nhe-rf1 protein rescues f508-cftr function

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NHE-RF1 protein rescues ∆F508-CFTR function

Florian BOSSARD*‡, Amal ROBAY*§‡, Gilles TOUMANIANTZ*¶, Shehrazade

DAHIMENE*, Frédéric BECQ°, Jean MEROT*, Chantal GAUTHIER*¶

‡ The first two authors contributed equally to this work.

* Inserm U533, Institut du Thorax, Faculté de Médecine, F-44035 Nantes, France.

° CNRS UMR 6187, I.P.B.C., Université de Poitiers, F-86022 Poitiers, France.

¶ Faculté des Sciences et Techniques, Université de Nantes, F-44322 Nantes, France.

§ Present address: Children's hospital of Philadelphia, Abramson research center, Philadelphia,

Pa, 19104, USA

Running head: ∆F508-CFTR and NHE-RF1

Contact information: Chantal Gauthier, l’Institut du Thorax, Inserm U533, Faculté de

Médecine, F-44035 Nantes, France; Phone : +33-2-40-08-33-94 ; Fax : +33-2-40-41-29-50

E-mail: [email protected]

of 38Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 19, 2007). doi:10.1152/ajplung.00445.2005

Copyright © 2007 by the American Physiological Society.

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Abstract

In Cystic Fibrosis (CF), the ∆F508-CFTR anterograde trafficking from the endoplasmic

reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of

∆F508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study

for CF treatment. Recent studies have demonstrated that PDZ containing proteins play an

essential role in determining polarized plasma membrane expression of ionic transporters.

Here, we have hypothesized that the PDZ containing protein NHE-RF1, which binds to the

carboxy terminus of CFTR, rescues ∆F508-CFTR expression in the apical membrane of

epithelial cells. The plasmids encoding ∆F508-CFTR and NHE-RF1 were intranuclearly

injected in A549 or MDCK cells and ∆F508-CFTR channel activity was functionally assayed

using SPQ fluorescent probe. Cells injected with ∆F508-CFTR alone presented a low chloride

channel activity whereas its co-expression with NHE-RF1 significantly increased both the

basal and forskolin-activated chloride conductances. This last effect was lost with ∆F508-

CFTR deleted of 13 last amino acids or by injection of a specific NHE-RF1 antisense

oligonucleotide, but not by NHE-RF1 sense oligonucleotide. Immunocytochemical analysis

performed in MDCK cells transiently transfected with ∆F508-CFTR further revealed that

NHE-RF1 specifically determined the apical plasma membrane expression of ∆F508-CFTR

but not that of a trafficking defective mutant potassium channel (KCNQ1). These data

demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-

RF1, can rescue ∆F508-CFTR activity.

Keywords: Cystic Fibrosis, ∆F508-CFTR, NHE-RF1, polarized expression, traffic.

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Introduction

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a cAMP-activated

chloride (Cl-) channel, is expressed in a wide variety of epithelial cells, including airway and

kidney (10, 42). Mutations in the CFTR gene lead to the genetic disease cystic fibrosis (CF), a

lethal autosomal recessive disorder. The major disease-causing mutation of CF is a deletion of

phenylalanine at position 508 (∆F508) in the cytoplasmic domain known as the first

nucleotide binding fold (NBF1). This mutation accounts for nearly 70% of the CF alleles (46)

and approximately, 90% of CF patients carry at least one ∆F508-CFTR allele (6). Nascent

wild-type CFTR (wtCFTR) is translated and core-glycosylated in the endoplasmic reticulum.

A fraction (25-40%) of the core-glycosylated CFTR protein (25, 49) is transported to the

Golgi apparatus, where glycosylation is further processed in mature CFTR targeted to the

plasma membrane (5). In contrast to wtCFTR, nearly all ∆F508-CFTR is retained in the

endoplasmic reticulum as a core-glycosylated form, which is sorted to the ubiquitin-

proteasome degradation pathway instead of the Golgi apparatus (5, 19). However, when

∆F508-CFTR is located at the plasma membrane, it is functional and presents a partial cAMP-

activated Cl- channel activity (9). Thus, identification of strategies aimed at increasing ∆F508-

CFTR anterograde trafficking and channel activity to the apical plasma membrane of airway

epithelial cells would have important therapeutic implications for treating CF.

PDZ domains are modular protein interaction domains that play a role in protein targeting

and protein complex assembly. These domains of ~90 amino acids are known by the acronym

of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90,

the Drosophila septate junction protein Discs-large, and the tight junction protein ZO-1 (18).

PDZ-containing proteins are typically involved in the assembly of supramolecular complexes

that perform localized signaling functions at particular subcellular locations. Also, it was

established that PDZ domain-based interactions are essential for polarized distribution of

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numerous membrane proteins in neurons and epithelial cells (21, 40). Organization around a

PDZ-based scaffold allows the stable localization of interacting proteins and enhances the rate

and fidelity of signal transduction within the complex. Some PDZ-containing proteins are

more dynamically regulated in distribution and may also be involved in the trafficking of

interacting proteins within the cell (40).

The last four amino acids of CFTR (Asp-Thr-Arg-Leu) constitute a consensus sequence

known to bind to PDZ domain proteins. The Na+/H+ exchanger regulatory factor isoform 1,

NHE-RF1 (also called EBP50, ERM-binding phosphoprotein 50), which contains two PDZ

domains, is able to bind to the C-terminus of CFTR through its PDZ1 domain (41, 47),

whereas the PDZ2 domain remains available to interact with other proteins (30), including

another CFTR channel when NHE-RF1 is bound to ezrin (23). Because NHE-RF1 associates

with ezrin, which binds to the regulatory subunit of protein kinase A (PKA)(12), it has been

hypothesized that NHE-RF1 anchors CFTR to the cytoskeleton at a subapical compartment,

targeting PKA near CFTR (41). NHE-RF1 plays a key role in the polarization of CFTR to the

apical plasma membrane in epithelial cells (32). NHE-RF1 binding to CFTR also increases

the open probability of CFTR channel (38). In addition, deletion of the PDZ interacting

domain of CFTR reduced its half-life at the apical membrane of polarized epithelial cells by

decreasing CFTR recycling from endosomes. This suggests that NHE-RF1 presents an apical

membrane retention motif (3).

Based on these data, the aim of the present study was to determine the consequences of

NHE-RF1 overexpression on the trafficking and Cl- channel activity of the ∆F508-CFTR

protein in a recombinant system. We demonstrated that NHE-RF1 overexpression was able to

restore the Cl- channel activity of ∆F508-CFTR in non polarized (A549) as well as in

polarized epithelial cells (MDCK). This effect was dependent on direct NHE-RF1 and CFTR

interactions as, ∆F508-CFTR missing its 13 C-terminal amino-acids was not activated by

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NHE-RF1 overexpression. This result was associated with an increased apical membrane

expression of ∆F508-CFTR in MDCK cells.

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Experimental procedures

Cell culture. The human lung epithelial-derived cell line (A549) was provided by the

American Type Culture Collection (Rockville, MD/USA). A549 cells were cultured as

previously reported (2). Madin-Darby Canine Kidney (MDCK) type II epithelial cells were

grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal

bovine serum (FBS). All cells were cultured at 37 ºC in a 5% CO2 humidified atmosphere.

Plasmids. Transgene cDNAs were subcloned into pcDNA3 or pcb6 mammalian expression

vectors under the control of a cytomegalovirus promoter. The pcDNA3-CFTR and pcDNA3-

∆F508-CFTR plasmids (a gift from J. Ricardo, Lisbon, Portugal) encode the human wtCFTR

and the mutated ∆F508-CFTR proteins respectively. To construct the ∆F508-K1468X mutant

plasmid, the KpnI/HpaI restriction fragment of the pcDNA3-∆F508-CFTR plasmid,

containing the ∆F508 mutation, was cloned in frame in pcDNA3-CFTR-K1468X. The double

mutation was verified by sequencing.

The pcDNA3-NHE-RF1 plasmid (a gift from V. Ramanesh, Massachusetts, USA) encodes the

human NHE-RF1 protein. The pcb6–E1Q1 plasmid encodes a human fusion protein KCNE1-

KCNQ1 exposing an extracellular VSV tag. KCNQ1 and KCNE1 are an apical potassium

channel and its transmembrane regulatory subunit respectively, they are responsible for the IKs

potassium current. The pcb6–P117L plasmid encodes a mutant of KCNE1-KCNQ1 presenting

a trafficking defect (7).

PCR experiments. MDCK cells were grown to confluence and total RNAs were extracted

following the modified guanidinium-thiocyanate-phenol-chloroform method. Total cDNAs

were synthetized by the murine Moloney leukaemia virus reverse transcriptase (Gibco BRL,

Villiers-Le-Bel, France). PCR products were generated using specific NHE-RF1 (5’-

GAGACCAAGCTGCTGGTG-3’ sense and 5’-GGCCAGGGAGATGTTGAAG-3’

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antisense) and CFTR primers (5’-AATGTAACAGCCTTCTGGGAG-3’ sense and 5’-

GTTGGCATGCTTTGATGAC-3’ antisense) targeting homologous regions of canine and

human sequences. NHE-RF1 or CFTR encoding plasmids were used as positive controls, and

H2O as a negative control.

Sense and antisense oligonucleotides for NHE-RF1. The sense and antisense

oligonucleotides purchased from Genosis (U.K.), were 5'-ATGAGCGCGGACGCAGCGGC-

3' and 5'-GCCGCTGCGTCCGCGCTCAT-3', respectively. The antisense oligoprobe was

designed to match the NHE-RF1 initiation ATG position (underlined sequence).

Intranuclear injection of plasmids. For functional assays, cells were microinjected with

plasmids [30 µg/ml for human wild type CFTR (wtCFTR) and ∆F508-CFTR and 50 µg/ml

for human NHE-RF1 and sense and antisense oligonucleotides of NHE-RF1] at 1 day after

plating on glass coverslips (Nunclon; InterMed Nunc, Roskilde, Denmark). Our protocol to

intranuclearly microinject individual cells has been reported in details elsewhere (29).

Plasmids were diluted in an injection buffer made of 50 mM N-2-hydroxyethylpiperazine-N’-

2-ethanesulfonic acid (HEPES), 50 mM NaOH, and 40 mM NaCl, pH 7.4. Fluorescein

isothiocyanate (FITC)-labelled dextran (0.5%) was added to the injection medium to visualize

successfully microinjected cells.

Plasmid transfection. For immunocytochemical assays, cells were transiently transfected with

indicated plasmids following JetPEI manufacturer’s protocol (Polyplus Transfection, Illkirch,

France).

Fluorescence measurements of chloride efflux. The Cl- channel activity of CFTR was

assessed using the halide-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl)

quinolinium (SPQ; Molecular probes, Leiden, Netherlands). Twenty-four hours post-

injection, cells were loaded with the intracellular SPQ dye by incubation in Ca2+-free

hypotonic (50% dilution) medium containing 10 mM SPQ at 37°C for 15 minutes. The

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coverslips were mounted on the stage of an inverted microscope (Nikon Diaphot, Japon)

equipped for fluorescence and illuminated at 360 nm. The emitted light was collected at 456

nm by a high-resolution image intensifier coupled to a video camera (Extended ISIS camera

system; Photonic Science, Roberts-bridge, UK) connected to a digital image processing board

controlled by FLUO software (Imstar, France). Cells were maintained at 37°C and

continuously superfused with an extracellular solution containing 145 mM NaCl, 4 mM KCl,

1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES and 5 mM glucose, pH 7.4. A microperfusion

system allowed local application and rapid change of the different experimental mediums.

Iodate (I-) and nitrate (NO3-) containing mediums were identical to the extracellular solution

except that I- and NO3- replaced Cl- as the dominant extracellular anions. All extracellular

mediums also contained 10 µM bumetanide in order to inhibit the Na+/K+/2Cl- co-

transporter. Single-cell fluorescence intensity was measured from digital image processing

and displayed against time. Fluorescence intensity was standardized according to the equation

F = (F-Fo) / Fo × 100, where F is the relative fluorescence and Fo is the fluorescence intensity

measured in the presence of I-. The membrane permeability to halides was determined as the

rate of SPQ dequenching upon perfusion with nitrates. At least three successive data points

were collected immediately after the NO3- containing medium application, and then fitted

using a linear regression analysis. The slope of the straight line reflecting the membrane

permeability to halides (p in min-1), was used as an index of CFTR activity.

Immunocytochemical assays. To evaluate the cell polarity of A549 and MDCK cells, they

were grown to confluence on glass coverslips. They were then washed with PBS and fixed in

paraformaldehyde 4% for 20 minutes at room temperature. After washing with PBS, the fixed

cells were permeabilized with 0,2% triton X-100 (Sigma, Saint Quentin Fallavier, France) in

PBS and incubated with anti-ZO-1 monoclonal antibody (1:20 dilution, Biogenesis, Poole,

UK) for 1 hour at room temperature. This antiserum was revealed with Alexa Fluor 488-

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conjugated secondary antibody (1:1000 dilution, Molecular Probes, Leiden, Netherlands) for

1 hour at room temperature.

To determine the cellular localization of NHE-RF1 and CFTR, MDCK cells were transiently

transfected with pcDNA3-CFTR alone, pcDNA3-∆F508-CFTR plasmid alone or in association

with the human pcDNA3-NHE-RF1 plasmid following standard protocol for JetPEI (Polyplus

Transfection, Illkirch, France). After 24 hours, cells were washed with PBS and fixed with

paraformaldehyde 4%. Cells were immunolabelled with primary monoclonal antibody raised

against the first extracellular loop of human CFTR (MATG 1031, 1:20 dilution, Transgene,

Stasbourg, France) and Alexa Fluor 488-conjugated secondary antibody (1:1000 dilution).

Cells were then also permeabilized and incubated with anti-human NHE-RF1 monoclonal

antibody (1:1000 dilution, BD Transduction Laboratories, Le-Pont-de-Claix, France) and

Alexa Fluor 594-conjugated secondary antibody as described above. Cells were mounted with

glycerol 50% in PBS and analysed by confocal microscopy.

Control experiments were performed using the wild-type, trafficking competent VSV-

KCNE1-KCNQ1 (E1Q1) potassium channel and the trafficking defective VSV-KCNE1-

KCNQ1-P117L mutant (P117L) that contain an extracellular VSV tag. (7). MDCK cells were

transiently transfected with pcb6-E1Q1 alone, pcb6-P117L alone or in association with

pcDNA3-NHE-RF1 following standard protocol for JetPEI. After 24 hours, to detect

membrane expression of the KCNE1-KCNQ1 or P117L proteins, non-permeabilized cells

were immunolabelled with anti-VSV monoclonal antibody (1:500 dilution, Sigma, Saint

Quentin Fallavier, France) as previously described. Cells were then fixed with

paraformaldehyde 4%, permeabilized and incubated with anti-NHE-RF1 antibody and Alexa

Fluor 594-conjugated secondary antibody. Alternatively, to detect intracellular expression of

the trafficking mutant P117L, the cells were fixed with paraformaldehyde 4%, permeabilized

and immunolabelled using an antibody raised against the intracellular C-terminal tail of

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KCNQ1 (1:500 dilution, Santa Cruz, CA, USA) and anti-NHE-RF1 antibody as described

above. Cells were mounted with glycerol 50% in PBS and analyzed by confocal microscopy.

Fluorescence intensity quantitation. Imaging conditions and acquisition were identical for all

experimental conditions. The fluorescence intensity of apical CFTR staining was assessed

using Metamorph software (Universal Imaging, Media, PA).

Drugs. Intracellular adenosine 3’,5’-cyclic monophosphate (cAMP) was increased with a

mixture containing 10 µM forskolin plus 100 µM 3-isobutyl-1-methylxanthine (IBMX) (both

from Sigma, Saint Quentin Fallavier, France). For SPQ experiments, drugs were dissolved in

dimethylsulfoxide (DMSO; Sigma), so that final concentration of the solvent was less than

0.1%.

Statistical analysis. Data are expressed as the means ± s.e.m. of n number of experiments.

The statistical significance of a drug effect versus baseline was assessed using a paired

Student’s t test. The significant difference between two experimental conditions was assessed

using an impaired Student’s t test.

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Results.

The polarity of A549 and MDCK cells grown on glass coverslips was assessed by

immunocytochemistry using the tight junction associated protein ZO-1 (Zonula occludens-1)

as a marker. Confocal microscopy analysis showed a discontinuous staining localized at cell

contacts in A549 cells when using a focal plane parallel to the cellular monolayer (Fig. 1A).

On the other hand, a typical polyedric staining surrounding MDCK cells was observed (Fig.

1B). Furthermore, as expected for polarized epithelial cells, transversal (xz) sections revealed

that ZO-1 was specifically localized in the apex of MDCK lateral membranes (Fig. 1D),

whereas no polarized distribution was observed in A549 membranes (Fig. 1C). Together,

these data indicate that MDCK but not A549 cells undergo consistent morphological

polarization when grown to confluence on glass coverslips.

PCR experiments performed on total mRNA extracts revealed that both A549 and MDCK

cells endogenously expressed NHE-RF1 but not CFTR transcripts (Fig. 2).

In A549 cells microinjected with the plasmid encoding wtCFTR, the basal CFTR permeability

to halides (p) was 0.15±0.01 min-1 (Fig. 3). The application of 10 µM forskolin, a direct

activator of adenylyl cyclase, plus 100 µM IBMX, a phosphodiesterase inhibitor, increased

the p value by 7-fold (P<0.001 vs. p baseline; Fig. 3). The co-expression of NHE-RF1 with

wtCFTR significantly increased the basal CFTR channel activity as well as the forskolin-

induced activation of CFTR (P<0.001 vs. basal activity obtained in cells expressing only

wtCFTR; Fig. 3). In MDCK cells, the NHE-RF1 overexpression did not modify the basal

wtCFTR channel activity but significantly increased the forskolin-induced activation of CFTR

(P<0.001 vs. activity obtained in cells expressing only wtCFTR; Fig. 3).

On the other hand, A549 and MDCK cells expressing ∆F508-CFTR alone presented a very

low permeability to halides and a low but significant sensitivity to forskolin (Fig. 4).

However, when co-expressed with NHE-RF1, ∆F508-CFTR presented a significant basal

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channel activity. More interestingly, ∆F508-CFTR channel activity was then increased more

than 3-fold by forskolin (P<0.001 vs. activity obtained in cells expressing only ∆F508-CFTR;

Fig. 4) in both cell lines.

Antisense oligonucleotide strategy was used to ascertain the role of NHE-RF1 on CFTR

channel activity. An antisense oligonucleotide was designed to target the NHE-RF1 mRNA

and to block its translation. As illustrated in figure 4, NHE-RF1 antisense oligonucleotide

blocked the effects of NHE-RF1 on the basal and forskolin-induced activations of ∆F508-

CFTR in both A549 and MDCK cells. On the other hand, the sense oligonucleotide, used as a

control, did not affect ∆F508-CFTR activity responses to NHE-RF1 (Fig. 4).

To establish whether increased wild-type or ∆F508 mutant CFTR activity resulted from

improved membrane expression of the protein, immunocytochemistry studies were performed

using a monoclonal antibody raised against the first extracellular loop of the human CFTR

protein. MDCK cells grown on glass coverslips which exhibit a consistent morphological

polarization (Fig. 1) were used in these experiments. As illustrated in figure 5A and 5B, the

extracellular antibody specifically detected wtCFTR localized in the apical membrane of non

permeabilized MDCK cells, regardless of the presence or the absence of NHE-RF1. Moreover

wtCFTR and NHE-RF1 were at least partially co-localized in MDCK apical membranes (Fig.

5B, panels e and f). As expected, ∆F508-CFTR transfected alone was not detected at the cell

surface (Fig. 6A, panel a) but was readily detected in the apical membranes when co-

transfected with NHE-RF1 (Fig. 6A, panels b and c). Furhtermore, both exogenous proteins

were apically localized (Fig. 6A, panels c and f). Fluorescence quantification showed that

apical immunostainings of wtCFTR were not significantly different in the presence and in the

absence of NHE-RF1 (Fig. 6B), whereas ∆F508-CFTR apical immunostainings were null but

reached the wild-type CFTR levels when co-expressed with NHE-RF1 (Fig. 6B).

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In order to establish the role of PDZ interactions in ∆F508-CFTR apical and functional

rescue, a ∆F508 mutant missing its PDZ interaction domain with NHE-RF1 was used. As

illustrated in figure 7, co-expression of ∆F508-K1468X-CFTR with NHE-RF1 in MDCK

cells did not result in any significant changes in the baseline or the forskolin-stimulated

chloride channel activity. To ascertain the effects of NHE-RF1 on CFTR trafficking did not

result from a global non specific rescue of ER-retained proteins or a non specific increase of

apical proteins expression, we analyzed its effect on the trafficking of an unrelated potassium

channel, KCNQ1. As illustrated in figure 8, the extracellularly tagged fusion protein KCNE1-

KCNQ1 but not its trafficking defective mutant (P117L) was readily detected in the apical

membranes of non permeabilized MDCK cells using extracellular anti-VSV antibody (Fig.

8A) (7). In these conditions, the P117L mutant was never detected at the plasma membrane

irrespective of the presence (Fig. 8B, panels b, e and h) or the absence of NHE-RF1 (Fig. 8B,

panels a, d and g). The P117L mutant detected using an anti-KCNQ1 antibody raised against

the cytoplasmic C-terminal tail of the protein (Fig. 8B, panel c), remained trapped in

intracellular compartments that did not co-localized with those of NHE-RF1 (Fig. 8B, panel

i).

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Discussion

This study demonstrates that the overexpression of the CFTR protein partner, NHE-RF1,

rescues ∆F508-CFTR activity. This conclusion is based on several lines of evidence. First,

cells injected with ∆F508-CFTR alone presented a low Cl- channel activity whereas the co-

expression with NHE-RF1 significantly increased both the basal and the forskolin-activated

∆F508-CFTR conductances. Second, these effects were abolished by a specific NHE-RF1

antisense oligonucleotide. Third, immunocytochemical analysis demonstrated an increased

∆F508-CFTR expression in the apical plasma membrane of the cells co-transfected with

NHE-RF1. Finally, the involvement of PDZ domains in such effect is strongly suggested by

the use of ∆F508-K1468X-CFTR, a ∆F508 mutant lacking the 13 last amino-acids involved in

PDZ interaction with NHE-RF1. In these last experimental conditions, the overexpression of

NHE-RF did not rescue the ∆F508-CFTR activity.

Our study was performed in heterologous systems (A549 and MDCK cells) overexpressing

∆F508-CFTR and NHE-RF1. Because CFTR channel is targeted to the apical membrane in a

number of epithelial cells including those of the airways, intestine and kidney (2, 10, 31, 42),

we first ascertained that MDCK cells were morphologically polarized in our experimental

conditions. Indeed, we show that the tight junction marker ZO-1 was localized in the upper

third of the lateral membranes of confluent MDCK cells grown on glass coverslips. This

observation is consistent with previous studies performed on cells growing on permeable

supports and indicates that MDCK cells grown in our experimental conditions exhibit a

significant morphological polarization to separate apical and basolateral membrane domains.

However, it is worth noting that in these latter conditions, the height of the cells remained less

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than when cells are grown on permeable support (43). Also, we show that both cell lines did

not express endogenous CFTR albeit they expressed endogenous NHE-RF1.

We demonstrate that NHE-RF1 overexpression produced an increase in wtCFTR basal

activity in A549 but not in polarized MDCK cells. The increase in wtCFTR basal activity

obtained in A549 cells is in agreement with Raghuram et al. (38), who have shown that NHE-

RF1 overexpression increases the Cl- channel activity of wtCFTR. Also, the absence of NHE-

RF1 effect on wtCFTR basal activity observed in MDCK cells is in accordance with three

other studies reporting that the apical and functional expression of wtCFTR in MDCK cells

does not necessitate NHE-RF1 binding (1, 17, 36). This corroborates our observation that

NHE-RF1 did not increase wtCFTR apical staining in MDCK cells. On the other hand, it has

been demonstrated that, in MDCK cells, NHE-RF1 is essential for polarized and functional

expression of wtCFTR (32, 33) even if it is not sufficient (27, 28). To make the scheme even

more complicated, Raghuram and colleagues further suggested that the regulation of CFTR

channel activity by NHE-RF1 would be biphasic: activated at low concentrations and

attenuated at high concentrations of NHE-RF1 (38). Nevertheless, we show here that, in both

cell lines, NHE-RF1 overexpression increased the forskolin-induced activation of wtCFTR. It

is well admitted that NHE-RF1 is bound to ezrin which is a PKA-anchoring protein (12)

leading to the formation of macromolecular complexes in which may be gathered CFTR,

ezrin and PKA. Thus, the NHE-RF1 overexpression may facilitate CFTR phosphorylation by

anchoring the regulatory subunits of the PKA in the vicinity of the Cl- channel (24, 34, 38,

44).

Very interestingly, we show that in A549 and MDCK cells, NHE-RF1 increased both the

basal and the forskolin-activated ∆F508-CFTR conductances. These effects were abolished by

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a specific NHE-RF1 antisense oligonucleotide but not by a sense oligonucleotide.

Furthermore, immunocytochemistry experiments showed that NHE-RF1 overexpression

modified ∆F508-CFTR localization in MDCK cells. When ∆F508-CFTR protein was

expressed alone, no plasma membrane staining was observed whereas ∆F508-CFTR co-

localized with NHE-RF1 in the apical cell membranes when co-expressed with the latter.

We show that ∆F508 rescue was critically dependent on its C-terminal PDZ ligand domain

suggesting that physical interaction with NHE-RF1 was a pre-requisite to its effect. Indeed,

∆F508-K1468X-CFTR exhibited a low basal Cl- channel activity which was insensitive to

forskolin in the presence of NHE-RF1. Our results corroborate the results recently published

by Guerra and colleagues (16). In the human cell line CFBE14o- endogenously expressing

∆F508-CFTR, transfection with vectors encoding wild-type mouse NHE-RF1 increased both

apical CFTR expression and apical PKA-dependent CFTR-mediated chloride efflux, whereas

transfection with mouse NHE-RF1 mutated in the binding groove of the PDZ domains or

truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-

dependent chloride efflux (16). Furthermore, the effect of NHE-RF1 on ∆F508-CFTR

trafficking in MDCK cells was specific, because NHE-RF1 did not rescue the plasma

membrane expression of another trafficking defective mutant potassium channel (KCNQ1;

Fig. 5B, panels b and c) (7). Also, the apical expression of NHE-RF1 in MDCK cells is

consistent with previous reports in human airway epithelial cells (41). On the basis of these

results, one can speculate on the mechanisms involved in the improvement of ∆F508-CFTR

trafficking by NHE-RF1. For example, NHE-RF1 overexpression may allow ∆F508-CFTR

protein to leave the endoplasmic reticulum and thus enable its maturation process to finally

facilitate its trafficking to the apical membrane. In addition, NHE-RF1 may also modulate the

recycling of ∆F508-CFTR between the apical plasma membrane and the recycling

compartments.

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The molecular mechanisms involved in the functional rescue of ∆F508-CFTR by NHE-RF1

remain unclear. Several studies suggest some putative roles of NHE-RF1. NHE-RF1 might

compete with another PDZ domain-containing protein known to bind and to inhibit CFTR,

CFTR-activated-ligand (CAL; 3, 4). NHE-RF1 might interact with chaperone proteins

involved in ∆F508-CFTR processing, like Hsc70/Hsp70, Hsp90 or calnexin (14, 26, 35, 37,

50) or with any of the co-chaperones involved in their regulation. NHE-RF1 may also

modulate the stoichiometry of CFTR Cl- channel and/or its association with specific signal

transduction pathways (23, 51). In addition, it has been established that NHE-RF1 can self-

associate through PDZ-PDZ interactions (15), and consequently acts as a scaffolding protein

in macromolecular complexes (38, 48). Finally, NHE-RF1 may participate to the differential

association of CFTR with β-adrenergic transduction pathways. Indeed, Naren and colleagues

have shown that CFTR is associated with a β2-adrenoceptor macromolecular signalling

complex (34). This observation has been strengthened by the study showing that the treatment

of primary human airway surface epithelial cells with a β2-adrenoceptor agonist, salmeterol,

increases the mature CFTR protein expression in a time-dependent fashion. This effect does

not result from the activation of the cAMP/PKA pathway, but involves the NHE-RF1 protein

(45). In addition, we have demonstrated that β3-adrenergic stimulation also increases CFTR

Cl- channel activity through a cAMP/PKA independent but mitogen-activated protein kinases

dependent transduction pathway (22, 39). Whether NHE-RF1 is also involved in the latter

transduction pathway remains to be elucidated.

Taken together, the present results demonstrate that NHE-RF1 overexpression is able to

restore a significant Cl- permeability in cells expressing ∆F508-CFTR. Considering that only

a small amount (10-15%) of ∆F508-CFTR activity is required to restore epithelial function

and moderate CF disease (11, 20), the improvement of CFTR function by NHE-RF1 may

have therapeutic implications.

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LCMP-00445-2005.R1

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Grants

This work was supported by grants from the Association "Vaincre la Mucoviscidose" and the

“Fondation Langlois”.

Acknowledgments

We thank Karine Laurent and Mortéza Erfanian for their expert technical assistance with

cell cultures.

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LCMP-00445-2005.R1

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Figure legends

FIG. 1. ZO-1 immunolocalization in confluent A549 and MDCK cells. A549 and MDCK

cells were grown to confluence on glass coverslips. Cells were fixed and immunostained with

Alexa Fluor 488-conjugated secondary antibody after incubation with monoclonal anti-ZO-1

antibody. A and B are longitudinal confocal micrographs of immunostained A549 and MDCK

cells respectively. C and D are transversal confocal micrographs of immunostained A549 and

MDCK cells respectively. Scale bars represent 10 µm.

FIG. 2. Endogenous expression of NHE-RF1 and CFTR mRNAs in A549 and MDCK

cells. PCR products were generated using primers specific for NHE-RF1 and CFTR and were

observed under UV radiation after 1% agarose gel electrophoresis.

-: negative control (H2O); +: positive control (pcDNA3-NHE-RF1 or pcDNA3-CFTR); NRT:

non reverse-transcribed cDNAs; RT: reverse-transcribed cDNAs.

FIG. 3. Activation of CFTR protein by NHE-RF1 overexpression in A549 and MDCK

cells. A549 and MDCK cells were micro-injected with wtCFTR (30 µg/ml) and NHE-RF1

(50 µg/ml) cDNAs. Membrane permeability to halides (p in min-1) was measured under

baseline conditions (open columns) and after the application of 10 µM forskolin (Fsk; filled

columns).

Data are the means ± s.e.m. of n cells. The significant effects of forskolin were analyzed by a

paired student's t test; ***: P< 0.001 vs. baseline.

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LCMP-00445-2005.R1

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FIG. 4. Activation of ∆F508-CFTR by NHE-RF1 overexpression in A549 and MDCK

cells. Cells were injected with ∆F508-CFTR (30 µg/ml) and NHE-RF1 (50 µg/ml) encoding

plasmids in the absence or the presence of sense or antisense oligonucleotides for NHE-RF1

(50 µg/ml). Membrane permeability to halides (p in min-1) was measured under baseline

conditions (open columns) and after the application of 10 µM forskolin (Fsk; filled columns).


paired student's t test; **: p<0.01 and ***: P< 0.001 vs. baseline. The significant influence of

the NHE-RF1 overexpression was determined by a two-way ANOVA; ##: p<0.01 and ###:

P<0.001 vs. cells only injected with ∆F508-CFTR. The significant influence of the antisense

and sense oligonucleotides was determined by a two-way ANOVA; °°: p<0.01 and °°°:

P<0.001 vs. cells injected with NHE-RF1. ∆F508: ∆F508-CFTR.

FIG. 5. Confocal imaging of MDCK cells transiently transfected with pcDNA3-CFTR

alone or in the presence of pcDNA3-NHE-RF1. (A) Twenty-four hours after transfection,

cells were fixed and immunostained with a monoclonal antibody raised against the first

extracellular loop of human CFTR and then incubated with a Alexa Fluor 488-conjugated

secondary antibody. (B) MDCK cells underwent the same treatment than in (A) but in

addition they were permeabilized and immunostained with monoclonal anti-human NHE-RF1

antibody which was visualized with Alexa Fluor 594-labelled secondary antibody.

Longitudinal and transversal confocal micrographs are annotated xy and xz, respectively.

AP: apical membrane; BM: basal membrane.

FIG. 6. Effects of the overexpression of NHE-RF1 on apical localization of ∆F508-

CFTR. (A) Confocal imaging of MDCK cells transiently transfected with pcDNA3-∆F508-

CFTR plasmid alone or in the presence of pcDNA3-NHE-RF1. Twenty-four hours after

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LCMP-00445-2005.R1

29

transfection, cells were fixed and immunostained with a monoclonal antibody raised against

the first extracellular loop of human CFTR and then incubated with a Alexa Fluor 488-

conjugated secondary antibody. In addition, MDCK were permeabilized and immunostained

with monoclonal anti-human NHE-RF1 antibody which was visualized with Alexa Fluor 594-

labelled secondary antibody. Longitudinal and transversal confocal micrographs are annotated

xy and xz, respectively (B) Quantitation of fluorescence intensity. The fluorescence intensity

of apical wtCFTR and ∆F508-CFTR staining was assessed using Metamorph software

(Universal Imaging, Media, PA). Data are the means ± s.e.m. of n cells. The significant

effects of NHE-RF1 were analyzed by a student's t test; ***: P< 0.001.

AP: apical membrane; BM: basal membrane; ∆F508: ∆F508-CFTR.

FIG. 7. Effect of NHE-RF1 overexpression on ∆F508-K1468X in MDCK cells. MDCK

cells were micro-injected with ∆F508-K1468X (30 µg/ml) and NHE-RF1 (50 µg/ml) cDNAs.

Membrane permeability to halides (p in min-1) was measured under baseline conditions (open

columns) and after the application of 10 µM forskolin (Fsk; filled columns).


paired student's t test.

FIG. 8. Confocal imaging of MDCK cells transiently transfected with pcb6-E1Q1 alone,

pcb6-P117L plasmid alone or in the presence of pcDNA3-NHE-RF1. (A) Twenty-four

hours after transfection, cells were immunostained with a monoclonal antibody raised against

the extracellular VSV tag of the KCNE1-KCNQ1 fusion protein and then incubated with a

Alexa Fluor 488-conjugated secondary antibody. (B panels a, b, d, e, g, h) MDCK cells

underwent the same treatment than in (A) but in addition they were permeabilized and

immunostained with monoclonal anti-human NHE-RF1 antibody which was visualized with

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LCMP-00445-2005.R1

30

Alexa Fluor 594-labelled secondary antibody. (B panels c, f, i) MDCK cells were fixed,

permeabilized and immunolabelled with a monoclonal antibody raised against the

cytoplasmic C-terminal tail of the KCNQ1 protein and then incubated with a FITC-

conjugated secondary antibody. They were also immunostained with monoclonal anti-human

NHE-RF1 antibody which was visualized with Alexa Fluor 594-labelled secondary antibody.

(A) Longitudinal and transversal confocal micrographs are annotated xy and xz, respectively.

(B) All panels are transversal confocal micrographs.

AP: apical membrane; BM: basal membrane.

of 38

Fig. 1

A549 MDCK

BA

DC

of 38

NHE-RF1 CFTR

- + NRTRT - + NRT

RT

MDCK

RT

NHE-RF1 CFTR

- + NRTRT - + NRT

A549

Fig. 2

of 38

p baselinep Fsk

Fig. 3

0.0

0.2

0.4

0.6

0.8

1.0

1.2p

(min

-1)

wtCFTR + NHE-RF1

A549***

***

n=25 n=34

0.0

0.2

0.4

0.6

0.8

1.0

1.2

p(m

in-1

)

+ NHE-RF1

MDCK

wtCFTR

***

n=19n=18

of 38

p baselinep Fsk

Fig. 4

0.0

0.1

0.2

0.3

0.4

p(m

in-1

) ###

###

**

**

°°°

°°

+ NHE-RF1 + NHE-RF1+ antisense

MDCK

***

∆F508

###

###

***

+ NHE-RF1+ sense

n=43 n=44 n=20 n=26

0.0

0.1

0.2

0.3

0.4

p(m

in-1

)

###

°°°

+ NHE-RF1 + NHE-RF1+ antisense

A549

##°°

***

∆F508 + NHE-RF1+ sense

###

##

***

n=24 n=29 n=15 n=19

*

of 38

Fig. 5

A

a

b

wtCFTR

+

NHE-RF1xz

xy

anti-CFTRantibody

anti-NHE-RF1antibody merge

c

d

e

f

10 µm

APBM

a

b

anti-CFTR antibody

10 µm

wtCFTR

AP

BMxz

xy

B

of 38

Fluo

resc

ence

inte

nsity

(arb

itrar

yun

its)

without NHE-RF1with NHE-RF1

0

5

10

15

20

25

wtCFTR ∆F508

***n=8 n=7 n=8 n=7

B

Aanti-CFTR antibody

anti-NHE-RF1 antibody

merge

∆F508

∆F508

+

NHE-RF1

c

a

b e

d

f

g

i

hxy

xz

xz

10 µm

AP

BM

AP

BM

Fig. 6

of 38

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14p

(min

-1)

p baselinep Fsk

∆F508-K1468X + NHE-RF1

n=43

MDCK

Fig. 7

of 38

xz

10 µm

AP

BM

anti-VSV antibody

xy

a

b

KCNE1-KCNQ1

Fig. 8

A

B

anti-KCNQ1 antibody

anti-NHE-RF antibody merge

10 µm

AP

BM

anti-VSV antibody

anti-NHE-RF1 antibody

merge

P117L

P117L + NHE-RF1

AP

BM

AP

BM

c

a

b e

d

f

g

i

h

P117L + NHE-RF1

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nhe-rf1 protein rescues f508-cftr function

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