1 Departments of Medicine and CF/Pulmonary Research and Treatment Center and 2 Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 ± 0.12 to 1.23 ± 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.
cystic fibrosis transmembrane regulator; protein kinase A; adenosine 3',5'-cyclic monophosphate; adenosine; compartmentalization; A kinase anchoring proteins
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INTRODUCTION |
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THE CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR (CFTR)
Cl channel is regulated by
phosphorylation/dephosphorylation of multiple conserved serines and
requires ATP binding and hydrolysis at two intracellular nucleotide
binding folds (17). Although multiple kinases and phosphatases
contribute to this complex regulation of CFTR, cAMP-dependent protein
kinase (PKA) is the dominant kinase activator of CFTR (24, 21, 34). The
signal transduction elements utilized for cAMP-mediated regulation of
endogenously expressed CFTR through PKA have not been fully
characterized. Two recent studies found that CFTR mediated
Cl
secretion in Calu-3 and T84 cells is
preferentially activated by the membrane-associated isoform of PKA,
PKAII (30, 31). This observation raised the possibility that
physiologic regulation of Cl
conductance is mediated
by PKA that is colocalized with CFTR. A principal mechanism for
membrane localization of PKA is high-affinity binding between a region
of the PKAII regulatory subunit (RII) and A kinase anchoring proteins
(AKAPs) (10). AKAP-localized PKAII regulates several ion channels,
including voltage-gated Na+ channels (27, 33), L-type
Ca2+ channels (18), Ca2+-dependent
K+ channels (13), and renal outer medulla K+ 1 channels (1).
We used CFTR's dependence on PKA activity and patch-clamp techniques to develop an assay that detects PKA holoenzyme in excised inside-out membrane patches. Using this assay, we tested the role of AKAPs in the functional association of PKA with isolated membranes. Moreover, we tested the role of anchored PKA in the activation of CFTR by the adenosine receptor coupled signaling pathway. Our results show that physiological regulation of CFTR by cAMP requires PKA that is functionally coupled to CFTR via an AKAP interaction.
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EXPERIMENTAL PROCEDURES |
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Cells. Human Calu-3 cells (ATCC HTT-55) were grown in MEM (GIBCO BRL) supplemented with 10% fetal bovine serum, 10% sodium pyruvate, 10% MEM non-essential amino acid solution (NEAA), and penicillin/streptomycin in an atmosphere of 95% air-5% CO2 at 37°C. Cells were passaged by 1:3 dilution every 5-7 days. For patch-clamp and whole cell voltage-clamp studies, 20,000 cells were seeded onto the center of plastic dishes (35 mm diameter, not coated) and used for experiments after 1-5 days.
Immunoblot analyses and RII overlays.
Calu-3 cells were grown to confluence on Transwell filters before
experiments were performed. Whole cell lysates prepared from the
monolayer cultures were separated into soluble and particulate fractions by centrifugation at 436,000 g for 15 min at 4°C,
and protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce; Rockford, IL). Equal
amounts of soluble and particulate fractions were resolved on SDS-PAGE
gels, transferred to Immobilon-P (Millipore), and analyzed by Western blot analysis as described (25). Rabbit anti-human PKA RI, RII
,
and catalytic subunits were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In control experiments, preincubation of antisera with blocking peptides specific for RI, RII, or the catalytic subunit
abolished the signals observed on Western blots (not shown).
Single channel studies.
Pipettes were pulled from glass capillaries (GC150TF-15, Clark
Electromedical Instruments) and filled with 160 mM
Tris · Cl and 30 mM sucrose (pH 7.0 adjusted with
Tris). The ionic composition of the bath buffer was the same. Under
these conditions, the resistance of an open pipette was about 4 M.
Slight negative pressure applied to the interior of a pipette touching
the cell membrane resulted in high-resistance seals, typically in the
range of 20-30 G
. CFTR Cl
channel activity
was recorded at ± 40 and 60 mV pipette potential as current jumps
with typical burstlike openings and flickery kinetics in the positive
voltage range (15, 16, 20). Under these conditions, the slope
conductance of CFTR in excised patches was ~7.0 pS.
Whole cell voltage-clamp studies.
The pipette solution (32) for intracellular dialysis was 40 mM
Tris · Cl; 100 mM potassium gluconate, 2 mM
MgCl2; 5 mM TES; 1 mM EGTA; and 0.1 mM CaCl2, 1 mM MgATP, 0.1 mM Na2GTP (pH 7.4). Ca2+ activity
was buffered to ~40 nM (1.0 mM EGTA and 0.1 mM CaCl2). The routine bath solution contained 150 mM Tris · Cl;
2 mM MgCl2; 1 mM CaCl2; 5 mM TES; 30 mM
sucrose; and 10 mM D-glucose (pH 7.4 with Tris). Patch
pipettes had a tip resistance of 2-3 M with these solutions.
CFTR-mediated whole cell conductance was studied as previously
described (32). In the standard protocol, voltage was clamped to
40 mV and stepped to 80 mV every 10 s as current was recorded.
Current voltage plots were obtained every 2 min from the current
recorded in response to a series of voltage steps from
40 mV,
covering the range from
100 to 100 mV in 20 mV increments. The
whole cell conductance was calculated by the slope of a linear fit of
the current-voltage relationship points from
100 mV and
40 mV. All membrane potential
(Vm) shown are clamp voltages
corrected for the measured junction potential of pipette and bath
solution (
5 mV).
Data acquisition and analysis. All hardware and software were from Axon Instruments. Single channel currents were filtered at 100 Hz, sampled at 1 kHz, and stored on the hard disk of a personal computer. Data were analyzed using Fetchan 6.0 and pStat 6.0. The product of CFTR channel number and open probability (NPo) was calculated for 90 s intervals, where N is the maximum number of channels observed, and Po is the single channel open probability (7). Whole cell voltage was clamped with Clampex 7.0. Whole cell currents were acquired at 500 Hz, filtered at 100 Hz, and analyzed with Clampfit 6.0. All the data were expressed as means ± SE. The Wilcoxon matched-pair sign-rank test and the Student's t-test were used for statistical analysis.
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RESULTS |
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To directly test the hypothesis that membrane-associated PKA holoenzyme can regulate CFTR, we isolated CFTR-containing Calu-3 cell membranes by patch excision and applied maneuvers designed to selectively stimulate PKA activity. In the initial series of experiments, cell-attached patches containing active CFTR were excised into bath solution that contained MgATP, and were allowed to run down for 5 min. Application of the cAMP analog CPT-cAMP (100 µM) to the bath activated CFTR channels in 5 of 18 excised patches (~28%, not shown). From this observation, we tentatively concluded that PKA holoenzyme was associated with at least some excised membrane patches. To study this apparent association in more detail, we refined the conditions of the assay by including activated PKC in the bath, which was shown by Jia et al. to be a condition that permitted efficient activation of CFTR in excised patches by exogenous PKA catalytic subunit (21).
Accordingly, we next tested excised Calu-3 patches for PKA holoenzyme
by excising CFTR containing patches into a bath that contained 1 mM
MgATP and 2 nM activated PKC. In these experiments, we also compared
the effects of CPT-cAMP on CFTR in the absence and presence of the
specific PKA inhibitor peptide (PKI, 10 µM). After rundown for 5 min
in patches incubated without PKI, 100 µM CPT-cAMP stimulated CFTR in
13 of 20 patches tested (~65%). The mean NPo for
all patches in this series increased from 0.36 ± 0.12 before to 1.23 ± 0.57 (n = 20, P < 0.0025, sign-rank test) after
the addition of 100 µM CPT-cAMP (Fig.
1, A and C).
In contrast, when 10 µM PKI was continuously present in the bath,
CPT-cAMP had no effect on NPo of CFTR (0.19 ± 0.15 before to 0.21 ± 0.15 after, n = 11, P > 0.1, sign-rank test; Fig. 1, B and C). This stimulation of CFTR by CPT-cAMP and its block by PKI shows that CFTR is
regulated by PKA activity that remains associated with most membrane
patches after excision.
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These observations are consistent with recent reports that CFTR is
selectively regulated by PKAII, the isoform of PKA that is typically
membrane associated (30, 31). Because PKAII associates with membrane
surfaces of subcellular compartments by binding to AKAPs, we next
studied the distribution of PKA regulatory subunit isoforms in Calu-3
cell fractions and tested for the presence of AKAPs by RII overlay. PKA
RI was detected in the soluble fraction of Calu-3 cells, whereas RII
was enriched in the particulate fraction (Fig.
2A). Biotinylated RII subunit bound
to ~15 distinct bands present in the Calu-3 cell particulate
fraction, as well as to several soluble Calu-3 cell proteins (Fig.
2B, left). The specificity of binding of RII to AKAPs
in such overlay assays is typically demonstrated by competition with a
synthetic peptide, HT-31, which is modeled on the RII binding domain of
human thyroid AKAP (6). RII binding to Calu-3 cell proteins was
completely blocked by 4 µM HT-31 peptide (Fig. 2B,
right), but not by a control peptide, HT-31P (not shown) (6).
Thus Calu-3 cell membranes contained both RII and multiple RII binding
proteins, i.e., putative AKAPs.
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We reasoned that the disruption of the RII-AKAP interaction by HT-31
might provide a test of whether or not the PKA holoenzyme activity we
detected functionally in excised membrane patches was bound to AKAPs.
Accordingly, we tested the effect of 10 µM HT-31 on the activation of
CFTR by CTP-cAMP. With 10 µM HT-31 in the bath solution, CPT-cAMP had
no effect on NPo (from 0.39 ± 0.14 before to 0.39 ± 0.13 after, n = 29, P > 0.5, sign-rank test; Fig.
3, A and C). In contrast,
in the presence of 10 µM HT-31P, a control peptide in which a proline
substitution was introduced to decrease its affinity for endogenous
RII, CPT-cAMP markedly increased CFTR NPo (from
0.27 ± 0.09 before to 1.48 ± 0.64 after, n = 34, P < 0.002, sign-rank test; Fig. 3, B and C). In
previous studies utilizing these peptides, this pattern of results was
interpreted as disruption of binding between PKA regulatory subunit and
AKAPs (1, 37). Thus PKA holoenzyme functionally coupled to CFTR is
specifically associated with isolated membrane patches of Calu-3 cells
by binding to one or more AKAPs.
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Adenosine receptors coupled to adenylate cyclase were recently reported
to activate CFTR as efficiently as forskolin, but generated only 20%
as much increase in total cAMP (8). Because our excised patch data
showed AKAPs to mediate both close physical association and functional
coupling of PKA and CFTR, we hypothesized that AKAPs could be important
in the efficient regulation of CFTR by adenosine receptors. To test
this possibility, we compared the effects of HT-31 and HT-31P on CFTR
mediated whole cell Cl currents in Calu-3 cells
(Fig. 4). We applied adenosine to the bath
at 1 µM, the approximate half-maximal effective dose for adenosine
activated Cl
secretion in Calu-3 cells in Ussing
chamber studies (not shown). Both basal [105.3 ± 21.4 pA/pF/100 mV;
reversal potential (Rp) = 29.6 ± 1.6 mV] and
adenosine stimulated (221.5 ± 39.5 pA/pF/100 mV,
Rp = 28.0 ± 1.4 mV, n = 15)
currents had reversal potentials (ECl) close to
32 mV and were unaffected by the addition of 3 mM DIDS (not
shown), consistent with CFTR mediated Cl
current
(32). Cells dialyzed with HT-31 (10 µM) had virtually no increase in
current in response to adenosine, whereas cells dialyzed with the
control peptide, HT-31P (10 µM), had a similar increase in whole cell
current in response to adenosine as the control cells. These results
implicate anchored PKA holoenzyme in the physiologic regulation of CFTR
by showing that disruption of the AKAP-PKA interaction breaks the
coupling of adenosine receptors to activation of CFTR.
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DISCUSSION |
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Our studies reveal that PKA holoenzyme is associated with
CFTR-containing Calu-3 cell membranes, and that it is tightly coupled to the regulation of CFTR Cl channel activity. We
based our investigation on the premises that patch excision is a way to
directly isolate CFTR-containing cell membranes and that CFTR
NPo acts as a real time reporter of PKA activity in
these excised patches. We speculated that this approach would reliably
detect membrane-associated PKA because CFTR NPo is
tightly regulated by PKA, and because highly specific and well
established maneuvers are available to manipulate PKA activity. Because
the principal hallmark of PKA holoenzyme is the activation of catalytic
subunit caused by binding of cAMP at its tandem binding sites on each
regulatory subunit (3), we first tested the effect on CFTR
NPo of adding a cAMP analog to the cytoplasmic face
of excised membrane patches. To ensure saturation of cAMP binding sites
in excised membrane patches, we used a large excess of CPT-cAMP (100 µM), a cAMP analog that is both lipophilic and resistant to
phosphodiesterases (26). The fundamental observation in our study is
that exposure of excised membrane patches to CPT-cAMP, in the presence
of MgATP, increased CFTR NPo in most excised
patches. This observation is primary evidence that PKA holoenzyme
remained associated with membrane patches displaced from cells by
excision, and was physically close enough to CFTR to affect its gating.
Importantly, the CPT-cAMP-induced change in CFTR gating was completely
inhibited by PKI, a specific peptide inhibitor of PKA. Inhibition by
PKI is a second hallmark of PKA activity (3). Because both CPT-cAMP and
PKI are highly selective reagents that exert opposite effects on PKA
activity, it is clear that PKA holoenzyme remains in membrane patches
after excision, and furthermore, that this PKA is functionally coupled to CFTR.
A second important conclusion from our studies is that after physical
isolation of Calu-3 membranes into an essentially infinite bath, the
PKA activity in excised patches is associated with AKAPs. Our RII
overlay experiments detected ~15 RII binding proteins in the
particulate fraction of Calu-3 cells. This assay is based on RII
binding to a 20-30 amino acid sequence present in AKAPs that forms
an -helical amphipathic domain (9, 10). This AKAP domain binds with
high affinity to several isoleucines present in the amino terminus of
the Type II isoform of RII (10). The amino terminal sequence of RII is
not present in RI, although it has been recently reported that RI can
bind AKAPs with lower affinity than RII (5). The high affinity
interaction between RII and the
-helical domain of AKAPs is
disrupted by HT-31, a 24 amino acid synthetic peptide based on the
-helical domain first identified in human thyroid AKAP (6). Our
observation that HT-31, but not the control peptide HT-31P, prevented
activation of CFTR by CPT-cAMP in excised membrane patches indicates
that the PKA activity that regulates CFTR in excised membrane patches was bound by a highly specific molecular mechanism. By similar reasoning, this result also constitutes formal evidence that one or
more of the AKAPs present in Calu-3 membranes are retained in excised
patches and are required for PKA to regulate CFTR.
The third major finding in our study is that the AKAP-PKA interaction
must be intact for activation of CFTR by adenosine receptors. It was
recently reported that A2 receptors stimulated CFTR-mediated Cl conductance more efficiently than forskolin (8).
A2 receptors generate cAMP by coupling to adenylate cyclase through
stimulatory G protein (Gs), but few other specifics of the
signal transduction elements linking A2 receptors to activation of CFTR
are known (8, 23). We found that the activation of CFTR mediated
Cl
current by 1 µM adenosine, a concentration we
selected to mimic a physiologic stimulus, was specifically abolished by
dialyzing cells with HT-31. This finding suggests minimally that
functional coupling of PKA to CFTR by AKAPs is not a phenomenon
detected only in excised membrane patches, but is a requisite step in
the signal transduction path between adenosine receptors and CFTR in
whole cells. Importantly, these whole cell data reveal that functional
coupling of PKA to CFTR is important at physiologic levels of cAMP.
The role of membrane-associated PKA for physiologic regulation of CFTR by cAMP suggests that AKAPs may be important in compartmentalization of signal transduction mechanisms in epithelia. There is growing awareness that such compartmentalization, which has been studied for several years in neural specializations, may play a prominent role in how the functions of polarized epithelial cells are regulated (11). We, and others, have reported that CFTR binds, via its conserved COOH terminus, the subapical PDZ scaffold protein, EBP50 (29, 35). Whereas no specific function has been identified for the CFTR-EBP50 interaction, it is attractive to speculate that it could mediate some aspect of CFTR regulation. In fact, EBP50 binds to ezrin, an actin binding protein, which is prominently involved in cytoskeletal reorganization (12). Moreover, ezrin was reported to function as an AKAP (12), in vitro. These separate observations prompt additional speculation that the binding of CFTR to EBP50 represents secondary binding of CFTR to an AKAP, which could be relevant to our present results. It is important to point out, however, that there is so far no evidence that ezrin functions as an AKAP in cells, or if it does, whether it is an AKAP that is important for regulation of CFTR.
Through the identification and characterization of individual apical AKAPs that mediate regulation of CFTR by compartmentalized PKA, it may be possible to learn the molecular identity of additional signaling elements important for the regulation of CFTR. For example, CFTR is coactivated by an unknown isoform of PKC, and is dephosphorylated by one or more protein phosphatases that have not been definitively identified (14, 24). Several AKAPs that have already been characterized have been found to anchor not only PKAII, but other signal transduction elements including PKC and phosphatases (22). Moreover, CFTR appears to affect the function of numerous other ion channels or transporters that can be present in the apical membrane of epithelia (28). Full characterization of an AKAP that anchors signal transduction elements that regulate CFTR could help define molecular or functional linkages between CFTR and other channels or transporters that appear to be coordinately regulated with CFTR.
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ACKNOWLEDGEMENTS |
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This study was supported by Cystic Fibrosis Foundation Grant CFF9710, and National Heart, Lung, and Blood Institute Grants HL-42384 and HL-533094.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Huang, 6021 Thurston Bowles Bldg., Campus Box 7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (E-mail: Pingbo_Huang{at}med.unc.edu).
Received 10 August 1999; accepted in final form 27 September 1999.
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