1 Renal Unit, Massachusetts General Hospital East, Charlestown 02129; 2 Department of Medicine, Harvard Medical School, and 3 Experimental Medicine, Brigham and Women's Hospital, Boston 02115; and 4 Renal Section, Boston Medical Center, Boston, Massachusetts 02118
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ABSTRACT |
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Previous studies
have indicated a role of the actin cytoskeleton in the regulation of
the cystic fibrosis transmembrane conductance regulator (CFTR) ion
channel. However, the exact molecular nature of this regulation is
still largely unknown. In this report human epithelial CFTR was
expressed in human melanoma cells genetically devoid of the filamin
homologue actin-cross-linking protein ABP-280 [ABP()]. cAMP stimulation of ABP(
) cells or
cells genetically rescued with ABP-280 cDNA [ABP(+)] was
without effect on whole cell Cl
currents. In
ABP(
) cells expressing CFTR, cAMP was also without effect on
Cl
conductance. In contrast, cAMP induced a 10-fold
increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell
Cl
currents of ABP(+)/CFTR(+) cells. Further, in
cells expressing both CFTR and a truncated form of ABP-280 unable to
cross-link actin filaments, cAMP was also without effect on CFTR
activation. Dialysis of ABP-280 or filamin through the patch pipette,
however, resulted in a DPC-inhibitable increase in the whole cell
currents of ABP(
)/CFTR(+) cells. At the single-channel level,
protein kinase A plus ATP activated single Cl
channels only in excised patches from ABP(+)/CFTR(+) cells.
Furthermore, filamin alone also induced Cl
channel
activity in excised patches of ABP(
)/CFTR(+) cells. The present
data indicate that an organized actin cytoskeleton is required for
cAMP-dependent activation of CFTR.
ABP-280; cystic fibrosis transmembrane conductance regulator; actin cytoskeleton; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) is an anion-selective channel whose dysfunction leads to
the onset of cystic fibrosis (1, 2, 29). CFTR activation is normally
elicited by stimulation of the cAMP pathway and protein kinase A (PKA)
activation. This is thought to be consistent with the fact that several
PKA-dependent phosphorylation sites have been found in CFTR. Other
regulatory mechanisms, however, have also been implicated in CFTR
regulation (8, 11, 30). Recent studies (4, 27) have determined a
regulatory role of actin in the activation of CFTR by the cAMP pathway
targeting PKA activation. In those studies, partial disruption of the
actin cytoskeleton with cytochalasin D induced activation of CFTR
Cl channel activity in the absence of PKA activation
(27). Furthermore, extended treatment with cytochalasin D (6-9 h)
to collapse the actin cytoskeleton completely prevented CFTR activation
by direct addition of PKA (27). However, PKA-insensitive CFTR function was readily restored by addition of exogenous actin, which is consistent with the presence of potential actin-binding domains in CFTR
(27). This raises the possibility for the actin cytoskeleton to
directly interact with and regulate CFTR (27). The ubiquitous and
abundant distribution of actin, however, may contribute against the
idea of this molecule behaving as a conventional "ligand" in the
regulation of CFTR, since actin filaments can take several conformations within the cytoplasm. We have previously demonstrated, for example, that a distinct form of "short" actin filaments may be responsible for CFTR activation, similar to that reported in previous studies with epithelial Na+ channels (26) and the
Na+-K+-ATPase (3).
The actin-binding protein ABP-280 and its muscle isoform filamin induce
the orthogonal cross-linking of actin filaments into three-dimensional
networks (20). Although deriving from different genes, both
actin-cross-linking protein isoforms are >70% identical and are thus
expected to have functional similarities (14, 15). Human melanoma cells
devoid of the actin-binding protein ABP-280 [ABP()]
display an impaired motility and a dysfunctional actin organization but
recover both a normal cytoskeletal phenotype and functional properties
by transfection of the full-length ABP-280 cDNA [ABP(+)
cells] (10). Furthermore, ABP(
) cells are unable to elicit
a normal cell volume regulatory response due to their inability to
modulate ion channel activity (7). Genetically rescued ABP(+) cells,
however, recover both the ability to regulate cell volume and the
ability to modulate ion channel function. Moreover, the dynamics of
actin filament organization are also relevant for CFTR regulatory
mechanisms, because further addition of filamin had an inhibitory
effect on its ion channel function (27). Therefore, in the present
study it was hypothesized that ABP(
) and ABP(+) cells were
excellent models to further assess the regulatory role of actin
filament organization in CFTR regulation. Our studies indicate that
cross-linked actin networks organized by the interaction between
ABP-280 and actin are essential for CFTR to function as an anion channel.
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MATERIALS AND METHODS |
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Human Melanoma Cell Lines
Human melanoma cells, grown as previously described (10), were originally derived from the transfection of a parent ABP(Patch-Clamp Techniques
Whole cell currents. Patch pipettes were made of KG-33 glass capillaries (Garner Glass, Claremont, CA). Actual currents and command voltages were obtained and driven with a Dagan 3900 amplifier (Dagan, Minneapolis, MN) using a 1-GSingle-channel currents. Actual currents and command voltages
were obtained and driven with a PC-501 patch-clamp amplifier (Warner
Instruments, Hamden, CT) using a 10-G feedback resistance in the
head stage as previously reported (9, 25). Signals were filtered at
1,000 Hz with an eight-pole Bessel filter (Frequency Devices,
Haverhill, MA). Data were acquired, digitized, and stored in a hard
disk of a personal computer through a TLL interface (Tecmar) until
further analysis with pCLAMP 6.0.3 (Axon Instruments, Foster City, CA).
Patch pipette and bathing solutions were as indicated for the whole
cell current experiments, containing either MgCl2 (70 mM)
or N-methylglucamine chloride (140 mM). Following our previous
studies on the role of the actin cytoskeleton in CFTR function (27),
whole cell and single-channel experiments were conducted at room
temperature (22 °C).
Detection of ABP-280 and CFTR in Melanoma Cells by Western Blot Analysis
The presence or absence of CFTR and ABP-280 were determined in control and transfected human melanoma cells using immunoblot analysis. Cells were harvested by washing with ice-cold Ca2+-free PBS and were scraped, centrifuged, and resuspended in ice-cold lysis buffer [1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris · HCl (pH 7.5), 1 mM EGTA, 0.25 mM sodium vanadate, 10 µg/ml phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin], scraped, and then frozen atActin-Binding Proteins
Muscle filamin from chicken gizzard (Sigma), ~1 mg/ml stock solution in water, was diluted 200-fold into either the patch pipette or the chamber. Nonmuscle filamin, ABP-280, purified from rabbit alveolar macrophages as previously described (17), was a kind gift from Dr. John H. Hartwig (Brigham and Women's Hospital, Boston, MA).Other Reagents
The cAMP stimulatory cocktail contained 8-bromoadenosine 3',5'-cyclic monophosphate (500 µM), IBMX (200 µM), and forskolin (10 µM). The ClPermeability-to-Selectivity Ratio Calculations
The ATP over Cl
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Similarly, the permeability-to-selectivity ratio of different anions
(PCl/PY) was calculated
using a modified equation such that
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RESULTS |
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Effect of ABP-280 Expression on the Whole cell Currents of CFTR-Expressing Human Melanoma Cells
To determine the role of ABP-280 in CFTR activation, the effect of a cAMP-stimulatory cocktail was assessed on ABP(
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The cAMP-activated Cl currents of ABP(+)/CFTR(+)
cells were readily inhibited by DPC [0.5 mM; 10.4 ± 2.25 nS/cell (n = 8) vs. 2.31 ± 0.78 nS/cell (n = 4) for
cAMP-activated and after DPC, respectively, P < 0.01; Fig.
3], indicative of the presence of CFTR-associated Cl
channel activity as previously
reported (21, 28, 29). In two of seven experiments, the whole cell
Cl
currents of ABP(+)/CFTR(+) cells were
spontaneously activated (6.10 ± 0.80 nS/cell), although they were
further activated by cAMP addition (12.8 ± 4.20 nS/cell).
Spontaneously active Cl
currents were inhibitable by
DPC (data not shown).
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Anion Selectivity of the cAMP-Activated Pathway
The anion permeability-to-selectivity ratio of the cAMP-activated whole cell conductance of ABP(+)/CFTR(+) cells was ClEffect of Intracellular ABP-280 Dyalisis on the Whole cell Currents
of CFTR-Expressing ABP() Cells
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Single-Channel Currents of CFTR-Expressing Human Melanoma Cells
The effect of ABP-280 on the cAMP-dependent activation of CFTR was also assessed at the single-channel level. Addition of PKA (10 µg/ml), in the presence of ATP (1 mM), to the cytoplasmic side of excised, inside-out patches of either ABP(
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Effect of Exogenous ABP-280 on CFTR Activation in ABP()
Cells
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DISCUSSION |
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The actin cytoskeleton plays a functional role in the cAMP response of epithelial cells (12, 26). The vasopressin-mediated increase in Na+ channel activity of A6 epithelial cells, for example, is mediated by the cAMP activation of PKA, which in turn modulates Na+ channel activity (25). This response was prevented by disruption of actin filament organization but was reestablished by addition of exogenous actin (26).
cAMP stimulation is also a paradigm for the activation of CFTR in epithelial tissues. However, despite the fact that CFTR contains multiple sites for phosphorylation by PKA, little is known about the role of PKA-mediated phosphorylation on the CFTR activation process itself. Recent studies from our laboratory indicated that a proper cAMP-dependent activation of CFTR requires an organized actin cytoskeleton (27). In those studies, it was demonstrated that cytoskeletal disruption with cytochalasin D blunted completely the cAMP-mediated activation of CFTR, and, only in the presence of an organized actin cytoskeleton does PKA induce CFTR-associated ion channel activity. However, actin-cytoskeleton-modifying agents, including cytochalasin D and phalloidin, do not diminish the total pool of actin but instead modify the structural arrangement of three-dimensional actin networks. Therefore, in this report it was postulated that specific changes in actin network conformations may also modify CFTR activation. The present study was an attempt to initiate a characterization of the role of three-dimensional actin structures on the functional interaction between the cAMP/PKA pathway and the CFTR activation process. The data in this report indicate that not only actin is necessary for a proper cAMP-mediated activation of CFTR but that proteins conveying specific three-dimensional actin network conformations are also necessary for this response to be properly accomplished. Although the molecular mechanism for this functional interaction between CFTR and the actin cytoskeleton is still largely unknown, it is clear that actin and actin-binding proteins are required in this process. Thus the actin filamental activation of CFTR may require the presence of vicinal scafolding proteins to elicit the activation process. One possibility may entail the role of actin networks in helping either PKA and/or other adjacent proteins interact with the ion channel. Recent studies have suggested, for example, that the actin-binding protein ezrin may interact with CFTR via the postsynaptic density disc-large ZO-1 domains of ERM-binding phosphoprotein 50 (EBP50), through the CFTR-conserved sequence DTRL in the COOH terminus of the channel protein (32). This is in agreement with previous studies indicating that another ion transport protein, the Na+/H+ exchanger, is associated with the EBP50 homologue NHE-RF, which confers cAMP sensitivity to the transporter (19, 35). In this context, it is possible that actin-binding proteins may actually help dissociate the PKA catalytic subunits from the anchored regulartory units of the inactive complex, thus conveying a compartmentalized dimension to the PKA activation process. Several reports have already established a regulatory role of the actin cytoskeleton in the dissociation of regulatory and catalytic subunits of PKA I (23) and II isoforms (13). Nevertheless, the cytoskeletal regulation of CFTR may be also independent of PKA activation. Previous evidence on the activation of cardiac CFTR by the anti-COOH antibody raised against the CTRL sequence of the channel would suggest that this process actually requires actin cytoskeletal integrity but is elicited in the absence of PKA activation (4). However, several CFTR reconstitution studies have previously determined that, in the likely absence of actin, PKA is still capable of inducing conformational changes to elicit a functional CFTR. Future studies are required to assess the nature of the modulation by both PKA and the actin cytoskeleton, which may work in concert to elicit a functional CFTR.
The present study focused on the ability of cross-linked actin networks
to elicit a proper cAMP response driving a functional CFTR. Filamin and
its homologue actin-binding protein (ABP-280) are homodimeric proteins
with a molecular mass of ~540 kDa and are known to cross-link actin
filaments (16, 31), generating three-dimensional F-actin networks that
behave as intracellular gels (34). ABP-280 also links the actin
cytoskeleton to the plasma membrane, thus enabling a functional
interaction with plasma membrane structures including receptors (22,
24). Although the ability of these proteins to cross-link actin relies
on dimeric binding to more than one actin filament, it is the angle
between the actin filaments that may be relevant for their final
conformation. The -actinin homodimers, for example, tightly bind
actin into bundles instead of orthogonal cross-linked actin filaments
formed by filamin. Interestingly, previous studies from our laboratory have shown that, while filamin inhibits (9),
-actinin activates (5)
epithelial Na+ channel activity, thus suggesting that the
spatial arrangement of cross-linked filaments is also relevant in the
regulation of a particular ion channel response.
The presence of apically added filamin/ABP-280 has been observed to be
a tonic inhibitor of epithelial ion channel activity. Filamin was
previously shown to inhibit spontaneous (9) as well as PKA-activated
(26) and actin-activated (9) Na+ channels in epithelial
cells. Further, ABP-280 and filamin inhibited spontaneous
K+ channel activity in human melanoma cells (7) and the
PKA-mediated activation of CFTR (27). In agreement with these findings,
cells lacking a functional ABP-280 are unable to volume regulate, due to a dysfunctional and constitutive K+ channel activation
(7). Genetically rescued melanoma cells transfected with the ABP-280
cDNA, in contrast, have a lower basal K+ permeability and
recover the ability to elicit cell volume regulation (7). Therefore, in
the present study the effect of ABP-280 on CFTR function was evaluated
in ABP() and ABP(+) melanoma cell lines transfected with CFTR.
cAMP only activated CFTR in the presence of a functional ABP-280. In
close agreement with previous studies on the effect of cytochalasin D,
however, we found that cAMP and PKA did not induce CFTR ion channel
activity in the ABP(
)/CFTR(+) cells. This in itself confirmed
that the three-dimensional nature of the actin cytoskeleton is
essential for proper regulation of ion channel activity. This
phenomenon was further confirmed both by transfection of ABP-280 in
CFTR-expressing ABP(
) cells and by intracellular dialysis of
ABP-280 in ABP(
)/CFTR(+) cells. The data in this report
strengthen our previous studies, indicating that actin filament
organization is a key component for a proper PKA response in vivo,
since cells whose actin cytoskeleton was collapsed by a 6- to 9-h
exposure to cytochalasin D (27) or by the lack of ABP-280 (this report)
were insensitive to cAMP stimulation under whole cell conditions and
most clearly to direct addition of PKA under excised conditions.
The present data are thus most consistent with a particular structural conformation of actin in the proper PKA-dependent regulatory mechanism of CFTR. It is important, however, to indicate that all three relevant proteins involved in this interface, namely, CFTR, actin, and ABP-280, are substrates for phosphorylation. Further studies, such as specific mutations of the proteins involved, will be required, therefore, to assess the specific molecular steps of this functional interface.
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ACKNOWLEDGEMENTS |
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We thank Dr. Thomas P. Stossel (Experimental Medicine, Hematology-Oncology Division, Brigham and Women's Hospital, Boston, MA) for his thorough review of the original manuscript. We are also grateful to Dr. John Hartwig (Experimental Medicine, Hematology-Oncology Division, Brigham and Women's Hospital, Boston, MA) for providing ABP-280.
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FOOTNOTES |
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These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48040 (H. F. Cantiello).
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: H. F. Cantiello, Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: cantiello{at}helix.mgh.harvard.edu).
Received 13 August 1998; accepted in final form 22 July 1999.
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