Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moyer, B. D.
Right arrow Articles by Stanton, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moyer, B. D.
Right arrow Articles by Stanton, B. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 34, 21759-21768, August 21, 1998


Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells*

Bryan D. Moyerab, Johannes Loffingac, Erik M. Schwiebertde, Dominique Loffing-Cuenia, Patricia A. Halpina, Katherine H. Karlsona, Iskandar I. Ismailovdf, William B. Gugginog, George M. Langfordh, and Bruce A. Stantonai

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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 (10-5 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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1.   Whole cell patch-clamp analyses examining GFP-CFTR function as a cAMP-activated Cl- channel. NIH-3T3 fibroblasts were transiently transfected with pGFP-CFTR (n = 4) (A) or pCFTR (n = 5) (B). Cells were treated with 8-bromo-cAMP (250 µM) and CPT-cAMP (250 µM) (filled circles), DIDS (100 µM) (open circles), and glybenclamide (100 µM) (×) applied to the extracellular bathing solution in a paired and sequential manner, and current (I)-voltage (V) curves were generated. I-V curves were linear, and CFTR Cl- currents were not time- or voltage-dependent, in accordance with previous reports (29, 68, 69). Mock- or non-transfected cells exhibited currents similar to transfected cells following glybenclamide treatment. Error bars were often smaller than the symbols used to plot the data. C, comparison of whole cell Cl- currents at -100 and +100 mV in cells expressing GFP-CFTR (filled bars) and CFTR (open bars). p > 0.05 for GFP-CFTR compared with CFTR at both voltages.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Comparison of electrophysiological properties of GFP-CFTR and CFTR Cl- channels
Channels were synthesized in Xenopus oocytes, and microsomes were incorporated into planar lipid bilayers for recordings (32, 33). p > 0.05 for GFP-CFTR compared with CFTR for each parameter.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 2.   Planar lipid bilayer analyses comparing CFTR and GFP-CFTR Cl- channel function. A, current traces from bilayers containing CFTR Cl- channels (left) and GFP-CFTR Cl- channels (right) activated with protein kinase A catalytic subunit (1.85 ng/ml) and ATP (100 µM) (top) and treated with DIDS (100 µM) (middle), and DPC (300 µM) (bottom) in a paired and sequential manner. DIDS and DPC were added to both cis and trans membrane faces. Holding potential was + 100 mV. Channel opening is indicated by upward current deflections. B, representative I-V curves (n = 4-5) demonstrating linear, non-rectifying nature of CFTR (left) and GFP-CFTR (right) Cl- channels.

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.


View larger version (58K):
[in this window]
[in a new window]
  		Fig. 3.   Localization and effect of cAMP on distribution of GFP-CFTR in living cells grown on glass coverslips. Three-dimensional reconstructions of confocal fluorescence micrograph xy sections. A, before cAMP treatment; GFP-CFTR fluorescence is depicted in green. B, 5 min following cAMP treatment; GFP-CFTR fluorescence is depicted in red. C, merge of A and B. Yellow depicts regions where GFP-CFTR colocalizes before and after cAMP treatment. Most GFP-CFTR fluorescence colocalizes at the basolateral plasma membrane region, suggesting negligible translocation of GFP-CFTR to the cell surface. Similar results were observed in cells treated for up to 60 min.


View larger version (129K):
[in this window]
[in a new window]
  		Fig. 4.   Localization of GFP-CFTR and CFTR in MDCK cells grown on permeable supports. A, confocal fluorescence micrographs (xy plane) of MDCK cells, stably transfected with GFP-CFTR, beginning at the apical membrane (section 1, upper left) and ending at the basal membrane (section 9, lower right) were acquired in 1.0-µm increments. GFP-CFTR fluorescence is shown in green, and nuclei, stained with propidium iodide, are shown in red. Diffuse red staining corresponds to cytoplasmic RNA. GFP-CFTR is predominantly localized to the apical plasma membrane region (1st three sections) above cell nuclei. Although all cells are not visibly expressing GFP-CFTR, higher laser power settings reveal that most cells exhibit GFP-CFTR fluorescence. B, confocal fluorescence micrograph (xz plane) showing distribution of GFP-CFTR along the apical-basal axis. GFP-CFTR is predominantly localized to a 2-3-µm thick band at the apical pole, consistent with expression in the apical membrane and sub-apical vesicles. Arrow in optical section 4 of A indicates plane of vertical section. C, confocal fluorescence micrograph (xz plane) of MDCK cells, transiently expressing CFTR (with no GFP tag), showing distribution of CFTR along the apical-basal axis. CFTR, stained with an antibody against the R domain and shown in blue, is polarized to the apical membrane region. AP, apical membrane; BM, basal membrane. Scale bar is 10 µm.


View larger version (104K):
[in this window]
[in a new window]
  		Fig. 5.   Confocal fluorescence micrographs (xz sections) of MDCK cells on permeable supports demonstrating polarization of GFP-CFTR to the apical membrane region. A, GFP-CFTR fluorescence is green. Basolateral membranes, stained for Na/K-ATPase, are red. GFP-CFTR is predominantly expressed at the apical cell pole. B, GFP-CFTR fluorescence is green. Tight junctions, stained for ZO-1, are red. GFP-CFTR is polarized to the apical membrane region in a plane parallel with tight junctions. AP, apical membrane; BM, basal membrane. Scale bars are 10 µm.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Comparison of Isc in parental and GFP-CFTR expressing MDCK cells
Isc was measured across MDCK monolayers as described previously (34). cAMP-stimulating mixture (100 µM CPT-cAMP, 100 µM isobutylmethylxanthine, and 20 µM forskolin) was applied to both apical and basolateral bathing compartments. DPC (10 mM) was applied to the apical bathing compartment.

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).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   Distribution of GFP-CFTR fluorescence along the apical-basal axis in MDCK cells grown on permeable supports and treated with cAMP-stimulating mixture (10 min, black bars or 60 min, white bars) or vehicle (60 min, hatched bars). GFP-CFTR fluorescence in fixed cells was quantitated in 1-µm confocal optical sections along the apical (section 1) to basal (section 10) axis. GFP-CFTR distribution did not change following cAMP treatment. For each treatment, 3 z series, each with 15-25 cells per field, were analyzed from each of four separate monolayers. p > 0.05 for vehicle compared with 10 or 60 min cAMP treatment for each section. AP, apical membrane; BM, basal membrane.


View larger version (112K):
[in this window]
[in a new window]
  		Fig. 7.   Confocal micrographs examining localization of GFP-CFTR in longitudinal cryosections of MDCK cells grown on permeable supports and treated with vehicle (A, C, and E) or cAMP-stimulating mixture (10 min; B, D, and F). A and B, fluorescence micrographs. GFP-CFTR fluorescence is green. Nuclei, stained with propidium iodide, are red. C and D, differential interference contrast micrographs showing MDCK cells and permeable supports. E, overlay of A and C. F, overlay of B and D. cAMP does not stimulate detectable GFP-CFTR trafficking to the apical membrane region. Diffuse intracellular GFP-CFTR fluorescence corresponds to GFP-CFTR expression in the ER which can often be closely apposed to plasma membranes. Scale bar is 10 µm.

Because of the limited resolution of the confocal microscope in the vertical dimension (which corresponds to 0.5 µm under optimal scanning conditions), it is conceivable that cAMP stimulates the trafficking of GFP-CFTR, localized in sub-apical vesicles less than 0.5 µm from the apical surface, to the apical plasma membrane. To address this possibility, we biotinylated apical cell-surface GFP-CFTR in monolayers treated with vehicle or cAMP-stimulating mixture using biotin-LC-hydrazide, a membrane-impermeant reagent which labels glycoproteins on sugar residues previously oxidized with sodium periodate. Biotinylations were performed at 4 °C to inhibit membrane trafficking and ensure that only cell-surface GFP-CFTR was labeled. Fig. 8A shows that the amount of surface-biotinylated GFP-CFTR on apical plasma membranes did not increase following 10 or 60 min of cAMP treatment. Densitometric analyses, which referenced biotinylated GFP-CFTR to total cell lysate GFP-CFTR (Fig. 8B), to account for filter to filter variability in GFP-CFTR expression levels, confirmed these observations. Similar results were obtained in a stably transfected cell line expressing 20-fold less GFP-CFTR and in cells not induced with sodium butyrate, which express 30-fold less GFP-CFTR, indicating that results were independent of GFP-CFTR expression levels.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 8.   A and B, Western blot analyses examining amount of apical membrane-biotinylated GFP-CFTR in MDCK cells grown on permeable supports and treated with vehicle or cAMP-stimulating mixture. A, apical surface-biotinylated GFP-CFTR. B, one-fourth of total cell lysate GFP-CFTR. Lane 1, 10-min vehicle; lane 2, 10-min cAMP; lane 3, 60-min cAMP; lane 4, no biotinylation; lane 5, no periodate oxidation; lane 6, parental, untransfected MDCK cells. GFP-CFTR was detected with CFTR C-terminal antibody. Identical banding patterns were obtained with GFP monoclonal antibody. *, high molecular weight GFP-CFTR; C, 240-kDa mature-glycosylated GFP-CFTR band C; and B, 210-kDa core-glycosylated GFP-CFTR band B. Densitometric analysis of apical membrane GFP-CFTR: 2-min cAMP = 1.04 ± 0.12 (n = 4), 10-min cAMP = 0.97 ± 0.14 (n = 9), and 60-min cAMP = 0.95 ± 0.20 (n = 4) compared with vehicle-treated cells. p > 0.05 for vehicle compared with all cAMP-treated monolayers. C, endocytosis and recycling of GFP-CFTR. Cells were treated with vehicle (closed circles) or cAMP mixture (open circles), and the fraction of GFP-CFTR remaining at the apical surface was determined by a two-step biotinylation procedure as described under "Experimental Procedures." Because biotinylation efficiency was variable in different experiments, the fraction of apical membrane GFP-CFTR at each time point was normalized to apical membrane GFP-CFTR at time 0, which is defined as 1. p > 0.05 for vehicle compared with cAMP-treated monolayers at all time points. n = 5 or 6 monolayers for each time point and treatment. D, glycosidase digestion of GFP-CFTR. Lane 1, Endo H control (no enzyme); lane 2, Endo H digestion; lane 3, PNGase F control (no enzyme); lane 4, PNGase F digestion; lane 5, lysate treated with 10% Nonidet P-40 for 1 h at room temperature. Control lysates, incubated for 1 h at room temperature without 10% Nonidet P-40, are shown in lanes 1 and 3. GFPCFTR was detected with CFTR C-terminal antibody. A indicates 200-kDa unglycosylated GFP-CFTR band A. E, confocal fluorescence micrograph (xz plane) of MDCK monolayer biotinylated on apical surface and stained with Texas Red-avidin. Biotinylated glycoproteins are restricted to the apical cell surface demonstrating that tight junctions were intact and that biotin did not have access to intracellular or basolateral membrane proteins. AP, apical membrane. BM, basal membrane. Scale bar is 10 µm.

It is conceivable that cAMP equally affects the rate of endocytosis of GFP-CFTR from the apical membrane and the rate of recycling of endosomes containing GFP-CFTR to the apical membrane such that the net cell-surface GFP-CFTR pool remains constant. To examine this possibility, GFP-CFTR internalization and recycling were followed in the presence and absence of cAMP using a two-step biotinylation procedure. Cells were placed on ice, and apical membrane glycoproteins were first oxidized with sodium periodate. Cells were then incubated at 37 °C for various times in the presence and absence of cAMP to allow GFP-CFTR internalization (at early time points) or internalization and recycling (at later time points). Cells were returned to ice, and periodate-oxidized GFP-CFTR molecules at the apical surface were next labeled with biotin-LC-hydrazide. As shown in Fig. 8C, 15% of GFP-CFTR was endocytosed from the apical membrane following a 3-min incubation at 37 °C in the presence or absence of cAMP. This time point reflects removal of GFP-CFTR from the apical membrane and accumulation in an endosomal pool. Longer incubations at 37 °C, which allow further internalization of apical membrane GFP-CFTR as well as recycling of GFP-CFTR internalized at earlier time points, did not change the amount of labeled GFP-CFTR at the apical membrane, and cAMP did not significantly affect the number of labeled apical membrane GFP-CFTR Cl- channels. These findings suggest that after 3 min, the rate of apical membrane GFP-CFTR internalization is equivalent to the rate of endosomal GFP-CFTR recycling such that net apical membrane GFP-CFTR remains constant.

Surface-biotinylated GFP-CFTR migrated as two bands at 240 and 300 kDa (Fig. 8A, labeled C and *, respectively), whereas GFP-CFTR from cell lysates migrated as three bands at 210, 240, and 300 kDa (Fig. 8B, labeled B, C, and *, respectively). These bands correspond to core-glycosylated (210 kDa; band B), mature-glycosylated (240 kDa; band C), and detergent-induced high molecular mass (300 kDa and as discussed below) GFP-CFTR. Endo H, which cleaves sugar residues from N-linked glycoproteins in pre-Golgi organelles and the cis-Golgi apparatus, had no apparent effect on the mobility of the 240-kDa band but increased the mobility of the 210-kDa band, which migrated at 200 kDa (band A) following Endo H digestion (Fig. 8D). PNGase F, which cleaves sugar residues from N-linked glycoproteins regardless of their localization in the secretory pathway, increased the mobility of both the 240- and the 210-kDa bands, which migrated as a single band at 200 kDa following PNGase F digestion (Fig. 8D). Detection of core-glycosylated GFP-CFTR by Western blotting but lack of substantial GFP-CFTR fluorescence in the endoplasmic reticulum (ER) by confocal microscopy in xz vertical scans using low laser power settings is likely due to dilution of GFP fluorescence throughout the extensive tubulovesicular elements of the ER. GFP-CFTR was detected in the ER by confocal microscopy using higher laser power settings. The predicted molecular masses of GFP (27 plus 3 kDa of linker sequence) fused to core-glycosylated (150 kDa) and mature glycosylated (180 kDa) forms of CFTR are 180 and 210 kDa, respectively. Thus, GFP-CFTR fusion proteins run approximately 30 kDa larger than anticipated in SDS-PAGE analyses. Glycoproteins, including CFTR, frequently migrate slower than predicted in SDS-PAGE because SDS does not bind sugar moieties and, as a result, migration toward the positive electrode is hindered (36, 39). The absence of the 210-kDa form of GFP-CFTR in surface-biotinylated samples indicates that cell integrity was not compromised and that biotinylation reagent was not accessible to the cell interior during these experiments. We confirmed this and verified that tight junctions were intact during the procedure by labeling biotinylated cell-surface glycoproteins with Texas Red-avidin. Texas Red-avidin fluorescence was restricted to the apical cell surface (Fig. 8E).

The high molecular weight GFP-CFTR band (indicated by * in Fig. 8) reacted with both GFP and CFTR C-terminal antibodies, suggesting that it was not due to nonspecific antibody binding. Previous studies have documented high molecular weight forms of CFTR, which migrate 60-100 kDa larger than anticipated, in SDS-PAGE analyses (40, 41). We have determined that this high molecular weight band is likely due to interaction of GFP-CFTR with non-ionic detergent used for cell lysis, as previously reported (41). Treatment of cell lysates with the non-ionic detergent Nonidet P-40 converted mature and core-glycosylated GFP-CFTR bands into the high molecular weight species (Fig. 8D). Detergent-induced degradation of mature and core-glycosylated forms is unlikely because total GFP-CFTR remained constant as determined by densitometry.

Control experiments, which included monolayers that were not biotinylated, monolayers that were not oxidized with sodium periodate, and monolayers from parental, untransfected MDCK cells, were all negative for apical membrane CFTR immunoreactivity in Western blot analyses (Fig. 8A). These results demonstrate that GFP-CFTR was not binding nonspecifically to streptavidin-agarose beads and that all CFTR immunoreactivity was derived from exogenously expressed GFP-CFTR protein. Our inability to detect endogenous CFTR in MDCK type I cells is likely due to low expression levels (36).

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 alpha -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).

                              
View this table:
[in this window]
[in a new window]
 
Table III
Effect of microtubule disruption on Isc in MDCK cells
Isc was recorded in monolayers treated with 33 µM lumicolchicine or colchicine for 5-7 h. cAMP mixture was applied as in Table II.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Crawford, I., Maloney, P. C., Zeitlin, P. L., Guggino, W. B., Hyde, S. C., Turley, H., Gatter, K. C., Harris, A., and Higgins, C. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9262-9266[Abstract/Free Full Text]
  2. Denning, G. M., Ostedgaard, L. S., Cheng, S. H., Smith, A. E., and Welsh, M. J. (1992) J. Clin. Invest. 89, 339-349
  3. Stanton, B. A. (1997) Wien Klin. Wochenschr. 109, 457-564[Medline] [Order article via Infotrieve]
  4. Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S., and Tsui, L.-C. (1989) Science 245, 1066-1073[Abstract/Free Full Text]
  5. Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J., and Smith, A. E. (1991) Cell 66, 1027-1036[CrossRef][Medline] [Order article via Infotrieve]
  6. Tabcharani, J. A., Chang, X.-B., Riordan, J. R., and Hanrahan, J. W. (1991) Nature 352, 628-631[CrossRef][Medline] [Order article via Infotrieve]
  7. Hug, M. J., Thiele, I. E., and Greger, R. (1997) Pfluegers Arch. 434, 779-784[CrossRef][Medline] [Order article via Infotrieve]
  8. Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196[Abstract/Free Full Text]
  9. Lukacs, G. L., Segal, G., Kartner, N., Grinstein, S., and Zhang, F. (1997) Biochem. J. 328, 353-361
  10. Morris, R. G., Tousson, A., Benos, D. J., and Schafer, J. A. (1998) Am. J. Physiol. 274, F300-F314[Abstract/Free Full Text]
  11. Lehrich, R. W., Aller, S. G., Webster, P., Marino, C. R., and Forrest, J. N., Jr. (1998) J. Clin. Invest. 101, 737-745[Medline] [Order article via Infotrieve]
  12. Tousson, A., Fuller, C. M., and Benos, D. J. (1996) J. Cell Sci. 109, 1325-1334[Abstract]
  13. Prince, L. S., Tousson, A., and Marchase, R. B. (1993) Am. J. Physiol. 264, C491-C498[Abstract/Free Full Text]
  14. Husted, R. F., Volk, K. A., Sigmund, R. D., and Stokes, J. B. (1995) J. Clin. Invest. 95, 644-650
  15. Vandorpe, D., Kizer, N., Ciampolillo, F., Moyer, B., Karlson, K., Guggino, W. B., and Stanton, B. A. (1995) Am. J. Physiol. 269, C683-C689[Abstract/Free Full Text]
  16. Hanaoka, K., Devuyst, O., Schwiebert, E. M., Wilson, P. D., and Guggino, W. B. (1996) Am. J. Physiol. 270, C389-C399[Abstract/Free Full Text]
  17. Engelhardt, J. F., Yankaskas, J. R., Ernst, S. A., Yang, Y., Marino, C. R., Boucher, R. C., Cohn, J. A., and Wilson, J. M. (1992) Nature 2, 240-248
  18. Gerdes, H.-H., and Kaether, C. (1996) FEBS Lett. 389, 44-47[CrossRef][Medline] [Order article via Infotrieve]
  19. Lippincott-Schwartz, J., and Smith, C. L. (1997) Curr. Biol. 7, 631-639
  20. Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Trends Biochem. Sci. 20, 448-455[CrossRef][Medline] [Order article via Infotrieve]
  21. Zhang, G., Gurtu, V., and Kain, S. R. (1996) Biochem. Biophys. Res. Commun. 227, 707-711[CrossRef][Medline] [Order article via Infotrieve]
  22. Schwiebert, E. M., Morales, M. M., Devidas, S., Egan, M. E., and Guggino, W. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2674-2679[Abstract/Free Full Text]
  23. Kaether, C., and Gerdes, H.-H. (1995) FEBS Lett. 369, 267-271[CrossRef][Medline] [Order article via Infotrieve]
  24. Lim, C. R., Kimata, Y., Oka, M., Nomaguchi, K., and Kohno, K. (1995) J. Biochem. (Tokyo) 118, 13-17[Abstract/Free Full Text]
  25. Anderson, M. P., Rich, D. P., Gregory, R. J., Smith, A. E., and Welsh, M. J. (1991) Science 251, 679-682[Abstract/Free Full Text]
  26. Moyer, B. D., and Stanton, B. A. (1997) Expressions 4, 6 (Invitrogen company newsletter)
  27. Gekle, M., Wunsch, S., Oberleithner, H., and Silbernag, S. (1994) Pfluegers Arch. 428, 157-162[CrossRef][Medline] [Order article via Infotrieve]
  28. Schwiebert, E. M., Egan, M. E., Hwang, T.-H., Fulmer, S. B., Allen, S. S., Cutting, G. R., and Guggino, W. B. (1995) Cell 81, 1063-1073[CrossRef][Medline] [Order article via Infotrieve]
  29. Schwiebert, E. M., Flotte, T., Cutting, G. R., and Guggino, W. B. (1994) Am. J. Physiol. 266, C1464-C1477[Abstract/Free Full Text]
  30. Goldin, A. L. (1992) Methods Enzymol., 266-279
  31. Pérez, G., Lagrutta, A., Adelman, J. P., and Toro, L. (1994) Biophys. J. 66, 1022-1027[Medline] [Order article via Infotrieve]
  32. Jovov, B., Ismailov, I. I., Berdiev, B. K., Fuller, C. M., Sorscher, E. J., Dedman, J. R., Kaetzel, M. A., and Benos, D. J. (1995) J. Biol. Chem. 270, 29194-29200[Abstract/Free Full Text]
  33. Jovov, B., Ismailov, I. I., and Benos, D. J. (1995) J. Biol. Chem. 270, 1521-1528[Abstract/Free Full Text]
  34. Kizer, N. L., Lewis, B., and Stanton, B. A. (1995) Am. J. Physiol. 268, F347-F355[Abstract/Free Full Text]
  35. Lisanti, M. P., Le Bivic, A., Sargiacomo, M., and Rodriguez-Boulan, E. (1989) J. Cell Biol. 109, 2117-2127[Abstract/Free Full Text]
  36. Mohamed, A., Ferguson, D., Seibert, F. S., Cai, H., Kartner, N., Grinstein, S., Riordan, J. R., and Lukacs, G. L. (1997) Biochem. J. 322, 259-265
  37. Cliff, W. H., Schoumacher, R. A., and Frizzell, R. A. (1992) Am. J. Physiol. 262, C1154-C1160[Abstract/Free Full Text]
  38. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991) Science 253, 202-205[Abstract/Free Full Text]
  39. Westermeier, R. (1993) Electrophoresis in Practice, pp. 167-188, VCH Publishers, Inc., New York
  40. Marshall, J., Fang, S., Ostegaard, L. S., O'Riordan, C. R., Ferrara, D., Amara, J. F., Hoppe, H., IV, Scheule, R. K., Welsh, M. J., Smith, A. E., and Cheng, S. H. (1994) J. Biol. Chem. 269, 2987-2995[Abstract/Free Full Text]
  41. Ostedgaard, L. S., and Welsh, M. J. (1992) J. Biol. Chem. 267, 26142-26149[Abstract/Free Full Text]
  42. Bradbury, N. A., and Bridges, R. J. (1994) Am. J. Physiol. 267, C1-C24[Abstract/Free Full Text]
  43. Breitfeld, P. P., McKinnon, W. C., and Mostov, K. E. (1990) J. Cell Biol. 111, 2365-2373[Abstract/Free Full Text]
  44. Maples, C. J., Ruiz, W. G., and Apodaca, G. (1997) J. Biol. Chem. 272, 6741-6751[Abstract/Free Full Text]
  45. Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718[Abstract/Free Full Text]
  46. Lukacs, G. L., Chang, X.-B., Bear, C., Kartner, N., Mohamed, A., Riordan, J. R., and Grinstein, S. (1993) J. Biol. Chem. 268, 21592-21598[Abstract/Free Full Text]
  47. D'Andrea, L., Lytle, C., Matthews, J. B., Hofman, P., Forbush, B., III, and Madara, J. L. (1996) J. Biol. Chem. 271, 28969-28976[Abstract/Free Full Text]
  48. Torres, R. J., Altenberg, G. A., Cohn, J. A., and Reuss, L. (1996) Am. J. Physiol. 271, C1574-C1582[Abstract/Free Full Text]
  49. Jilling, T., and Kirk, K. L. (1997) Int. Rev. Cytol. 172, 193-241[Medline] [Order article via Infotrieve]
  50. Howard, M., Jilling, T., DuVall, M., and Frizzell, R. A. (1996) Kidney Int. 49, 1642-1648[Medline] [Order article via Infotrieve]
  51. Howard, M. B., Watkins, S. C., and Frizzell, R. A. (1996) Ped. Pulmonol. 13, (suppl.) 231-232
  52. Marples, D., Knepper, M. A., Christensen, E. I., and Nielsen, S. (1995) Am. J. Physiol. 269, C655-C664[Abstract/Free Full Text]
  53. Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Marumo, F., and Sasaki, S. (1995) J. Biol. Chem. 270, 10384-10387[Abstract/Free Full Text]
  54. Rice, J. E., Livingstone, C., and Gould, G. W. (1996) Biochem. Soc. Trans. 24, 540-546[Medline] [Order article via Infotrieve]
  55. Hansson, G. C., Simons, K., and van Meer, G. (1986) EMBO J. 5(3), 483-489
  56. Richardson, J. C. W., Scalera, V., and Simmons, N. L. (1981) Biochim. Biophys. Acta 673, 26-36[Medline] [Order article via Infotrieve]
  57. Valentich, J. D. (1981) Ann. N. Y. Acad. Sci. 372, 384-405[Medline] [Order article via Infotrieve]
  58. Nichols, G. E., Lovejoy, J. C., Borgman, C. A., Sanders, J. M., and Young, W. W., Jr. (1986) Biochim. Biophys. Acta 887, 1-12[Medline] [Order article via Infotrieve]
  59. Marshall, J., Molloy, R., Moss, G. W. J., Howe, J. R., and Hughes, T. E. (1995) Neuron 14, 211-215[CrossRef][Medline] [Order article via Infotrieve]
  60. Makhina, E. N., and Nichols, C. G. (1998) J. Biol. Chem. 273, 3369-3374[Abstract/Free Full Text]
  61. Grabner, M., Dirksen, R. T., and Beam, K. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1903-1908[Abstract/Free Full Text]
  62. Gottlieb, T. A., Gonzalez, A., Rizzolo, L., Rindler, M. J., Adesnik, M., and Sabatini, D. D. (1986) J. Cell Biol. 102, 1242-1255[Abstract/Free Full Text]
  63. Rindler, M. J., Ivanov, I. E., Plesken, H., Rodriguez-Boulan, E., and Sabatini, D. D. (1984) J. Cell Biol. 98, 1304-1319[Abstract/Free Full Text]
  64. Ling, B. N., Zuckerman, J. B., Lin, C., Harte, B. J., McNulty, K. A., Smith, P. R., Gomez, L. M., Worrell, R. T., Eaton, D. C., and Kleyman, T. R. (1997) J. Biol. Chem. 272, 594-600[Abstract/Free Full Text]
  65. Stutts, M. J., Canessa, C. M., Olsen, J. C., Hamrick, M., Cohn, J. A., Rossier, B. C., and Boucher, R. C. (1995) Science 269, 847-850[Abstract/Free Full Text]
  66. McNicholas, C. M., Guggino, W. B., Schwiebert, E. M., Hebert, S. C., Giebisch, G., and Egan, M. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8083-8088[Abstract/Free Full Text]
  67. Kunzelmann, K., Kiser, G. L., Schreiber, R., and Riordan, J. R. (1997) FEBS Lett. 400, 341-344[CrossRef][Medline] [Order article via Infotrieve]
  68. Berger, H. A., Anderson, M. P., Gregory, R. J., Thompson, S., Howard, P. W., Maurer, R. A., Mulligan, R., Smith, A. E., and Welsh, M. J. (1991) J. Clin. Invest. 88, 1422-1431
  69. Tilly, B. C., Winter, M. C., Ostedgaard, L. S., O'Riordan, C., Smith, A. E., and Welsh, M. J. (1992) J. Biol. Chem. 267, 9470-9473[Abstract/Free Full Text]

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. M. Bomberger, R. L. Barnaby, and B. A. Stanton
The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells
J. Biol. Chem., July 10, 2009; 284(28): 18778 - 18789.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. Ferrari, A. Veligodskiy, U. Berge, M. S. Lucas, and R. Kroschewski
ROCK-mediated contractility, tight junctions and channels contribute to the conversion of a preapical patch into apical surface during isochoric lumen initiation
J. Cell Sci., November 1, 2008; 121(21): 3649 - 3663.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
S. Ye, D. P. MacEachran, J. W. Hamilton, G. A. O'Toole, and B. A. Stanton
Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif
Am J Physiol Cell Physiol, September 1, 2008; 295(3): C807 - C818.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. M. Haggie and A. S. Verkman
Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging
J. Biol. Chem., August 29, 2008; 283(35): 23510 - 23513.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Cebotaru, N. Vij, I. Ciobanu, J. Wright, T. Flotte, and W. B. Guggino
Cystic Fibrosis Transmembrane Regulator Missing the First Four Transmembrane Segments Increases Wild Type and {Delta}F508 Processing
J. Biol. Chem., August 8, 2008; 283(32): 21926 - 21933.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Moreau-Marquis, J. M. Bomberger, G. G. Anderson, A. Swiatecka-Urban, S. Ye, G. A. O'Toole, and B. A. Stanton
The {Delta}F508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability
Am J Physiol Lung Cell Mol Physiol, July 1, 2008; 295(1): L25 - L37.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. K. Berdiev, E. Cormet-Boyaka, A. Tousson, Y. J. Qadri, H. M. J. Oosterveld-Hut, J. S. Hong, P. A. Gonzales, C. M. Fuller, E. J. Sorscher, G. L. Lukacs, et al.
Molecular Proximity of Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel Assessed by Fluorescence Resonance Energy Transfer
J. Biol. Chem., December 14, 2007; 282(50): 36481 - 36488.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
O. Granio, C. Norez, K. J. D. Ashbourne Excoffon, P. H. Karp, M. Lusky, F. Becq, P. Boulanger, J. Zabner, and S.-S. Hong
Cellular Localization and Activity of Ad-Delivered GFP-CFTR in Airway Epithelial and Tracheal Cells
Am. J. Respir. Cell Mol. Biol., December 1, 2007; 37(6): 631 - 639.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
B. K. Panama, M. McLerie, and A. N. Lopatin
Heterogeneity of IK1 in the mouse heart
Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3558 - H3567.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Swiatecka-Urban, L. Talebian, E. Kanno, S. Moreau-Marquis, B. Coutermarsh, K. Hansen, K. H. Karlson, R. Barnaby, R. E. Cheney, G. M. Langford, et al.
Myosin Vb Is Required for Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator in Rab11a-specific Apical Recycling Endosomes in Polarized Human Airway Epithelial Cells
J. Biol. Chem., August 10, 2007; 282(32): 23725 - 23736.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
D. P. MacEachran, S. Ye, J. M. Bomberger, D. A. Hogan, A. Swiatecka-Urban, B. A. Stanton, and G. A. O'Toole
The Pseudomonas aeruginosa Secreted Protein PA2934 Decreases Apical Membrane Expression of the Cystic Fibrosis Transmembrane Conductance Regulator
Infect. Immun., August 1, 2007; 75(8): 3902 - 3912.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Swiatecka-Urban, S. Moreau-Marquis, D. P. MacEachran, J. P. Connolly, C. R. Stanton, J. R. Su, R. Barnaby, G. A. O'Toole, and B. A. Stanton
Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells
Am J Physiol Cell Physiol, March 1, 2006; 290(3): C862 - C872.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
B. K. Panama and A. N. Lopatin
Differential polyamine sensitivity in inwardly rectifying Kir2 potassium channels
J. Physiol., March 1, 2006; 571(2): 287 - 302.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Guerra, T. Fanelli, M. Favia, S. M. Riccardi, G. Busco, R. A. Cardone, S. Carrabino, E. J. Weinman, S. J. Reshkin, M. Conese, et al.
Na+/H+ Exchanger Regulatory Factor Isoform 1 Overexpression Modulates Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression and Activity in Human Airway 16HBE14o- Cells and Rescues {Delta}F508 CFTR Functional Expression in Cystic Fibrosis Cells
J. Biol. Chem., December 9, 2005; 280(49): 40925 - 40933.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Swiatecka-Urban, A. Brown, S. Moreau-Marquis, J. Renuka, B. Coutermarsh, R. Barnaby, K. H. Karlson, T. R. Flotte, M. Fukuda, G. M. Langford, et al.
The Short Apical Membrane Half-life of Rescued {Delta}F508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of {Delta}F508-CFTR in Polarized Human Airway Epithelial Cells
J. Biol. Chem., November 4, 2005; 280(44): 36762 - 36772.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
E. Regan-Klapisz, I. Sorokina, J. Voortman, P. de Keizer, R. C. Roovers, P. Verheesen, S. Urbe, L. Fallon, E. A. Fon, A. Verkleij, et al.
Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction
J. Cell Sci., October 1, 2005; 118(19): 4437 - 4450.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. P. O'Sullivan, M. D. Linden, A. L. Frelinger III, M. R. Barnard, M. Spencer-Manzon, J. E. Morris, R. O. Salem, M. Laposata, and A. D. Michelson
Platelet activation in cystic fibrosis
Blood, June 15, 2005; 105(12): 4635 - 4641.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Cheng, H. Wang, and W. B. Guggino
Regulation of Cystic Fibrosis Transmembrane Regulator Trafficking and Protein Expression by a Rho Family Small GTPase TC10
J. Biol. Chem., February 4, 2005; 280(5): 3731 - 3739.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. Reigada and C. H. Mitchell
Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport
Am J Physiol Cell Physiol, January 1, 2005; 288(1): C132 - C140.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
J. M. Younger, H.-Y. Ren, L. Chen, C.-Y. Fan, A. Fields, C. Patterson, and D. M. Cyr
A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase
J. Cell Biol., December 20, 2004; 167(6): 1075 - 1085.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. Sugita, H. Kongo, and Y. Shiba
Molecular Dissection of the Butyrate Action Revealed the Involvement of Mitogen-Activated Protein Kinase in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis
Mol. Pharmacol., November 1, 2004; 66(5): 1248 - 1259.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
F. Bilan, V. Thoreau, M. Nacfer, R. Derand, C. Norez, A. Cantereau, M. Garcia, F. Becq, and A. Kitzis
Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity
J. Cell Sci., April 15, 2004; 117(10): 1923 - 1935.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Cheng, H. Wang, and W. B. Guggino
Modulation of Mature Cystic Fibrosis Transmembrane Regulator Protein by the PDZ Domain Protein CAL
J. Biol. Chem., January 16, 2004; 279(3): 1892 - 1898.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. P. Kowalski and G. B. Pier
Localization of Cystic Fibrosis Transmembrane Conductance Regulator to Lipid Rafts of Epithelial Cells Is Required for Pseudomonas aeruginosa-Induced Cellular Activation
J. Immunol., January 1, 2004; 172(1): 418 - 425.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X.-M. Zhang, X.-T. Wang, H. Yue, S. W. Leung, P. H. Thibodeau, P. J. Thomas, and S. E. Guggino
Organic Solutes Rescue the Functional Defect in {Delta}F508 Cystic Fibrosis Transmembrane Conductance Regulator
J. Biol. Chem., December 19, 2003; 278(51): 51232 - 51242.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
T. A. Brindikova, N. Bourcier, B. Torres, D. Pchejetski, M. Gekle, G. V. Maximov, V. Montminy, P. A. Insel, S. N. Orlov, and P. Isenring
Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter
Am J Physiol Cell Physiol, December 1, 2003; 285(6): C1445 - C1453.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
C. A. Bertrand and R. A. Frizzell
The role of regulated CFTR trafficking in epithelial secretion
Am J Physiol Cell Physiol, July 1, 2003; 285(1): C1 - C18.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
T. A. Tucker, K. Varga, Z. Bebok, A. Zsembery, N. A. McCarty, J. F. Collawn, E. M. Schwiebert, and L. M. Schwiebert
Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids
Am J Physiol Cell Physiol, March 1, 2003; 284(3): C791 - C804.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Huang, T. Dudez, I. Scerri, M. A. Thomas, B. N. G. Giepmans, S. Suter, and M. Chanson
Defective Activation of c-Src in Cystic Fibrosis Airway Epithelial Cells Results in Loss of Tumor Necrosis Factor-alpha -induced Gap Junction Regulation
J. Biol. Chem., February 28, 2003; 278(10): 8326 - 8332.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
N. A. Ameen, C. Marino, and P. J. I. Salas
cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo
Am J Physiol Cell Physiol, February 1, 2003; 284(2): C429 - C438.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
L. Fu and E. Sztul
Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator
J. Cell Biol., January 21, 2003; 160(2): 157 - 163.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S.-X. Wang, M. Ikeda, and W. B. Guggino
The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression
J. Biol. Chem., January 17, 2003; 278(4): 2713 - 2722.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. Estell, G. Braunstein, T. Tucker, K. Varga, J. F. Collawn, and L. M. Schwiebert
Plasma Membrane CFTR Regulates RANTES Expression via Its C-Terminal PDZ-Interacting Motif
Mol. Cell. Biol., January 15, 2003; 23(2): 594 - 606.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Peter, K. Varga, Z. Bebok, C. M. McNicholas-Bevensee, L. Schwiebert, E. J. Sorscher, E. M. Schwiebert, and J. F. Collawn
Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression
J. Biol. Chem., December 13, 2002; 277(51): 49952 - 49957.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
J. B. Lyczak and G. B. Pier
Salmonella enterica Serovar Typhi Modulates Cell Surface Expression of Its Receptor, the Cystic Fibrosis Transmembrane Conductance Regulator, on the Intestinal Epithelium
Infect. Immun., November 1, 2002; 70(11): 6416 - 6423.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Swiatecka-Urban, M. Duhaime, B. Coutermarsh, K. H. Karlson, J. Collawn, M. Milewski, G. R. Cutting, W. B. Guggino, G. Langford, and B. A. Stanton
PDZ Domain Interaction Controls the Endocytic Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator
J. Biol. Chem., October 11, 2002; 277(42): 40099 - 40105.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. M. Haggie, B. A. Stanton, and A. S. Verkman
Diffusional Mobility of the Cystic Fibrosis Transmembrane Conductance Regulator Mutant, Delta F508-CFTR, in the Endoplasmic Reticulum Measured by Photobleaching of GFP-CFTR Chimeras
J. Biol. Chem., May 3, 2002; 277(19): 16419 - 16425.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
W. S. Marshall, E. M. Lynch, and R. R. F. Cozzi
Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water
J. Exp. Biol., May 1, 2002; 205(9): 1265 - 1273.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. S. Gelman, E. S. Kannegaard, and R. R. Kopito
A Principal Role for the Proteasome in Endoplasmic Reticulum-associated Degradation of Misfolded Intracellular Cystic Fibrosis Transmembrane Conductance Regulator
J. Biol. Chem., March 29, 2002; 277(14): 11709 - 11714.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-S. Yoo, B. D. Moyer, S. Bannykh, H.-M. Yoo, J. R. Riordan, and W. E. Balch
Non-conventional Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator through the Early Secretory Pathway
J. Biol. Chem., March 22, 2002; 277(13): 11401 - 11409.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
W. S. Marshall, J. A. Howard, R. R. F. Cozzi, and E. M. Lynch
NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR
J. Exp. Biol., March 15, 2002; 205(6): 745 - 758.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Y. Chang, A. Di, A. P. Naren, H. C. Palfrey, K. L. Kirk, and D. J. Nelson
Mechanisms of CFTR regulation by syntaxin 1A and PKA
J. Cell Sci., February 15, 2002; 115(4): 783 - 791.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Cheng, B. D. Moyer, M. Milewski, J. Loffing, M. Ikeda, J. E. Mickle, G. R. Cutting, M. Li, B. A. Stanton, and W. B. Guggino
A Golgi-associated PDZ Domain Protein Modulates Cystic Fibrosis Transmembrane Regulator Plasma Membrane Expression
J. Biol. Chem., January 25, 2002; 277(5): 3520 - 3529.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. Loffing-Cueni, J. Loffing, C. Shaw, A. M. Taplin, M. Govindan, C. R. Stanton, and B. A. Stanton
Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line
Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1889 - C1897.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Z. Rappoport, M. S. Lipkowitz, and R. G. Abramson
Localization and topology of a urate transporter/channel, a galectin, in epithelium-derived cells
Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1926 - C1939.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. S. Rajan, M. E. Illing, N. F. Bence, and R. R. Kopito
Specificity in intracellular protein aggregation and inclusion body formation
PNAS, October 25, 2001; (2001) 181479798.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
B. L. Blazer-Yost, M. Butterworth, A. D. Hartman, G. E. Parker, C. J. Faletti, W. J. Els, and S. J. Rhodes
Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits
Am J Physiol Cell Physiol, August 1, 2001; 281(2): C624 - C632.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
U. Schwahn, N. Paland, S. Techritz, S. Lenzner, and W. Berger
Mutations in the X-linked RP2 gene cause intracellular misrouting and loss of the protein
Hum. Mol. Genet., May 1, 2001; 10(11): 1177 - 1183.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
R. Maitra, C. M. Shaw, B. A. Stanton, and J. W. Hamilton
Increased functional cell surface expression of CFTR and {Delta}F508-CFTR by the anthracycline doxorubicin
Am J Physiol Cell Physiol, May 1, 2001; 280(5): C1031 - C1037.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. Milewski, J. Mickle, J. Forrest, D. Stafford, B. Moyer, J Cheng, W. Guggino, B. Stanton, and G. Cutting
A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane
J. Cell Sci., January 2, 2001; 114(4): 719 - 726.
[Abstract] [PDF]

Home page
 NeuroscientistHome page
T. E. Hughes
Looking at Receptors: What Have Fluorescent Receptors and Channels Told Us?
Neuroscientist, October 1, 2000; 6(5): 371 - 379.
[Abstract] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. Howard, X. Jiang, D. B. Stolz, W. G. Hill, J. A. Johnson, S. C. Watkins, R. A. Frizzell, C. M. Bruton, P. D. Robbins, and O. A. Weisz
Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells
Am J Physiol Cell Physiol, August 1, 2000; 279(2): C375 - C382.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
A. A. Gerceker, T. Zaidi, P. Marks, D. E. Golan, and G. B. Pier
Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa and Salmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor
Infect. Immun., February 1, 2000; 68(2): 861 - 870.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Tsukita and S. Yonemura
Cortical Actin Organization: Lessons from ERM (Ezrin/Radixin/Moesin) Proteins
J. Biol. Chem., December 3, 1999; 274(49): 34507 - 34510.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
B. D. Moyer, D. Loffing-Cueni, J. Loffing, D. Reynolds, and B. A. Stanton
Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells
Am J Physiol Renal Physiol, August 1, 1999; 277(2): F271 - F276.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. L. Ferguson and W. F. Flintoff
Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion
J. Biol. Chem., June 4, 1999; 274(23): 16269 - 16278.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
A. M. Kachinsky, S. C. Froehner, and S. L. Milgram
A PDZ-containing Scaffold Related to the Dystrophin Complex at the Basolateral Membrane of Epithelial Cells
J. Cell Biol., April 19, 1999; 145(2): 391 - 402.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
J. A. Johnston, C. L. Ward, and R. R. Kopito
Aggresomes: A Cellular Response to Misfolded Proteins
J. Cell Biol., December 28, 1998; 143(7): 1883 - 1898.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H.-L. Ji, M. L. Chalfant, B. Jovov, J. P. Lockhart, S. B. Parker, C. M. Fuller, B. A. Stanton, and D. J. Benos
The Cytosolic Termini of the beta - and gamma -ENaC Subunits Are Involved in the Functional Interactions between Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel
J. Biol. Chem., September 1, 2000; 275(36): 27947 - 27956.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. D. Moyer, M. Duhaime, C. Shaw, J. Denton, D. Reynolds, K. H. Karlson, J. Pfeiffer, S. Wang, J. E. Mickle, M. Milewski, et al.
The PDZ-interacting Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is Required for Functional Expression in the Apical Plasma Membrane
J. Biol. Chem., August 25, 2000; 275(35): 27069 - 27074.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. S. Rajan, M. E. Illing, N. F. Bence, and R. R. Kopito
Specificity in intracellular protein aggregation and inclusion body formation
PNAS, November 6, 2001; 98(23): 13060 - 13065.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moyer, B. D.
Right arrow Articles by Stanton, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moyer, B. D.
Right arrow Articles by Stanton, B. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement