas PKA usually failed to activate CFTR
Cl channels when patches were excised and continuously
exposed to 1 mM MgATP for 10 min. When patches were excised
for 10 min and allowed to become refractory to PKA, responsiveness
could be fully restored by adding PKC and the lipid cofactor
DiC8 to the cytoplasmic side in the continued presence of
PKA (Fig. 1B). This stimulation after the addition of PKC
and DiC8 was elicited by the PKA already present in the
bath rather than by PKC itself, because addition of PKC and
DiC8 after the same delay had no effect in the absence of
PKA (Fig. 1C). Moreover, CFTR responsiveness to PKA did not decline if PKC was present during the 10-min period following excision
(Fig. 1D). Taken together, these results suggest that PKA
phosphorylation alone is not a sufficient stimulus to open the CFTR
channel; constitutive phosphorylation by PKC is also required.
Fig. 1.
Responsiveness of CFTR channels to PKA
catalytic subunit under various conditions. Forskolin (5 µM) and MgATP (1 mM) were present
continuously in the bath. Recordings started in the cell-attached
configuration with a pipette voltage of 30 mV. Data were filtered at
50 Hz (eight-pole Bessel filter). The interruption in each trace
indicates the time during which it was excised and pipette voltage was
changed to +30 mV. Downward deflections represent inward
currents carried by Cl ions flowing into the pipette. PKA
(200 units/ml), PKC (3.78 nM) and DiC8 (5 µM) were added to the cytoplasmic side at the arrow. A, stimulation of CFTR activity elicited
by the addition of PKA within 2 min of rundown. B, inability
of PKA to stimulate CFTR channels when added 10 min after excision and
subsequent stimulation after addition of PKC and DiC8.
C, trace showing that PKC and DiC8 had no effect
on channel activity in the absence of PKA when added after the same
time interval as in B. Note that at least two channels were
active in this patch in the cell-attached configuration. D,
presence of PKC and DiC8 during the 10-min period after
excision prevents the loss of responsiveness to PKA (compare with trace
B).
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
Effects of PKA on
po. A, time dependence of PKA
activation of CFTR channels in three excised patches. Experiments were
carried out in bath solution containing forskolin (5 µM) and MgATP (1 mM). The number of channels was determined at
the end of each experiment by locking them open using AMP-PNP. Patches were excised at time 0 and exposed to PKA after 1 (
), 5 (
), or 10 (
) minutes, as indicated by the arrows. Note the loss of PKA responsiveness with time after excision. B,
po determined after PKA addition at various
times following excision as shown in A. C,
po of CFTR channels and their response to PKA
when added 10 min after excision in the absence () or presence ()
of PKC (3.78 nM) and DiC8 (5 µM).
Values are mean ± S.E. (bars); n = 3-4 patches. Conditions were the same as in Fig. 1.
[View Larger Version of this Image (18K GIF file)]
Fig. 3.
Mechanism of the PKC dependent response to
PKA: an effect on N or
po? Effect of PKC on the number of
functional channels in the patch (A) and
po (B). PKA was added to the bath 5 min after excision, and PKC and DiC8 were added 10 min
later. AMP-PNP (1 mM) was added at the end of each
experiment to lock functional channels in the open burst state.
n = 6 patches; *, p < 0.01; **,
p < 0.005. C, mean duration of open bursts
(To) and interbursts (Tc) during
exposure of the patches to PKA or to both PKA and PKC. Note that only
the interburst duration is affected by PKC potentiation. D,
inhibiting constitutive PKC phosphorylation reduces the
po response to PKA without reducing the
N. This recording, which is from a patch containing >40
channels, begins immediately after the patch was excised from a cell
that had been pretreated with chelerythrine for 5 min. Addition of PKA
stimulated CFTR channel activity (see expanded trace) but caused only a
slight increase in macroscopic current. Nevertheless, subsequent
addition of AMP-PNP gradually increased the current by at least
20-fold, consistent with the locking open of a large number of channels
that initially had low open probability (po <0.05)
during stimulation by PKA alone. Adding PKC and DiC8 did not cause additional channels to lock. Conditions were the same as in
Fig. 1.
[View Larger Version of this Image (22K GIF file)]
channels in 15 of 15 cell-attached patches.
Similarly, under control conditions, cpt-cAMP (100 µM)
activated whole cell CFTR chloride currents in 6 of 6 cells with a time
to half-maximal activation of 2.1 ± 0.6 min under control
conditions but in other experiments had no effect within 12 min when
cells were pretreated with chelerythrine (Fig. 5).
Moreover, in further studies, a selective inhibitor of the
Ca2+-dependent 
and 1
isoforms of PKC (Gö6976; Refs. 28-30) also prevented acute
activation of CFTR whole cell Cl currents by cpt-cAMP
(Fig. 5), although partial activation of CFTR was observed after
prolonged treatment with cpt-cAMP in the presence of either
chelerythrine or Gö6976. The time to half-maximal activation of
this delayed response was 23.9 ± 6.2 min in chelerythrine-treated cells and 21.5 ± 5.1 min in Gö6976-treated cells, a lag
that was approximately 10-fold longer than for control cells, which had
been treated with cpt-cAMP in the absence of PKC inhibitors (p = 0.0022 and 0.0022, respectively; Mann-Whitney
U test; Fig. 5, D and E). The delayed
response was abrupt, suggesting it may be caused by sudden activation
of an alternative pathway that circumvents inhibition of PKC activity.
Similar results were obtained when chelerythrine-treated cells were
exposed to 8-bromo-cAMP rather than cpt-cAMP (data not shown). By
contrast, a delayed response did not develop during very long
recordings in the absence of cAMP analogs (range, 31-50 min;
n = 3). These results suggest that the inhibition of
PKC can eventually be overcome by activation of an alternative pathway,
which restores the responsiveness of CFTR channels to PKA.
Fig. 4.
Effect of chelerythrine pretreatment on
forskolin-activated CFTR channels in cell-attached patches. Cells
were exposed to chelerythrine chloride (1 µM) for 30 min
before obtaining cell-attached patches. The Npo
of CFTR channels was compared during stimulation of control or
chelerythrine-treated cells by 5 µM forskolin. *, p < 0.001 compared with control conditions (sign rank
test).
[View Larger Version of this Image (10K GIF file)]
Fig. 5.
Effect of inhibiting endogenous PKC activity
on the response of CFTR channels to cpt-cAMP in BHK cells.
A, whole cell currents recorded under control conditions,
during stimulation by cpt-cAMP (100 µM), and following
superfusion of the cell with cpt-cAMP-free solution for 32 min. Data
were low pass-filtered at 100-500 Hz using a three-pole Bessel filter
and sampled at 200-1000 Hz. B, steady state current-voltage
relationships for the whole cell currents shown in A. C,
effect of cpt-cAMP on whole cell currents recorded at +60 mV under
control conditions () or in the presence of the PKC inhibitors
Gö6976 (
) or chelerythrine chloride (). , mean ± S.E. (bars) of six control cells. D, delay between addition of cpt-cAMP and half-maximal stimulation of the whole
cell current (T0.5) under control conditions
(open bar, n = 6) and in the presence of chelerythrine
(CHEL) or Gö6976 (hatched bars, n = 6 for each inhibitor). E, rate of CFTR current activation
(measured between 10 and 90% of maximal response) under control
conditions (open bar) and in the presence of chelerythrine (CHEL) or Gö6976 (hatched bars, n = 6 for each inhibitor). *, significant difference from control
(p < 0.0022 using the Mann-Whitney U
test).
[View Larger Version of this Image (27K GIF file)]
secretion in native
epithelia is complicated because PKC has multiple sites of action,
including inhibition of basolateral potassium conductance.
Nevertheless, there are several reports of phorbol ester stimulation of
Cl secretion in airway and other epithelia (32-36) that
might be explained by basal PKA activity combined with the permissive
effect of PKC described here.
1.0 (38, 39). Exposure to PKC increased
the po without altering the number of functional
(i.e. PKA-responsive and locked by AMP-PNP) channels in the
patches. This result was most obvious when CHO patches containing only
a few channels were used, since the probability that all the functional
channels would be open simultaneously was highest under these
conditions.
, I,
II, and 
isozymes) contains a putative
Ca2+ binding site, which is lacking in the
"Ca-independent" group (
, 
, 
, 
, and µ isozymes).
Another "atypical" group (including the 
and 
isozymes) is
structurally distinct and poorly characterized. Based on its
sensitivity to Gö6976, the permissive regulation in CHO and BHK
cells appears to be mediated by endogenous Ca-dependent or I forms. Nevertheless, PKA responsiveness in excised
patches was restored by the addition of rat brain PKC, which consists mainly of the Ca-dependent II isozyme, and
the role of PKC isozymes in CFTR regulation may vary according to cell
type.
cell currents suddenly
became responsive to cpt-cAMP in the presence of PKC inhibitors after a
delay of more than 20 min. Since channels in excised patches did not
regain responsiveness to PKA during long exposures (>30 min) in the
absence of PKC, the surrogate pathway that overcomes PKC inhibition in
whole cell patches is probably not membrane-delimited. In whole cell
studies, similar responses were observed with two structurally
unrelated PKC inhibitors, further evidence that inhibition of acute
cAMP stimulation is a bona fide effect on PKC. Also, PKA responsiveness
did not increase gradually in whole cell patches in a manner consistent
with the loss of inhibitor potency. Rather, the delayed response
developed abruptly after 20 min, as if the dependence on PKC was
relieved by activation of an alternate signaling pathway.
Supported by the Respiratory Health Network of Centers of
Excellence and a fellow of the Canadian Lung Association/Medical Research Council and the Canadian Cystic Fibrosis Foundation.
§
Supported by a fellowship from the Montréal Chest
Institute.
¶
A Medical Research Council scientist. To whom correspondence
should be addressed. Tel.: 514-398-8320; Fax: 514-398-7452; E-mail: hanrahan{at}physio.mcgill.ca.
1
The abbreviations used are: CFTR, cystic
fibrosis transmembrane conductance regulator; PKA, protein kinase A;
PKC, protein kinase C; po, open probability;
N, number of functional channels; CHO, Chinese hamster
ovary; BHK, baby hamster kidney; TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; AMP-PNP, 5-adenylyl ,-imidodiphosphate; DiC8,
1,2-dioctanoylglycerol (8:0); pF, picofarad; cpt-cAMP,
chlorophenylthio-cAMP.
2
C. J. Mathews, J. A. Tabcharani, and J. W. Hanrahan, manuscript in preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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