EDITORIAL FOCUS
Actin filament organization is required for proper cAMP-dependent activation of CFTR

Adriana G. Prat1,2, C. Casey Cunningham2,3, G. Robert Jackson Jr.1, Steven C. Borkan4, Yihan Wang4, Dennis A. Ausiello1,2, and Horacio F. Cantiello1,2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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(-) cell line (M2) with an LK444 expression vector encoding for resistance to G418, which either did or did not contain the cDNA for full-length ABP-280 (10). ABP(-) and ABP(+) cells were further transfected with the pREP vector encoding for resistance to hygromycin, which either did or did not contain the full-length wild-type CFTR cDNA. After previously described standard transfection procedures (10) were performed, CFTR-expressing clones were selected and individually cultured with hygromycin (200 µg/ml)-containing medium. Four cell lines were thus obtained with all possible combinations of presence or absence of ABP-280 and CFTR, namely, ABP(-)/CFTR(-), ABP(+)/CFTR(-), ABP(-)/CFTR(+), and ABP(+)/CFTR(+) cells. CFTR was also expressed in human melanoma cells expressing a mutated form of ABP-280 that binds to membrane proteins but is specifically truncated at the self-association domain of the molecule, thus rendering the protein unable to cross-link actin filaments into gel networks. Melanoma cells transfected with this mutated form of ABP-280 [ABP-Trunc(+)/CFTR(+)] expressed a protein missing up to 250 amino acids from the COOH-terminal end of ABP-280. Briefly, truncated ABP-280 cDNA was obtained by using controlled Exo III endonuclease digestion of the full-length ABP-280 cDNA truncated from 80 to 1,100 bp deleted from the 3'-end.

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-GOmega feedback resistor in the head stage. Data were acquired, digitized, and stored as indicated below. Holding potentials refer to the patch pipette. The patch pipette and bathing solution was (in mM) 140 NaCl, 5.0 KCl, 0.8 MgCl2, 1.2 CaCl2 and 10 (HEPES), pH 7.4. In some experiments, NaCl in the pipette (140 mM) was replaced by MgCl2 (70 mM), all other solutes remaining the same. To determine anion selectivity of the cAMP-activated whole cell currents, the bathing NaCl solution was replaced with solutions containing HEPES (10 mM) and equimolar concentrations of either NaBr, NaI, or sodium gluconate, pH 7.4. Whenever indicated, the patch pipette was filled up to one-third of its height with MgATP (100 mM, pH 7.4, adjusted with N-methylglucamine) as previously described (8, 27, 28). The rest of the pipette was backfilled with the NaCl-containing solution as described above.

Single-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-GOmega 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 at -80°C. Samples were thawed on ice, and a whole cell lysate was obtained by passing each sample through a 26-gauge needle (×15 passes). Protein content was determined with the BCA protein assay (Pierce, Rockford, IL). CFTR and ABP-280 samples were separated by a 7.5% SDS-polyacrylamide gel and a 4-20% SDS-polyacrylamide gradient gel, respectively. Samples were then transferred to polyvinylidene difluoride membranes and blocked for 1 h with 5% dried milk also containing 0.5% nonimmune goat serum in 50 mM Tris · Hcl, pH 7.6, 141 mM NaCl, and 0.2% Tween 20 (TBST). Blots were probed with one of two monoclonal antibodies directed against CFTR (MAb 13-1 or MAb 24-1 from Genzyme, Cambridge, MA; 0.5-1 µg/ml) or ABP-280 (1 µg/ml), diluted in TBST containing 1% BSA, for 24-48 h at 4°C. Binding of primary antibody was detected with a horseradish peroxidase-based enzyme-linked chemiluminescence system (Kirkegaard & Perry, Gaithersburg, MD).

Actin-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 Cl- channel blocker diphenylamine-2-carboxylate (DPC; Fluka Chemical, Ronkonkoma, NY) was kept in a 20 mM stock solution in ethanol. DIDS (Sigma) was kept in a 10 mM stock solution in distilled water and used at a final concentration of 500 µM. The catalytic subunit of the cAMP-dependent protein kinase (PKA; Sigma) was used at a final concentration of 10 µg/ml. The monoclonal antibody MAb 13-1 (Genzyme) raised against the R-domain of CFTR was used at a final concentration of 2.92 µg/ml. Whenever indicated, a heat-inactivated antibody was prepared by incubating the antibody at 100 °C for 30 min.

Permeability-to-Selectivity Ratio Calculations

The ATP over Cl- permeability-to-selectivity ratio (PATP/PCl) was calculated with a derivation of the Goldman-Hodgkin-Katz equation (18) from the cAMP-stimulated Cl- and ATP currents obtained under asymmetrical conditions, such that
<IT>P</IT><SUB>ATP</SUB>/<IT>P</IT><SUB>Cl</SUB> = {<IT>z</IT><SUP>2</SUP><SUB>Cl</SUB> × Cl<SUB>o</SUB> × [1 − exp(<IT>az</IT><SUB>A</SUB><IT>V</IT><SUB>r</SUB>)]}

/{<IT>z</IT><SUP>2</SUP><SUB>A</SUB> × A<SUB>i</SUB> × exp(<IT>az</IT><SUB>A</SUB><IT>V</IT><SUB>r</SUB>) × [1 − exp(<IT>az</IT><SUB>Cl</SUB><IT>V</IT><SUB>r</SUB>)]}
where a = -F/RT, zA is the valence of ATP, Ai is the concentration of ATP (100 mM), and Vr is the reversal potential of the whole cell currents under asymmetrical conditions.

Similarly, the permeability-to-selectivity ratio of different anions (PCl/PY) was calculated using a modified equation such that
<IT>P</IT><SUB>Cl</SUB>/<IT>P</IT><SUB>Y</SUB> = {<IT>z</IT><SUP>2</SUP><SUB>Y</SUB> × Y<SUB>o</SUB> × [1 − exp(<IT>az</IT><SUB>Cl</SUB><IT>U</IT>)]}

/{<IT>z</IT><SUP>2</SUP><SUB>Cl</SUB> × Cl<SUB>i</SUB> × exp(<IT>az</IT><SUB>Cl</SUB><IT>U</IT>) × [1 − exp(<IT>az</IT><SUB>Y</SUB><IT>U</IT>)]}
where a = -F/RT, zCl is the valence of Cl- (-1), Cli is the concentration of intracellular Cl- (149 mM), zY is the valence of the substituted anion (-1 for either Br-, I-, or gluconate), and Yi is the concentration of extracellular anion (140 mM). U is the difference between the calculated reversal potential (EY, -1.59 mV) and the observed reversal potential (Vr) under asymmetrical conditions.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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(-) and ABP(+) melanoma cells also expressing CFTR. Addition of cAMP cocktail to either the ABP(-) cells also lacking CFTR [ABP(-)/CFTR(-)] or cells transfected with ABP-280 alone [ABP(+)/CFTR(-)] was without effect on the whole cell Cl- currents [0.67 ± 0.19 nS/cell (n = 7) vs. 0.31 ± 0.11 nS/cell (n = 6) and 1.50 ± 0.72 nS/cell (n = 3) vs. 0.81 ± 0.50 nS/cell (n = 3) for control and cAMP conditions, respectively; Fig. 1, A and B]. Whole cell currents from CFTR-expressing ABP(-) cells [ABP(-)/CFTR(+)] were also insensitive to the cAMP stimulatory cocktail [0.91 ± 0.31 nS/cell (n = 9) vs. 1.68 ± 0.67 nS/cell (n = 9); Figs. 1C and 2A]. Addition of cAMP stimulatory cocktail to CFTR-expressing, ABP(+) cells, [ABP(+)/CFTR(+)], in contrast, induced a 1,280% increase in the whole cell Cl- currents [0.85 ± 0.36 nS/cell (n = 11) vs. 10.4 ± 2.25 nS/cell (n = 8), P < 0.001; Figs. 1D and 2B]. However, cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments [ABP-Trunc(+)/CFTR(+) cells] were also insensitive to cAMP stimulation [2.56 ± 0.65 nS/cell (n = 10) vs. 2.59 ± 1.00 nS/cell (n = 6); Fig. 2C]. One experiment out of seven tested in ABP-Trunc(+)/CFTR(+) cells showed cAMP activated whole cell Cl- currents (from 2.9 to 11.2 nS/cell), indicating a maximal response similar to that of ABP(+)/CFTR(+) cells.





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Fig. 1.   Effect of cAMP on basal Cl- currents of cystic fibrosis transmembrane conductance regulator (CFTR) transfected human melanoma cells. Representative whole cell currents obtained between +100 and -100 mV in symmetrical Cl- (140 mM) in ABP(-)/CFTR(-) (A), ABP(+)/CFTR(-) (B), ABP(-)/CFTR(+) (C), and ABP(+)/CFTR(+) (D) cells, before (top trace) and after (middle trace) addition of a cAMP-stimulatory cocktail. cAMP-activated whole cell currents from ABP(+)/CFTR(+) cells were readily inhibited by diphenylamine-2-carboxylate (DPC; 0.5 mM; bottom traces). Data are representative of 3-9 experiments for the various cell types.





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Fig. 2.   Effect of cAMP on Cl- whole cell currents of CFTR-transfected human melanoma cells. Current-voltage relationships from the whole cell currents of ABP(-)/CFTR(+) (A), ABP(+)/CFTR(+) (B), and ABP-Trunc(+)/CFTR(+) (C) cells were obtained before (open circle ) and after () addition of a cAMP-stimulatory cocktail in the presence of symmetrical Cl- (140 mM). Data are from 4-6 experiments. Whole cell currents (I, nA) were obtained between +100 and -100 mV. Vh, holding potential applied to the pipette electrode.

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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Fig. 3.   Effect of cAMP and DPC on Cl- and ATP whole cell currents of ABP(+)/CFTR(+) cells. Whole cell currents were obtained in symmetrical Cl- (140 mM) or in the presence of MgATP (100 mM) in the patch pipette and bathing NaCl (140 mM). The whole cell conductance was calculated before (left bar) and after (middle bar) addition of a cAMP-stimulatory cocktail and after further addition of DPC (500 µM) to the bathing solution (right bar). Data are from 11, 8, and 4 experiments for control, cAMP, and cAMP + DPC, respectively. * P < 0.001 and ** P < 0.01.

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 Cl- (1.0) > Br- (0.88) >=  I- (0.81) >> gluconate (0.53), determined by the shift in reversal potential (n = 4-5, P < 0.05, for all ratios) when extracellular Cl was replaced with other anions. This is in agreement with previous data of wild-type CFTR, which follow almost the same permeability-to-selectivity ratio series (33). Another indication for CFTR to be activated in the CFTR(+)/ABP(+) cells comes from the permeability to cellular ATP observed, in agreement with previous studies (27, 28). Under asymmetrical ATP/Cl conditions (100 mM MgATP in pipette, 140 mM NaCl in bath), ATP-permeable whole cell currents were also observed after activation with the cAMP cocktail [1.22 ± 0.37 nS/cell (n = 6) vs. 5.70 ± 1.30 nS/cell (n = 3), P < 0.02, for control and cAMP activated, respectively]. The Cl- and ATP currents were simultaneously inhibited by bathing DPC [5.70 ± 1.30 nS/cell (n = 3) vs. 1.80 ± 1.30 nS/cell (n = 2)]. Further, the reversal potential of the cAMP-activated ATP/Cl- currents indicated an ATP/Cl- permeability-to-selectivity ratio of 0.40, in agreement with values previously reported for CFTR-expressing cells (28) and purified CFTR (6). In summary, the cAMP stimulatory cocktail only activated CFTR-mediated whole cell currents in the ABP-280-expressing [ABP(+)/CFTR(+)] cells.

Effect of Intracellular ABP-280 Dyalisis on the Whole cell Currents of CFTR-Expressing ABP(-) Cells

To further assess the regulatory nature of actin filament organization on the cAMP-dependent activation of CFTR in human melanoma cells, the effect of the actin-cross-linking protein ABP-280 was assessed in ABP(-)/CFTR(+) cells by dialysis of either filamin (0.1-20 nM) or the nonmuscle isoform ABP-280 (2 nM) from the patch pipette (Fig. 4, A and B). In the presence of intracellular ABP-280, addition of cAMP stimulatory cocktail induced CFTR activation similar to that observed in ABP(+)/CFTR(+) cells in five of six experiments (Fig. 4A). When filamin was intracellularly dialyzed from the patch pipette, a dose-response effect was observed. cAMP-dependent activation of the whole cell currents of ABP(-)/CFTR(+) cells was observed in five of five experiments between 0.5 and 20 nM intracellular filamin, whereas no effect was detected when using 0.1 nM filamin (n = 3, Fig. 4B). Taken together, these data further suggest that CFTR is expressed in the plasma membrane of the cells but requires the presence of an actin-cross-linking protein to be sensitive to the cAMP pathway. The presence of CFTR in ABP(-)/CFTR(+) cells was further determined by Western blotting, indicating that the level of CFTR expression was similar to that of ABP(+)/CFTR(+) cells (Fig. 4C). CFTR(-) cells displayed no specific labeling for this channel protein (Fig. 4C).




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Fig. 4.   Effect of intracellular ABP-280 on basal Cl- currents of ABP(-)/CFTR(+) cells. A: whole cell currents at +100 mV were obtained in symmetrical NaCl (140 mM) in the presence (open circle ) or the absence () of ABP-280 (2 nM) in the patch pipette and followed as a function of time. Arrows indicate addition of cAMP stimulatory cocktail and DPC. Data are from a representative experiment for each condition from 5 and 4 experiments for ABP-280 and control, respectively. B: cAMP-dependent whole cell Cl- currents at +100 mV were also obtained in the presence of intracellular filamin (0.1-20 nM; triangle ). Results were compared with those obtained in the presence of ABP-280 (2 nM) in the patch pipette (). Data are from 8 and 5 experiments for filamin and ABP-280, respectively. C: detection of ABP-280 and CFTR in human melanoma cells. Presence or absence of CFTR and ABP-280 were determined in control and transfected human melanoma cells as described in MATERIALS AND METHODS. The CFTR and ABP-280 studies were each performed on a single gel but are shown separated to improve clarity. Immunolabeling of CFTR (top) was conducted in membranes isolated from ABP(+)/CFTR(+) (left lane), ABP(-)/CFTR(+) (middle lane), and ABP(-)/CFTR(-) (right lane) cells. Data are representative of 3 experiments.

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(-)/CFTR(-) (Fig. 5A) or ABP(+)/CFTR(-) (Fig. 5B) cells had no effect on single ion channel activity (n = 3 for each condition). Furthermore, PKA plus ATP was also without effect on excised patches from ABP(-)/CFTR(+) cells (n = 3, Fig. 5C). However, addition of PKA plus ATP readily activated single Cl- channel currents in excised patches from ABP(+)/CFTR(+) cells (n = 6, Fig. 5D). The PKA-activated ion channels of ABP(+)/CFTR(+) cells had a conductance of 9.57 ± 0.58 pS (n = 12, Fig. 6A) in symmetrical Cl- (150 mM). The PKA-activated channels were inhibited by anti-CFTR antibodies (2.92 µg/ml, Fig. 6B) and by the Cl- channel blocker DPC (0.5 mM, Fig. 6C) but were insensitive to both a heat-inactivated antibody (2.92 µg/ml, Fig. 6B) and to DIDS (0.4 mM, n = 3, data not shown). The current-voltage relationship and the inhibitory effects of DPC and anti-CFTR antibodies, but not DIDS, were consistent with the functional fingerprinting of Cl--permeable CFTR-mediated single-channel currents. Addition of actin (1 mg/ml) to the cytoplasmic side of excised patches from ABP(+)/CFTR(+) cells also induced ion channel activity similar to PKA (data not shown).


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Fig. 5.   Effect of protein kinase A (PKA) on CFTR activation in ABP-280-expressing cells. Addition of PKA (10 µg/ml) + ATP (1 mM) to the cytoplasmic side of quiescent excised, inside-out patches from ABP(-)/CFTR(-) (A), ABP(+)/CFTR(-) (B), ABP(-)/CFTR(+) (C), and ABP(+)/CFTR(+) (D) cells induced and/or increased Cl- channel activity only in cells transfected with both CFTR and ABP-280. Holding potential was -80 mV. Channels in ABP(+)/CFTR(+) cells activated in clusters (see also Fig. 6B). Dashed line indicates the closed state of the ion channels. Traces obtained in symmetrical Cl- conditions (70 mM MgCl2/140 mM NaCl) are representative of 3 (A-C) and 6 (D) experiments.





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Fig. 6.   Single-channel currents of CFTR-mediated Cl- currents in human melanoma cells. A: single-channel currents (left) were obtained in symmetrical Cl- (140 mM N-methylglucamine chloride) from excised inside-out patches of ABP(+)/CFTR(+) cells. Current-voltage relationship (right) had a slope of 9.57 ± 0.58 pS (n = 12). B: addition of the anti-CFTR monoclonal antibody MAb 13-1 (2.92 µg/ml) to excised patches from ABP(+)/CFTR(+) cells (bottom trace) inhibited the PKA-stimulated ion channel activity (top trace). Heat-inactivated MAb 13-1 (2.92 µg/ml), however, was without effect on inhibiting the PKA-stimulated channels (middle trace). Data are representative of 6 experiments. C: PKA-activated single-channel Cl- currents (top trace) were inhibited by DPC (500 µM, bottom trace). Data are representative of 6 experiments.

Effect of Exogenous ABP-280 on CFTR Activation in ABP(-) Cells

Addition of filamin (30 nM) to the cytoplasmic side of excised patches from ABP(-)/CFTR(+) cells in the absence of PKA and ATP induced ion channel activity similar to that observed in ABP(+)/CFTR(+) cells in four of five experiments (Fig. 7). These data are in agreement with the whole cell currents and further support the requirement of cross-linked actin networks for proper activation of CFTR.


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Fig. 7.   Effect of filamin on CFTR activation in ABP(-)/CFTR(+) cells. Addition of filamin (30 nM) alone to excised patches from ABP(-)/CFTR(+) cells stimulated Cl- channel activity (compare top and bottom traces) in a manner similar to the PKA-stimulated ion channel activity of ABP(+)/CFTR(+) cells and previously reported Cl- channel activity (27). Channels also activated in clusters as in Fig. 5D. Traces are representative of 4 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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), alpha -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, M. P., D. P. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. Generation of cAMP-activated chloride current by expression of CFTR. Science 251: 679-682, 1991[Medline].

2.   Berger, H. A., M. P. Anderson, R. J. Gregory, S. Thompson, P. W. Howard, R. A. Maurer, R. Mulligan, A. E. Smith, and M. J. Welsh. Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel. J. Clin. Invest. 88: 1422-1431, 1991[Medline].

3.   Cantiello, H. F. Actin filaments stimulate Na+-K+-ATPase. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F637-F643, 1995[Abstract/Free Full Text].

4.   Cantiello, H. F. Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator. Exp. Physiol. 83: 505-514, 1996.

5.   Cantiello, H. F. Role of the actin cytoskeleton on epithelial Na+ channel regulation. Kidney Int. 48: 970-984, 1995[Medline].

6.   Cantiello, H. F., G. R. Jackson, Jr., C. F. Grosman, A. G. Prat, S. C. Borkan, Y.-H. Wang, I. L. Reisin, C. R. O'Riordan, and D. A. Ausiello. Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. 274 (Cell Physiol. 43): C799-C809, 1998[Abstract/Free Full Text].

7.   Cantiello, H. F., A. G. Prat, J. V. Bonventre, C. C. Cunningham, J. Hartwig, and D. A. Ausiello. Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells. J. Biol. Chem. 268: 4596-4599, 1993[Abstract/Free Full Text].

8.   Cantiello, H. F., A. G. Prat, I. L. Reisin, E. H. Abraham, L. B. Ercole, J. F. Amara, R. J. Gregory, and D. A. Ausiello. External ATP activates the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269: 11224-11232, 1994[Abstract/Free Full Text].

9.   Cantiello, H. F., J. Stow, A. G. Prat, and D. A. Ausiello. Actin filaments control epithelial Na+ channel activity. Am. J. Physiol. 261 (Cell Physiol. 30): C882-C888, 1991[Abstract/Free Full Text].

10.   Cunningham, C. C., J. B. Gorlin, D. J. Kwiatkowski, J. H. Hartwig, P. A. Janmey, H. R. Byers, and T. P. Stossel. Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255: 325-327, 1992[Medline].

11.   Dechecchi, M., A. Tamanini, G. Berton, and G. Cabrini. Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J. Biol. Chem. 268: 11321-11325, 1993[Abstract/Free Full Text].

12.   Ding, G., N. Franki, J. Condeelis, and R. M. Hays. Vasopressin depolymerizes F-actin in toad bladder epithelial cells. Am. J. Physiol. 260 (Cell Physiol. 29): C9-C16, 1991[Abstract/Free Full Text].

13.   Feliciello, A., Y. Li, E. Avvedimento, M. Gottesman, and C. Rubin. A-kinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus. Curr. Biol. 7: 1011-1014, 1997[Medline].

14.   Gariboldi, M., E. Maestrini, F. Canzian, G. Manenti, L. De Gregorio, S. Rivella, A. Chatterjee, G. Herman, N. Archidiacono, R. Antonacci, M. Pierotti, T. Dragani, and D. Toniolo. Comparative mapping of the actin-binding protein 280 genes in human and mouse. Genomics 21: 428-430, 1994[Medline].

15.   Gorlin, J., E. Henske, S. Warren, C. Kunst, M. D'Urso, G. Palmieri, J. Hartwig, G. Bruns, and D. Kwiatkowski. Actin-binding protein (ABP-280) filamin gene (FNL) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28. Genomics 17: 496-498, 1993[Medline].

16.   Hartwig, J., and D. Kwiatkowski. Actin-binding proteins. Curr. Opin. Cell Biol. 3: 87-97, 1991[Medline].

17.   Hartwig, J., and T. P. Stossel. Isolation and properties of actin, myosin, and a new actin binding protein in rabbit alveolar macrophages. J. Biol. Chem. 250: 5696-5705, 1975[Abstract].

18.   Hille, B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.

19.   Lamprecht, G., E. Weinman, and C. Yun. The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3. J. Biol. Chem. 273: 29972-29978, 1998[Abstract/Free Full Text].

20.   Matsudaira, P. Modular organization of actin crosslinking proteins. Trends Biol. Sci. 16: 87-92, 1991.

21.   McCarty, N. A., S. McDonough, B. N. Cohen, J. R. Riordan, N. Davidson, and H. A. Lester. Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates. J. Gen. Physiol. 102: 1-23, 1993[Abstract].

22.   Meyer, S., S. Zuerbig, C. Cunningham, J. Hartwig, T. Bissell, K. Gardner, and J. Fox. Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein Ibalpha . J. Biol. Chem. 272: 2914-2919, 1997[Abstract/Free Full Text].

23.   Moos, J., J. Peknicova, G. Geussova, V. Philimonenko, and P. Hozak. Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells. Mol. Reprod. Dev. 50: 79-85, 1998[Medline].

24.   Ohta, Y., T. P. Stossel, and J. H. Hartwig. Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fcgamma RI). Cell 67: 1-20, 1991[Medline].

25.   Prat, A. G., D. A. Ausiello, and H. F. Cantiello. Vasopressin and protein kinase A activate G protein-sensitive Na channels. Am. J. Physiol. 265 (Cell Physiol. 34): C218-C223, 1993[Abstract/Free Full Text].

26.   Prat, A. G., A. M. Bertorello, D. A. Ausiello, and H. F. Cantiello. Activation of epithelial Na+ channels by protein kinase A requires actin filaments. Am. J. Physiol. 265 (Cell Physiol. 34): C224-C233, 1993[Abstract/Free Full Text].

27.   Prat, A. G., Y.-F. Xiao, D. A. Ausiello, and H. F. Cantiello. cAMP-independent regulation of CFTR by the actin cytoskeleton. Am. J. Physiol. 268 (Cell Physiol. 37): C1552-C1561, 1995[Abstract/Free Full Text].

28.   Reisin, I. L., A. Prat, E. H. Abraham, J. F. Amara, R. J. Gregory, D. A. Ausiello, and H. F. Cantiello. The cystic fibrosis transmembrane conductance regulator (CFTR) is a dual ATP and chloride channel. J. Biol. Chem. 269: 20584-20591, 1994[Abstract/Free Full Text].

29.   Rich, D. P., M. P. Anderson, R. I. Gregory, S. H. Cheng, S. Paul, D. N. Jefferson, I. D. McCann, K. W. Klinger, A. E. Smith, and M. J. Welsh. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 347: 358-363, 1990[Medline].

30.   Schwiebert, E., N. Kizer, D. Gruenert, and B. Stanton. GTP-binding proteins inhibit cAMP activation of chloride channels in cystic fibrosis airway epithelial cells. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992[Abstract].

31.   Shizuta, Y., H. Shizuta, M. Gallo, P. Davies, and I. Pastan. Purification and properties of filamin, an actin binding protein from chicken gizzard. J. Biol. Chem. 251: 6562-6567, 1976[Abstract].

32.   Short, D., K. Trotter, D. Reczek, S. Kreda, A. Bretscher, R. Boucher, M. Jackson Stutts, and S. Milgram. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J. Biol. Chem. 273: 19797-19801, 1998[Abstract/Free Full Text].

33.   Tabcharani, J., P. Lindsell, and J. Hanrahan. Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels. J. Gen. Physiol. 110: 341-354, 1997[Abstract/Free Full Text].

34.   Weihing, R. R. The filamins: properties and functions. Can. J. Biochem. Cell Biol. 63: 397-413, 1985[Medline].

35.   Yun, C., G. Lamprecht, D. Forster, and A. Sidor. NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J. Biol. Chem. 273: 25856-25863, 1998[Abstract/Free Full Text].


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