From the Departments of a Physiology and h Biology, Dartmouth Medical School, Hanover, New Hampshire 03755, the g Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, and the d Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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The mechanism by which cAMP stimulates cystic
fibrosis transmembrane conductance regulator (CFTR)-mediated chloride
(Cl) secretion is cell type-specific. By using
Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we
tested the hypothesis that cAMP stimulates Cl
secretion
by stimulating CFTR Cl
channel trafficking from an
intracellular pool to the apical plasma membrane. To this end, we
generated a green fluorescent protein (GFP)-CFTR expression vector in
which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR
function in whole cell patch-clamp or planar lipid bilayer experiments.
In stably transfected MDCK type I cells, GFP-CFTR localization was
substratum-dependent. In cells grown on glass coverslips,
GFP-CFTR was polarized to the basolateral membrane, whereas in cells
grown on permeable supports, GFP-CFTR was polarized to the apical
membrane. Quantitative confocal fluorescence microscopy and surface
biotinylation experiments demonstrated that cAMP did not stimulate
detectable GFP-CFTR translocation from an intracellular pool to the
apical membrane or regulate GFP-CFTR endocytosis. Disruption of the
microtubular cytoskeleton with colchicine did not affect
cAMP-stimulated Cl
secretion or GFP-CFTR expression in
the apical membrane. We conclude that cAMP stimulates CFTR-mediated
Cl
secretion in MDCK type I cells by activating channels
resident in the apical plasma membrane.
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INTRODUCTION |
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The cystic fibrosis transmembrane conductance regulator
(CFTR),1 a cAMP-activated
chloride (Cl) channel, is targeted to the apical plasma
membrane region in many epithelial cells, including those in the kidney
(1-3), and is defective in the genetic disease cystic fibrosis (4).
Stimulation of CFTR-mediated Cl
secretion by cAMP has
been reported to occur by the following two mechanisms that are not
mutually exclusive: first, cAMP stimulates protein kinase A-mediated
phosphorylation and activation of CFTR Cl
channels
resident in the plasma membrane (5-7); and second, cAMP stimulates
trafficking of CFTR from an intracellular pool to the plasma membrane
while decreasing endocytic retrieval of CFTR from the plasma membrane
(8-11). The second mechanism is more controversial; in intestinal
epithelial cells, some investigators have found positive effects of
cAMP on CFTR trafficking to the apical membrane (12), whereas other
investigators have not (2, 13). In contrast, little is known about the
intracellular trafficking of CFTR or the mechanism(s) by which cAMP
stimulates CFTR-mediated Cl
secretion in kidney
epithelia. Because CFTR Cl
channels are expressed in all
nephron segments of the kidney (3) and are important for
transepithelial Cl
transport (14, 15) and enlargement of
renal cysts in polycystic kidney disease (16), it is important to
elucidate the role of cAMP in the regulation of CFTR in normal and
pathophysiological renal states.
The study of CFTR trafficking in many epithelial cells, including renal epithelia, is hampered by the low level of endogenous CFTR expression (1, 17). To begin to understand the trafficking of CFTR, we constructed a jellyfish green fluorescent protein (GFP)-CFTR expression vector in which GFP was ligated to the N terminus of wild-type CFTR, and we used GFP fluorescence to localize CFTR in living and fixed cells. GFP, a 27-kDa protein from the jellyfish Aequorea victoria, has emerged as an in vivo reporter protein for studying complex biological processes such as organelle dynamics and protein trafficking (18, 19). GFP generates a bright green fluorescence, is resistant to photobleaching, does not require any exogenous cofactors or substrates to fluoresce, and, when ligated to other proteins, generally does not alter fusion protein function or localization (18, 20).
The present study was conducted to test the hypothesis that cAMP
stimulates CFTR-mediated Cl secretion in mammalian kidney
epithelial cells by inducing a relocation of CFTR from intracellular
organelles to the apical plasma membrane. By using MDCK type I cells as
a model, we generated stable transfectants expressing full-length
GFP-CFTR fusion protein. By using quantitative confocal fluorescence
microscopy, cell-surface biotinylation, and short circuit current
(Isc) analyses, we demonstrate that the predominant
mechanism by which cAMP stimulates GFP-CFTR-mediated Cl
secretion is by activating channels resident in the apical membrane and
not by stimulating insertion of channels into the apical membrane or
inhibiting retrieval of channels from the apical membrane.
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EXPERIMENTAL PROCEDURES |
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GFP-CFTR and CFTR Expression Vectors
To construct the pGFP-CFTR mammalian expression vector, human
CFTR cDNA was excised from pBluescript II SK (Stratagene, La Jolla, CA) with AvaI, treated with Klenow fragment to
fill-in sticky ends, and ligated into SmaI-digested and calf
intestinal alkaline phosphatase-treated pS65T-GFP-C1
(CLONTECH, Palo Alto, CA). To maximize GFP
fluorescence, S65TGFP cDNA was exchanged for enhanced GFP cDNA
using AgeI/KpnI. Enhanced GFP is codon-optimized for expression in mammalian systems and exhibits 6-fold greater levels
of fluorescence than S65T-GFP (21). DNA sequence analysis of the
GFP-CFTR junction confirmed the intended reading frame. Proceeding from
the N to the C terminus, the resultant fusion protein consists of GFP,
a linker sequence of 23 amino acids, and CFTR. Based on the predicted
transmembrane topology of CFTR (4), GFP resides in the cytoplasmic
compartment. pCFTR expression vector (with no GFP tag) (22) was used to
compare the function of GFP-CFTR to CFTR in whole cell patch-clamp
experiments. To examine the effect of GFP on CFTR function in planar
lipid bilayer experiments, GFP-CFTR was subcloned from pGFP-CFTR into
pcDNA3.1 (Invitrogen, Carlsbad, CA) using NheI and
EcoRV (to generate pcDNA3.1 GFP-CFTR). Similarly, CFTR
was subcloned from pGFP-CFTR into pcDNA3.1 using Asp718
and XhoI (to generate pcDNA3.1 CFTR). These vectors allow synthesis of cRNA for expression in Xenopus
oocytes.
Cell Culture
MDCK type I cells were obtained from the American Type Tissue Collection (pass number 54, CCL-34, Rockville, MD) and grown on tissue culture-treated polystyrene flasks in minimum essential medium with Earle's salts (Life Technologies, Inc.) containing 10% fetal bovine serum (HyClone), 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine (Life Technologies, Inc.). Cells were grown in 5% CO2-balanced air at 37 °C. When confluent, cells were subcultured by trypsinization (0.05% trypsin, 0.53 mM EDTA in Hanks' balanced salt solution; Life Technologies, Inc.). For short circuit current and confocal microscopic experiments, cells were seeded at 50,000-75,000 cells/0.33 cm2, and for surface biotinylation experiments, cells were seeded at 200,000/4.5 cm2 on permeable Transwell filter bottom cups (Costar, Cambridge, MA). Cells were fed daily and used 5-7 days after seeding. Because GFP fluorescence is temperature-sensitive (23, 24), we grew all cells used for experiments at 33 °C to maximize GFP signal intensity. In preliminary experiments, we observed that culturing cells at reduced temperature did not affect cell growth or viability and did not alter GFP-CFTR localization compared with cells cultured at 37 °C.
NIH-3T3 fibroblasts, which do not express detectable levels of
endogenous CFTR Cl channels (25), were use in whole cell
patch-clamp experiments to examine whether GFP affected CFTR
Cl
channel function. Cells were grown in Dulbecco's
modified Eagle's high glucose medium (Life Technologies, Inc.) with
5% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml
streptomycin, 2 mM L-glutamine, and 0.2%
fungizone (Biofluids, Rockville, MD). Cells were seeded at 30-50%
confluency prior to transfection in 35-mm tissue culture dishes coated
with 1:20 diluted Vitrogen 100 (purified bovine dermal collagen;
Collagen Corp., Fremont, CA).
Transfection
NIH-3T3 fibroblasts were transiently transfected with LipofectAMINE PLUS (Life Technologies, Inc.) as per the manufacturer's instructions using 1 µg of pGFP-CFTR or pCFTR with 1 µg of pGFP (Green Lantern plasmid; Life Technologies, Inc.) and 1 µg pRL-CMV luciferase reporter vector (Promega, Madison, WI). The GFP plasmid was used to identify green fluorescent cells, which were likely co-transfected with the CFTR constructs, using a Nikon Eclipse TE200 inverted fluorescence microscope intrinsic to the patch-clamp system. The luciferase plasmid was used to assess relative transfection efficiency; all transfectants displayed equivalent levels of luciferase activity. Transfected cells were transferred to 33 °C for 2-3 days before patch-clamp recording.
To generate MDCK cells stably transfected with pGFP-CFTR, we first optimized transient transfection efficiency with the PerFect Lipid Transfection Kit (Invitrogen) according to the manufacturer's instructions (26). A T75 flask of MDCK cells was transfected with pGFP-CFTR plasmid. Twenty-four hours post-transfection, cells were selected with 300 µg/ml G418 (Life Technologies, Inc.) and fed every 4-5 days for 2 weeks in complete media containing G418. Surviving cell colonies were trypsinized, and single cells with bright GFP fluorescence were sorted into individual wells of 96-well plates using a FACStar PLUS flow cytometer (Becton Dickenson, San Jose, CA). GFP fluorescence was excited using the 488 nm line from an argon laser and collected with a 530/30 nm band pass filter. Clones were expanded and screened for GFP fluorescence by confocal fluorescence microscopy. Similar experimental results were found in MDCK C7 cells (generous gift of Dr. Hans Oberleithner) (27) stably transfected with GFP-CFTR. Following establishment of cell lines, G418 was reduced to 150 µg/ml and was removed 3-4 days prior to experimentation. MDCK stable transfectants were treated with 5 mM sodium n-butyrate (Sigma) for 15-18 h prior to experimentation to increase GFP-CFTR expression levels. Sodium butyrate was removed 2 h prior to experimentation.
Whole Cell Patch-Clamp
Whole cell patch-clamp recording of GFP-positive cells was
performed as described previously in detail (22, 28, 29). Only 50% of
pGFP-CFTR and pCFTR transfectants expressing visible levels of GFP
fluorescence responded to cAMP treatment with an increase in
Cl conductance. Non-responding cells, which were probably
transfected with pGFP but not pGFP-CFTR or pCFTR, exhibited currents
similar to non- or mock-transfected cells, and were excluded from data analysis.
Planar Lipid Bilayers
Single channel properties of GFP-CFTR and CFTR Cl
channels were studied in planar lipid bilayers. Stage V-VI
Xenopus oocytes were harvested and injected with 5 ng of
CFTR cRNA, 5 ng of GFP-CFTR cRNA, or 50 nl of water as described
previously (30). Membrane vesicles were prepared 48 h
postinjection following the method of Pérez et al.
(31). Thirty to forty oocytes in each group were washed and homogenized
in high K+/sucrose medium containing the following protease
inhibitors: aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), phenylmethylsulfonyl fluoride (100 µM), and
DNase I (2 µg/ml). Oocyte membranes were isolated by discontinuous
sucrose gradient density centrifugation and resuspended in 300 mM sucrose, 100 mM KCl, and 5 mM
MOPS (pH 6.8). Membrane vesicles were separated into 50-µl fractions
and stored at
80 °C until use. Planar lipid bilayers were made
from a phospholipid solution containing a 1:1 mixture of
diphytanoyl-phosphatidylethanolamine/diphytanoyl-phosphatidylserine (in
n-octane; final phospholipid concentration of 25 mg/ml).
Membrane vesicles were applied with a fire-polished glass rod to one
side (trans) of a preformed bilayer bathed with symmetrical 100 mM KCl, 10 mM MOPS-Tris (pH 7.4). Acquisition
and analysis of single channel recordings were performed as described
(32, 33). Channel activity was recorded in the presence of 1.85 ng/ml
protein kinase A catalytic subunit (gift of Dr. Gail Johnson,
University of Alabama) and 100 µM ATP.
Short Circuit Current
Short circuit current (Isc) was measured across MDCK
monolayers as described previously (34). In all experiments, amiloride (105 M) was present in the apical bath
solution to inhibit electrogenic Na+ absorption. Under
these conditions, cAMP-stimulated Isc across monolayers of
MDCK cells is referable to Cl
secretion.
Immunocytochemistry
Unless specifically stated otherwise, all steps were performed at room temperature in Ca2+/Mg2+-free PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 9 mM Na2HPO4 (pH 7.1)), and antibody incubations were for 1 h.
Na/K-ATPase-- Cells were washed, fixed, and permeabilized with ice-cold 100% MeOH for 10 min and washed with 0.3% Triton X-100 (Sigma) and 0.1% BSA (PBS-TB). Nonspecific binding sites were blocked with 8% BSA for 30 min, and cells were incubated with 10 µg/ml anti-Na/K-ATPase monoclonal antibody (IgG1) (Upstate Biotechnology, Lake Placid, NY) overnight at 4 °C. Cells were washed with PBS-TB and incubated with 1:100 goat anti-mouse Texas Red secondary antibody (Molecular Probes, Eugene, OR) for 3 h. Cells were washed in PBS-TB and mounted in 90% glycerol, 10% PBS containing 10 mg/ml n-propyl gallate (Sigma) to retard fading.
ZO-1--
Cells were washed, fixed, and permeabilized in 100%
acetone for 2 min at 20 °C and incubated in 10 µg/ml anti-ZO-1
rabbit polyclonal antibody (Zymed, So. San Francisco) in 1% BSA. Cells were washed, incubated with 1:100 goat anti-rabbit Texas Red secondary antibody (Molecular Probes), and mounted as above.
CFTR-- Cells were immunostained essentially as described previously (2) using 10-40 µg/ml anti-CFTR R domain (IgG1) or anti-CFTR C-terminal (IgG2a) monoclonal antibodies (Genzyme, Cambridge, MA).
Biotinylated Membranes-- Biotinylated monolayers were fixed in 3.0% paraformaldehyde for 30 min on ice and permeabilized with 0.1% Triton X-100 for 3 min, and nonspecific binding sites were blocked with 2% BSA. Biotinylated proteins were detected with 50 µg/ml Texas Red-avidin (Pierce) in 1% BSA for 30 min, washed, and mounted as above.
Control experiments in which cells were stained with nonspecific antibody of the appropriate isotype (for monoclonal antibodies), non-immune serum (for polyclonal antibodies), and/or secondary antibody only demonstrated the specificity of observed signals.Cryosectioning
MDCK cells grown on permeable supports were fixed in 3.0% paraformaldehyde in Ca2+/Mg2+-free PBS for 15 min at room temperature. Monolayers were excised with a razor blade, cut into thin strips, embedded in Tissue-Tek (Miles, Ellhart, IN), and frozen in liquid nitrogen-cooled liquid propane. Sections 5-7 µm in thickness were cut with a cryostat and examined by confocal microscopy.
Confocal Microscopy
Images were acquired using a Zeiss (Thornwood, NY) Axioskop microscope equipped with a laser scanning confocal unit (model MRC-1024, Bio-Rad), a 15-milliwatt krypton-argon laser, and a × 63 Plan Apochromat/1.4 NA or × 40 Plan Neofluor/1.3 NA oil immersion objective. GFP fluorescence was excited using the 488-nm laser line and collected using a standard fluorescein isothiocyanate filter set (530 ± 30 nm). Fluorescence associated with Texas Red-labeled secondary antibodies and propidium iodide was simultaneously excited using the 568-nm laser line and collected using a standard Texas Red filter set (605 ± 32 nm). Three-dimensional reconstructions were rendered using LaserSharp version 2.1A (Bio-Rad) software. Acquired images were imported into National Institutes of Health Image version 1.57 software (Bethesda, MD) for quantitation and into Adobe Photoshop version 3.0 for image processing and printing. For live cell microscopy, cells were mounted in a temperature-controlled, flow-through perfusion chamber (RC21-B Chamber, Warner Instrument Corp., Hamden, CT) at 37 °C in PBS (pH 7.4) containing 1 mM CaCl2 and 0.5 mM MgCl2 to maintain cell adherence, 25 mM HEPES to buffer pH changes, and 5.5 mM glucose as an energy source. During image acquisition, solution was not perfused over cells to minimize drift in the z-dimension.
For quantitative confocal microscopy, all images from the same z series were collected using the same values for laser power, photomultiplier gain, iris, and black level. Typically, three scans were Kalman averaged per z section and 10 z sections were collected at 1.0-µm increments beginning at the apical membrane and ending at the basal membrane. Care was taken to ensure that pixel saturation was less than 10% and that signal intensities were in the linear range of photomultiplier tube sensitivity.
Cell-surface Biotinylation
Biotinylation of apical cell-surface glycoproteins was performed as described by Lisanti et al. (35). To examine GFP-CFTR endocytosis and recycling, cells were incubated at 37 °C between sodium periodate and biotin-LC-hydrazide treatments as described by Prince et al. (8). Following biotinylation, monolayers were solubilized in lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40 and containing the Complete Protease Inhibitor mixture (Boehringer Mannheim)), scraped from filters, and spun at 14,000 × g for 4 min to pellet insoluble material. Less than 1% of GFP-CFTR remained in the insoluble pellet as determined by Western blotting. Aliquots of cell lysates were removed for SDS-PAGE analysis, and the remainder of the supernatants were brought to a volume of 900 µl with lysis buffer and precipitated with 100 µl of a 50% slurry of streptavidin-agarose beads (Pierce) overnight at 4 °C with end-over-end rotation. Beads were pelleted by brief centrifugation for 30 s at 14,000 × g and washed three times with lysis buffer. Biotinylated proteins were eluted by boiling for 5 min in 50 µl of Laemmli sample buffer (0.24 M Tris-HCl (pH 8.9), 16% glycerol, 0.008% bromphenol blue, 5.6% SDS, and 80 mM dithiothreitol).
Glycosidase Digestion
Cell lysates (30-40 µg protein) were digested with endoglycosidase H (Endo H, 1500 units) or peptide N-glycosidase F (PNGase F, 1500 units) (New England Biolabs, Beverly, MA) for 1 h at room temperature following the manufacturer's instructions, with the exception that lysates were not denatured prior to digestion. Denaturation induced GFP-CFTR aggregation and protein failed to enter separating gels.
SDS-PAGE and Western Blotting
Cell lysates and biotinylated proteins were separated on 4-15% Tris-HCl gradient gels (Bio-Rad) and transferred to polyvinylidene difluoride Immobilon membranes (Millipore, Bedford, MA). Membranes were blocked overnight at 4 °C in 5% non-fat dry milk in Tris-buffered saline, 0.02% Tween 20 and incubated with either GFP (1:1000) (CLONTECH) or CFTR C-terminal (1:1000) monoclonal antibodies followed by anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5,000-1:10,000; Amersham Pharmacia Biotech). Blots were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech) using Hyperfilm ECL (Amersham Pharmacia Biotech) and digitally scanned with a Silverscan III flatbed scanner (LaCie, Hillsboro, OR). Densitometric analysis of band intensities was performed with public domain NIH Image version 1.57 software.
Statistical Analyses
Differences between means were compared by either paired or unpaired two-tailed Student's t test as appropriate using Instat statistical software (GraphPad, San Diego, CA). Data are expressed as mean ± S.E. Statistical significance is ascribed for p < 0.05.
Other Materials
8-(4-Chlorophenylthio)-cAMP (CPT-cAMP, monosodium salt) was purchased from Boehringer Mannheim and Sigma. 8-Bromo-cAMP, isobutylmethylxanthine, forskolin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), glybenclamide, colchicine, lumicolchicine, and propidium iodide were purchased from Sigma. Diphenylamine carboxylic acid (DPC) was purchased from Fluka (Milwaukee, WI) and Sigma.
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RESULTS |
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GFP-CFTR Functions as a cAMP-activated Cl
Channel--
Because CFTR Cl
channel function is
necessary for normal CFTR trafficking (8), we performed experiments to
examine whether fusion of GFP to the N terminus of CFTR affected
function as a cAMP-activated Cl
channel. To this end, we
performed whole cell patch-clamp experiments on transiently transfected
NIH-3T3 fibroblasts, which express no detectable endogenous CFTR
Cl
channels (25). As shown in Fig.
1, cAMP-activated Cl
currents were similar in cells expressing GFP-CFTR and CFTR (with no
GFP tag). Currents were not sensitive to DIDS but were inhibited by
glybenclamide, consistent with established CFTR pharmacology (10, 29,
36). To compare the single channel properties of GFP-CFTR to CFTR (with
no GFP tag), we performed planar lipid bilayer experiments. Recordings
from channels synthesized in Xenopus oocytes and
incorporated into planar lipid bilayers demonstrated that GFP-CFTR
single channel conductance, chloride to iodide permeability ratio, and
blocker sensitivity (inhibition by DPC but not DIDS) were similar to
CFTR (Table I and Fig.
2). These parameters are similar to those
previously reported for CFTR channels in planar lipid bilayers (32,
33), in stably transfected cells (37, 38), and in apical membranes of
cells expressing endogenous CFTR (38). Water-injected oocytes did not
produce any CFTR-like Cl
channel activity. Taken
together, these findings indicate that fusion of GFP to the N terminus
of CFTR does not affect CFTR Cl
channel function.
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GFP-CFTR Localization Is Substratum-dependent--
To
generate a cell model to study CFTR trafficking in polarized kidney
epithelial cells, we stably transfected MDCK type I cells, which
express low levels of endogenous CFTR Cl channels (36),
with pGFP-CFTR expression plasmid. By using laser scanning confocal
fluorescence microscopy, GFP-CFTR was localized to the basolateral
plasma membrane region in cells grown on glass coverslips (Fig.
3). Acute treatment with cAMP did not stimulate detectable trafficking of GFP-CFTR to apical or basolateral membranes (Fig. 3). By contrast, GFP-CFTR fluorescence was
predominantly localized to the apical plasma membrane region and
sub-apical vesicles in stably transfected, fully polarized cells
cultured on permeable supports (Fig. 4,
A and B). Similar results were obtained in MDCK
cells transiently transfected with pCFTR (with no GFP tag) and stained
with R domain or C-terminal CFTR antibodies, indicating that fusion of
GFP to CFTR does not alter CFTR subcellular localization or trafficking
to the apical membrane (Fig. 4C). We confirmed that GFP-CFTR
was polarized to the apical membrane region by performing
double-labeling experiments in fixed cells. By using a monoclonal
antibody against the Na/K-ATPase to label basolateral membranes,
GFP-CFTR did not colocalize with the Na/K-ATPase (Fig.
5A). By using a polyclonal
antibody against ZO-1, a protein localized to the cytoplasmic face of
tight junctions, GFP-CFTR was expressed in a horizontal plane between
tight junctions (Fig. 5B). Because CFTR is polarized to the
apical plasma membrane of many epithelial cells in vivo (1,
2), we performed all subsequent trafficking experiments using cells
grown on permeable supports to simulate the physiologically relevant
situation.
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GFP-CFTR Mediates Transepithelial Cl
Secretion--
We performed short circuit current experiments to
examine whether GFP-CFTR functioned as an apical membrane,
cAMP-activated Cl
channel in polarized cells by measuring
transepithelial Cl
secretion across monolayers of
parental untransfected and stably transfected MDCK cells. In all
experiments, amiloride (10
5 M) was present in
the apical bath solution to inhibit electrogenic Na+
absorption. Under these conditions, cAMP-stimulated Isc is
referable to Cl
secretion. A cAMP-stimulating mixture
(100 µM CPT-cAMP, 100 µM isobutylmethylxanthine, and 20 µM forskolin) elicited a
rapid and small increase in Isc in parental, untransfected
MDCK cells (Table II), consistent with
activation of endogenous CFTR Cl
channels. In GFP-CFTR
stable transfectants, cAMP-stimulating mixture elicited a rapid and
large increase in Isc which reached a peak value at 2 min,
remained elevated for the duration of cAMP treatment (up to 20 min),
and decreased following treatment with DPC (10 mM), an
inhibitor of CFTR Cl
channels (29) (Table II). These data
demonstrate that GFP-CFTR functions as a cAMP-stimulated apical
membrane Cl
channel and mediates transepithelial
Cl
secretion in polarized MDCK type I cells.
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Effect of cAMP on GFP-CFTR Trafficking-- We next tested the hypothesis that cAMP increases Isc by stimulating CFTR trafficking from an intracellular pool to the apical plasma membrane. Monolayers were treated with cAMP-stimulating mixture for 10 min, at which time cAMP-stimulated Isc has peaked and reached an elevated state, and the distribution of GFP-CFTR fluorescence along the apical to basal axis was quantitated in optical sections using confocal fluorescence microscopy. Apical and basal cell-surface boundaries were identified by labeling surface glycoproteins with wheat germ agglutinin-Texas Red at 4 °C. As shown in Fig. 6, cAMP did not affect GFP-CFTR distribution. Approximately, 70% of GFP-CFTR fluorescence was localized to the apical membrane and sub-apical membrane regions, comprising the first three optical sections in Fig. 6, in vehicle and cAMP-treated monolayers. Similar results were obtained in cells treated with cAMP for 60 min. Qualitatively similar results were obtained using confocal microscopy to localize GFP-CFTR in longitudinal cryosections sectioned along the apical-basal axis (Fig. 7).
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Role of Microtubules in GFP-CFTR Function and
Localization--
Microtubules are frequently involved in the
trafficking of transport proteins to the plasma membrane upon agonist
stimulation (42). To examine the role of microtubules in
cAMP-stimulated GFP-CFTR Cl channel function and GFP-CFTR
localization to the apical membrane region, we treated monolayers for
5-7 h with colchicine to depolymerize the microtubular cytoskeleton or
lumicolchicine, an inactive colchicine analog that does not
depolymerize microtubules. GFP-CFTR function was measured by short
circuit current analysis, and GFP-CFTR localization was examined by
confocal fluorescence microscopy. Results from short circuit current
experiments (Table III) demonstrated that colchicine had no effect on basal or cAMP-stimulated peak and elevated
Isc. Similar results were obtained in parental,
untransfected MDCK cells, suggesting that microtubule disruption does
not affect endogenous CFTR function (Table III). Following short
circuit current experiments, monolayers were fixed and stained with a
monoclonal antibody against
-tubulin. Microtubules were
depolymerized in colchicine-treated but not in lumicolchicine-treated
monolayers; however, GFP-CFTR polarization to the apical membrane
region was unaltered by colchicine treatment (data not shown). This
observation was confirmed by quantitative confocal fluorescence
microscopy and apical surface biotinylation (densitometric analysis of
apical membrane GFP-CFTR in colchicine-treated cells = 0.90 ± 0.15 (n = 6) compared with lumicolchicine-treated
cells where apical GFP-CFTR was defined as 1). The small decrease in
biotinylated apical membrane GFP-CFTR following colchicine treatment is
attributed to slowed recycling of apical endosomes to the plasma
membrane in the absence of intact microtubules (43, 44). Microtubule
depolymerization was independently verified by analyzing the ratio of
soluble (depolymerized) and insoluble (polymerized) tubulin fractions
by Western blotting. The insoluble:soluble tubulin ratio was 97:3
following lumicolchicine treatment compared with 2:98 following
colchicine treatment (n = 3). Taken together, these
findings suggest that both steady state GFP-CFTR localization at the
apical membrane region and acute activation of transepithelial
Cl
secretion mediated by GFP-CFTR Cl
channels are independent of an intact microtubular cytoskeleton. Furthermore, these findings suggest that the half-life of GFP-CFTR at
the apical membrane and in apical endosomes is long (greater than
7 h), in accordance with previous reports of CFTR half-life in
heterologous cells overexpressing CFTR and in epithelial cells expressing endogenous CFTR (45, 46).
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DISCUSSION |
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We have generated a cell model of MDCK epithelial cells stably
expressing a GFP-CFTR fusion protein which functions as a
cAMP-activated Cl channel and is targeted to the apical
plasma membrane in cells cultured on permeable supports. Quantitative
confocal fluorescence microscopy, apical surface biotinylation, and
short circuit current experiments showed that acute treatment with a
cAMP-stimulating mixture increased GFP-CFTR-mediated Cl
secretion by activating channels resident in the apical plasma membrane
and that cAMP-dependent activation and steady state
distribution of GFP-CFTR at the apical membrane region were independent
of an intact microtubular cytoskeleton. Our findings suggest that the
predominant mechanism by which cAMP activates transepithelial Cl
secretion in MDCK type I kidney epithelial cells is by
stimulating protein kinase A-mediated phosphorylation of CFTR
Cl
channels resident in the apical membrane. However, we
cannot exclude the possibility that cAMP stimulates trafficking of a small amount of GFP-CFTR to the apical membrane which is below the
detection limits of the techniques employed. Alternatively, cAMP may
control the trafficking of other regulatory proteins required for CFTR
Cl
channel activity, as has been suggested for the
Na-K-2Cl cotransporter (47).
Our findings are in agreement with numerous studies in epithelial and non-epithelial cells in which cAMP did not acutely stimulate CFTR trafficking to the plasma membrane (2, 7, 13, 48). Thus, it should not be considered dogma that CFTR traffics to the cell surface following cAMP treatment or that CFTR regulates its own trafficking. Instead, CFTR trafficking should be considered cell type-specific (49), because cAMP stimulates CFTR translocation to the plasma membrane in some cells (10-12, 50, 51) but not others (2, 7, 13, 48). In contrast to our findings, very recent reports have found positive effects of cAMP on CFTR trafficking in kidney epithelial cells. For instance, Morris et al. (10) recently reported that arginine vasotocin, a hormone which increases cellular cAMP levels, mobilized CFTR from an intracellular compartment to the apical membrane in amphibian kidney A6 cells. Similarly, in preliminary results, Howard et al. (50, 51) demonstrated that CFTR containing a FLAG-epitope tag trafficked to the apical plasma membrane following acute (10 min) forskolin treatment in MDCK type II cells. In reports quantitating surface expression of CFTR following cAMP treatment, plasma membrane CFTR increased 100% in T84 cells (12), 50-200% in HeLa cells (50), and 100-600% in MDCK type II cells (51). The detection systems used in the present study are sensitive enough to detect changes of these magnitudes.
We speculate that these conflicting results may be due to cell type-specific CFTR trafficking patterns. Agonist-stimulated trafficking of polytopic membrane transport proteins is often cell type-specific. For example, aquaporin-2 traffics from intracellular vesicles to the apical plasma membrane following treatment with cAMP-stimulating agents in collecting duct principal cells (52) but not in Xenopus oocytes (53). Similarly, insulin stimulates GLUT-4 trafficking from an intracellular pool to the plasma membrane in adipocytes and skeletal muscle but not in heterologous expression systems (54). The absence of agonist-stimulated protein trafficking in these systems has been attributed to cell-specific expression of signaling proteins and/or trafficking factors. Thus, we consider it likely that cAMP stimulates FLAG-CFTR trafficking to the apical membrane in MDCK type II cells but not GFP-CFTR trafficking to the apical membrane in MDCK type I cells, because of differential expression of trafficking proteins (i.e. SNAREs, annexins, Rab GTPases, etc.) and glycosphingolipids (55).
Because MDCK type I cells exhibit electrophysiological and
morphological properties similar to cells in the collecting duct (56,
57), whereas MDCK type II cells partially resemble cells in proximal
tubule (56) and thick ascending limb (58), it is conceivable that cAMP
relocates CFTR to the apical membrane in cells derived from proximal
tubule and/or thick ascending limb but not collecting duct. It is
unlikely that MDCK type I cells lack factors required to traffic
GFP-CFTR appropriately, as these cells express low levels of
endogenous, functional CFTR Cl channels in the apical
membrane (36). We have confirmed that the MDCK type I cells used in
this study express endogenous CFTR by reverse transcriptase-polymerase
chain reaction (data not shown). In contrast, MDCK type II cells do not
express detectable levels of CFTR by reverse transcriptase-polymerase
chain reaction, Western blotting, or functional analyses (36). We
consider it unlikely that GFP inhibits the ability of cAMP to stimulate
CFTR trafficking to the apical membrane, because GFP-CFTR and
exogenously expressed CFTR (without any GFP tag) were polarized to the
apical plasma membrane region under steady state conditions, and GFP
did not interfere with CFTR Cl
channel function in whole
cell patch-clamp and planar lipid bilayer experiments. In addition,
fusion of GFP to other ion channels does not inhibit protein
trafficking or function (59-61).
The initial internalization rate of GFP-CFTR from the apical membrane was 5% per min, similar to the internalization rate of CFTR in stably transfected Chinese hamster ovary cells (9) which do not traffic CFTR to the cell surface following cAMP treatment (7). In contrast, in T84 intestinal epithelial cells the initial rate of endogenous CFTR internalization was 50% per min (8), and CFTR endocytosis and trafficking to the apical membrane were regulated by cAMP (8, 12). Thus, similar to CFTR trafficking to the plasma membrane, CFTR endocytosis and recycling are also cell type-specific.
GFP-CFTR polarity was substratum-dependent. When cells were grown to confluency on glass coverslips, a condition in which MDCK cells do not adopt a fully polarized morphology (57), GFP-CFTR was sorted to the basolateral membrane domain. In contrast, when cells were grown as fully polarized monolayers on permeable supports, GFP-CFTR was sorted to the apical membrane domain. Coating glass coverslips with various extracellular matrix proteins, including collagen, fibronectin, and laminin, to promote cellular differentiation and polarization, did not alter basolateral GFP-CFTR localization.2 We speculate that substratum-dependent GFP-CFTR polarity is attributable to differences in cell differentiation in glass-grown versus filter-grown cells. Significant amounts (up to 50%) of membrane proteins which are distributed in a polarized fashion in cells grown on permeable supports are found on the "opposite" membrane domain in MDCK cells grown on glass coverslips (62, 63). Our findings emphasize the need to study trafficking of CFTR and other polarized membrane proteins in physiologically relevant settings. When using cultured epithelial cells as a model system, experiments should be performed using fully polarized monolayers grown on permeable supports. Examination of trafficking in non-physiological experimental systems (i.e. cells grown on glass coverslips or plastic dishes) may lead to conclusions that are not relevant to the in vivo situation.
In conclusion, our data support a model in which cAMP activates CFTR
Cl channels resident in the apical plasma membrane in
MDCK type I cells, a model of renal distal tubule, and collecting duct. Because CFTR functions not only as a Cl
channel, but also
as a regulator of other cAMP-responsive apical membrane ion channels
including the epithelial sodium channel (64, 65), an outwardly
rectifying chloride channel (28), and a renal potassium channel (66),
we speculate that apical membrane resident CFTR Cl
channels serve at least two functions in distal nephron: first, mediation of transepithelial Cl
transport (14, 15), and
second, regulation of sodium chloride reabsorption/secretion as well as
potassium secretion (64). Given that cAMP did not acutely stimulate
CFTR trafficking in this study, it is unlikely that CFTR regulates
these other ion channels by regulating their trafficking to the apical
membrane. It is more likely that CFTR regulates these channels by
membrane-delimited pathway(s) involving direct interactions (67) or
indirect autocrine signaling cascades (28). In this manner, apical
membrane resident CFTR may control overall electrolyte homeostasis in
renal distal tubule.
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ACKNOWLEDGEMENTS |
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Flow cytometry and confocal microscopy were performed at Dartmouth Medical School in the Herbert C. Englert Cell Analysis Laboratory, which was established by a grant from the Fannie E. Rippel Foundation. We gratefully acknowledge Bakhram Berdiev for performing planar lipid bilayer experiments as well as Ken Orndorff, Alice Givan, and Gary Ward for their assistance with confocal microscopy and flow cytometry. We thank Dr. Michael Caplan for helpful discussions, Dr. Bonnie Blazer-Yost for supplying MDCK C7 cells, and Dr. Duane Compton for assistance with preparation of soluble and insoluble cytoskeletal fractions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK-45881 and DK-51067. Flow cytometry and confocal microscopy performed at the Dartmouth Medical School, in the Herbert C. Englert Cell Analysis Laboratory, were supported in part by Core Grant CA 23108 of the Norris Cotton Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Supported by a pre-doctoral fellowship from the Dolores Zohrab Liebmann Foundation.
c Supported by a post-doctoral fellowship from the Swiss National Science Foundation.
e Supported by a New Investigator grant from the Cystic Fibrosis Foundation.
f Supported by Grant CFF Ismail9710 from the Cystic Fibrosis Foundation.
i To whom correspondence should be addressed: Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1775; Fax: 603-650-1130; E-mail: Bruce.A.Stanton{at}Dartmouth.edu.
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; GFP, green fluorescent protein; MDCK, Madin-Darby canine kidney; Isc, short circuit currentEndo H, endoglycosidase HPNGase F, peptide N-glycosidase FCPT-cAMP, 8-(4-chlorophenylthio)-cAMPDIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acidDPC, diphenylamine carboxylic acidER, endoplasmic reticulumPBS, phosphate-buffered salinePAGE, polyacrylamide gel electrophoresisBSA, bovine serum albuminMOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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