Transient Receptor Potential Canonical Channel 6 Links Ca 2+ Mishandling to Cystic Fibrosis...

Transient Receptor Potential Canonical Channel 6Links Ca21 Mishandling to Cystic FibrosisTransmembrane Conductance Regulator ChannelDysfunction in Cystic Fibrosis

Fabrice Antigny1, Caroline Norez1, Luc Dannhoffer1, Johanna Bertrand1, Dorothee Raveau1, Pierre Corbi2,Christophe Jayle2, Frederic Becq1, and Clarisse Vandebrouck1

1Institut de Physiologie et Biologie Cellulaires, Universite de Poitiers, Centre National de la Recherche Scientifique, and 2Service de Chirurgie

Cardiothoracique, Centre Hospitalier Universitaire La Miletrie, Poitiers, France

In cystic fibrosis (CF), abnormal control of cellular Ca21 homeostasisis observed. We hypothesized that transient receptor potentialcanonical (TRPC) channels could be a link between the abnormalCa21 concentrations in CF cells and cystic fibrosis transmembraneconductance regulator (CFTR) dysfunction. We measured the TRPCand CFTR activities (using patch clamp and fluorescent probes) andinteractions (using Western blotting and co-immunoprecipitation)in CF and non-CF human epithelial cells treated with specific andscrambled small interfering RNA (siRNA). The TRPC6-mediated Ca21

influx was abnormally increased in CF compared with non-CF cells.After correction of abnormal F508 deletion (del)-CFTR trafficking inCF cells, the level of TRPC6-dependentCa21 influxwasalsonormalized.In CF cells, siRNA–TRPC6 reduced this abnormal Ca21 influx. In non-CFcells, siRNA–TRPC6 reduced the Ca21 influx and activity wild-type (wt)-CFTR. Co-immunoprecipitation experiments revealed TRPC6/CFTRand TRPC6/F508 del-CFTR interactions in CF or non-CF epithelialcells. Although siRNA–CFTR reduced the activity of wt-CFTR in non-CF cells and of F508 del-CFTR in corrected CF cells, it also enhancedTRPC6-dependent Ca21 influx in non-CF cells, mimicking the resultsobtained in CF cells. Finally, this functional and reciprocal couplingbetween CFTR and TRPC6 was also detected in non-CF ciliatedhuman epithelial cells freshly isolated from lung samples. Thesedata indicate that TRPC6 and CFTR are functionally and reciprocallycoupled within a molecular complex in airway epithelial human cells.Because this functional coupling is lost in CF cells, the TRPC6-dependent Ca21 influx is abnormal.

Keywords: Ca21 signaling; CFTR rescue; Cl2 channel cystic fibrosis; TRPC

Calcium influx into the cytosol is temporally and spatiallytightly controlled by the activity of numerous plasma membraneion channels. An increasing number of pathologic situations areassociated with Ca21 signaling abnormalities. In most patientswith cystic fibrosis (CF), the deletion of F508 amino acid in thecystic fibrosis transmembrane conductance regulator (CFTR)gene product promotes the intracellular retention and degrada-tion of F508 deletion (del)-CFTR proteins (1). A particularlypuzzling aspect of the pathophysiology of this disease involvesCa21 signaling abnormalities. Indeed, the release of the endo-plasmic reticulum (ER) Ca21 store was found to be abnormallyincreased in human airway epithelial CF cells, whereas the

rescue of functional F508 del-CFTR by pharmacologic treatmentwas correlated with a normalization of Ca21 mobilization (2).Plasma membrane channels implicated in this Ca21 influx in-clude the transient receptor potential canonical (TRPC) chan-nels (3). Among them, the nonselective, Ca21-permeable TRPCcation channels are of particular interest. They are known toform divalent cation–selective and divalent cation–nonselectivecation channels (4). TRPC6 may contribute to the hypersecre-tion of mucus, a feature of many respiratory diseases (5).

Here, we explored the molecular mechanism of Ca21

signaling abnormalities in CF, and hypothesized that TRPCchannels could be a link between the abnormal Ca21 levelsobserved in CF cells and CFTR dysfunction. We identifiedTRPC6 as the major TRPC isoform, and investigated itsfunction in various human airway epithelial cells. We proposethat TRPC6 is a cation channel responsible for Ca21 influx intothe cytosol of epithelial cells, is modulated by CFTR, and isassociated with CFTR. Thus, TRPC6 as an essential componentof the receptor-activated Ca21 influx machinery must beconsidered in airway epithelial cells and in CF.

MATERIALS AND METHODS

Cell Lines

The human CF and non-CF tracheal-gland serous CF-KM4 and MM39cell lines were grown at 378C in 5% CO2 under standard cultureconditions, as previously described (6, 7). The CF-KM4 cells, trans-formed with the lentiviral vector expressing wild-type CFTR cDNA (8)(in this study, referred to as reverted CF-KM4), were generously givenby Professor Philippe Birembaut (Institut National de la Sante et de laRecherche U903, Reims University, Reims, France).

Freshly Isolated Ciliated Human Epithelial Cells

The study was approved by our local institutional Ethics Committee.Human lung tissue was obtained from three patients (one woman

aged 59 years, and two men aged 64 years) undergoing surgery for lungcarcinoma. After lobectomy, lung samples distant from the malignantlesion were quickly dissected. After removal of connective tissues, car-tilage, and smooth muscle tissues, epithelial tissues were cut into smallsegments and placed in culture medium (Dulbecco’s minimum essentialmedium/HAM-F12, supplemented with 5 mg/ml insulin, 7.5 mg/mltransferrin, 1 mM hydrocortisone, 2 mg/ml Endothelial Cell Growthsupplement, 25 ng/ml Epithelial Growth Factor, 3 3 1028 M triiodothy-ronine, 1 mM L-glutamine, and 100 mM/ml penicillin–streptomycin).Single epithelial cells were mechanically dissociated by passing thebronchial tissue repeatedly through fire-polished Pasteur pipettes.Dissociated ciliated human epithelial cells were plated onto culturedishes and allowed to adhere to the bottom of the dish for 6 hours.

Small Interfering RNA Transfection Protocol

For knockdown studies, cells were used at 60% confluence. Each smallinterfering RNA (siRNA) consisted of a pool of three target-specific20–25 nucleotide siRNA designed to knock down the expression of

(Received in original form September 21, 2009 and in final form February 15, 2010)

This work was supported by the French association Vaincre la Mucoviscidose, F.A.

and D.R. were supported by a studentship from Vaincre la Mucoviscidose, and

L.D. was supported by a postdoctoral grant from Vaincre la Mucoviscidose. J.B.

was supported by a studentship from the French association Mucovie.

Correspondence and requests for reprints should be addressed to Clarisse

Vandebrouck, Ph.D., Institut de Physiologie et Biologie Cellulaires, Universite

Poitiers, Centre National de la Recherche Scientifique, 40 Avenue du Recteur

Pineau, Poitiers F-86022, France. E-mail: [email protected]

Am J Respir Cell Mol Biol Vol 44. pp 83–90, 2011

Originally Published in Press as DOI: 10.1165/rcmb.2009-0347OC on March 4, 2010

Internet address: www.atsjournals.org

a specific gene of interest, i.e., siRNA–TRPC6 (sc-42672; Santa CruzBiotechnology, Santa Cruz, CA) or siRNA–CFTR (sc-35054; SantaCruz Biotechnology). Control siRNA (referred to here as scrambledsiRNA) was a nontargeted 20–25 nucleotide siRNA designed asa negative control (sc-37007; Santa Cruz Biotechnology). OptimalsiRNA transfections were performed using 0.5 g/ml of siRNA accord-ing to the protocols and transfection medium recommended by themanufacturer (Santa Cruz Biotechnology). Cells were incubated for 48hours before Ca21 imaging or Western blot analysis.

Extraction of TRPC mRNA and Reverse Transcription

Total RNA was extracted using RNABle (Eurobio, Les Ulis, France),according to the manufacturer’s protocol, and mRNA was reverse-transcribed to cDNA, as described elsewhere (9). The specific oligonu-cleotide primers used for each subtype of TRPC channels are presentedin Table 1.

Immunoprecipitation and Western Blot Analysis

Total proteins were obtained and quantified as previously described(10). For immunoprecipitation and Western blotting, we used a mono-clonal human anti-CFTR antibody (IgG2a clone 24-1; R & D Systems,Lille, France), a goat anti-TRPC6 polyclonal antibody (Santa CruzBiotechnology), or a rabbit anti-TRPC1 (Santa Cruz Biotechnology).Other details were described elsewhere (10).

Recording Calcium Signals

Cells were loaded with 3 mM Fluo-4 acetoxymethyl ester (Fluoroprobes/Interchim, Montlucxon, France) for 20 minutes at room temperature,and Ca21 activity was recorded using confocal laser scanning microscopy,as described elsewhere (2).

Single-Cell Measurement of CFTR by Fluorescence Imaging

Ion-channel functions were assessed by single-cell fluorescence imag-ing, using the potential-sensitive probe, bis-(1,3-diethylthiobarbituricacid)-trimethine oxonol (DiSBAC2(3); Molecular Probes, Leiden,Netherlands), as previously reported (11).

Immunofluorescence

We used cells incubated with the primary specific antibody for eachTRPCs isoform: rabbit anti-TRPC1 polyclonal antibody (1:300; SantaCruz Biotechnology) and goat anti-TRPC6 polyclonal antibody (1:300;Santa Cruz Biotechnology) for 2 hours at room temperature. Cellswere then incubated with the corresponding conjugated antibody. Incontrol samples, the primary antibody was omitted. Other details weredescribed elsewhere (10).

Patch-Clamp Recordings

Calcium channels were recorded via cell-attached patches using thepatch-clamp method, as previously described (12). The intrapipetttesolution contained (in mM): 110 CaCl2, 10 Hepes, and 0.1 DIDS (4.49-diisothyocyanato stilbene-2.29 disulfonic acid). DIDS was used to blockCl2 conductance. The bathing solution contained (in mM): 150 K-aspartate, 5 MgCl2, 10 EGTA, and 10 Hepes, pH 7.4.

Statistics

Results are expressed as the mean 6 SEM of n observations. Sets ofdata were compared using the Student t test. Differences were

considered statistically significant when P , 0.05. (in figure legends:ns, no significant difference; *P , 0.05, **P , 0.01, and ***P , 0.001).All statistical tests were performed using GraphPad (Prism, La Jolla,CA) version 4.0 for Windows (GraphPad Software).

Chemicals

We purchased 1-oleoly-2-acetyl-sn-glycerol (OAG), SKF96365 (SKF)thapsigargin, and gadolinium from Sigma-Aldrich (St. Louis, MO).Miglustat was from Toronto Research Chemicals (Toronto, Ontario,Canada). Forskolin and Genistein were from LC Laboratories(Woburn, MA). CFTRinh-172 was from Calbiochem (Nottingham,United Kingdom).

RESULTS

TRPC1 and TRPC6 Are the Main Endogenous TRPC Channels

in Human Airway Epithelial Cells

The expression profiles of isoforms TRPC1–TRPC7 wereanalyzed by the RT-PCR technique in human tracheal glandserous cells of non-CF (MM39) and CF (F508 del homozygous,CF-KM4) origins. Figure 1A shows that TRPC1, TRPC3, andTRPC6 mRNAs were detected in MM39 cells, and TRPC1,TRPC4, and TRPC6 mRNAs were detected in CF-KM4 cells.On the other hand, the TRPC2, TRPC5, and TRPC7 isoformswere not detected in either epithelial cell line. As a control,TRPC5 and TRPC7 were found in another tissue, i.e., rat brain(data not shown). TRPC2 is unique among TRPCs because itscomplete gene has been lost from the human genome, in whichits remnants constitute a pseudogene (13). In conclusion, onlyTRPC1 and TRPC6 are present in both CF and non-CF cells(Figure 1A). Their protein expression was confirmed by West-ern blot experiments (Figure 1B) and confocal immunofluores-cence microscopy imaging (Figure 1C). These results suggestthe expression of TRPC1 and TRPC6 isoforms in these humanairway epithelial cells.

TRPC6 Activity Is Abnormal in CF Airway Epithelial Cells

The TRPC1 and TRPC6 isoforms are regulated by differentpathways (14). TRPC1 activity is dependent on ER Ca21 de-pletion (15, 16), whereas TRPC6 is essentially described asa receptor-operated channel, and is directly activated by Diac-ylglycerol (DAG). Because we observed an abnormal ER Ca21

depletion in CF cells (2), we first compared global TRPC1 activityin CF versus non-CF cells, and in CF cells that had beenpharmacologically corrected with the corrector miglustat (17).We examined capacitative calcium entry (CCE), using Fluo-4acetoxymethyl ester probes (Figure 2A) or a patch-clamp tech-nique (Figures 2B and 2C), and ER Ca21 depletion was inducedby 10 mM thapsigargin (a blocker of the Sarco/EndoplasmicReticulum Calcium ATPase pump). We found no significantdifference in the Ca21 influx induced by CCE among CF, non-CF,and miglustat-corrected CF cells (Figure 2A), and no difference

TABLE 1. SPECIFIC PRIMERS FOR EACH TRANSIENT RECEPTOR POTENTIAL CANONICALCHANNEL SUBTYPE

Primer (Sense) Primer (Antisense) Base Pairs

TRPC1 59-ATGTATACAACGAGCTGTATCTTG-39 59-AGTCTTTGGTGAGGGAATGATG-39 525

TRPC2 59-TCTGGACCATGTTCGGTATG-39 59-GCTACCTCGCTTTGCAGTC-39 565

TRPC3 59-CTGCAAATGAGAGCTTTGGC-39 59-AACTTCCATTCTACATCACTGTC-39 388

TRPC4 59-ATTCATATACTGCCTTGTGTTG-39 59-GGTCAGCAATCAGTTGGTAAG-39 329

TRPC5 59-ACTTCTATTATGAAACCAGAGC-39 59-GCATGATCGGCAATAAGCTG-39 289

TRPC6 59-AAGACATCTTCAAGTTCATGGTC-39 59-TCAGCGTCATCCTCAATTTCC-39 322

TRPC7 59-GGATGCAGATGTGGAATGGAAG-39 59-CGTCATTTTCTCTGTCCACCTG-39 365

84 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 44 2011

in thapsigargin-dependent channel activity (Figures 2B and 2C).These findings indicate that TRPC1 is not affected in CF cells.

The activity of TRPC6 was then studied with the membrane-permeable DAG analogue OAG in CF-KM4 cells, MM39 cells,reverted CF-KM4 cells (stably transfected with wild-type CFTR),and CF-KM4 cells corrected after two different treatments: 2hours in the presence of 100 mM miglustat, or cultured at lowtemperature 278C for 24 hours (Figures 3A and 3B). Ca21

mobilization was measured for 10 minutes in CF-KM4 cells (areaunder the curve [AUC] 5 385 6 20 nM 3 10 minutes, n 5 160),and was found to be z 2-fold increased compared with MM39cells (AUC 5 204 6 7 nM 3 10 minutes, n 5 228) (Figures 3A and3B). The TRPC inhibitor SKF96365 (10 mM) decreased theOAG–Ca21 response by 60% in MM39 (n 5 35) and CF-KM4(n 5 28) cells (Figure 3B). Values produced by the global Ca21

mobilization induced by 100 mM OAG, as observed in miglustat-corrected-CF cells (AUC 5 218 6 13 nM 3 10 minutes, n 5 66), inlow temperature–corrected cells (AUC 5 204 6 13 nM 3 10minutes, n 5 72), and in reverted CF-KM4 cells (AUC 5 191 6

11 nM 3 10 minutes, n 5 100), were similar to non-CF values(Figure 3B). As expected, in the absence of extracellular Ca21, theOAG–Ca21 influx was abolished (Figure 3B, bar at far right). Thus,these results show that TRPC6 activity is exacerbated in CF cells,

but is reversible when the F508 del-CFTR abnormal function iseither corrected or reverted.

With single-channel, cell-attached patch-clamp recordings,we studied the activity of TRPC6 after the addition to the bathof 10 mM OAG. In basal condition (Figure 3C, left traces), wedetected sparse, spontaneous Ca21 channel activities in all celllines. After the addition to the bath of OAG, we recordeddownward deflections of the current, corresponding in our

Figure 1. Identification of transient receptor potential canonical

(TRPC) channels in cystic fibrosis (CF) and non-CF human tracheal-

gland epithelial cells. (A) The expression of TRPC1 and TRPC6 mRNAwas assessed by RT-PCR in CF-KM4 and MM39 cells. Each horizontal

bar indicates the TRPC isoform tested (left lane, cDNA; right lane, H2O).

bp, base pairs. (B) The expression of TRPC1 and TRPC6 proteins wasassessed by Western blot (WB) analysis of CF-KM4 and MM39 cells. (C )

Localization of TRPC1 and TRPC6 proteins was assessed by confocal

immunofluorescence on CF-KM4 and MM39 cells. Scale bar, 10mm.

Figure 2. Function of TRPC1 currents in human tracheal-gland serous

cells. (A) Histogram shows normalized area under the curve (AUC)

corresponding to store-operated Ca21 entry (SOCE) in CF-KM4 (n 5

117), MM39 (n 5 119), and corrected CF-KM4 (n 5 74) cells. The SOCEwas obtained after a 5-minute perfusion of 10 mM thapsigargin (TG) in

the presence of 2 mM extracellular Ca21, and in the presence or absence

of 10 mM SKF96365 (SKF). (B) Original cell-attached patch-clamp

recordings of single-channel inward currents at 260 mV after stimula-tion by 10 mM TG of CF-KM4 and MM39. The closed (c) and open

(o) states of the channel are marked to the right of each tracing.

(C ) Histogram shows mean channel NPo (corresponding to number ofchannel 3 open probability) in resting and TG-stimulated CF-KM4,

MM39, and corrected CF-KM4 cells. Corrected CF-KM4 cells correspond

to CF-KM4 cells treated for 2 hours with 100 mM of miglustat. Results are

presented as mean 6 SEM. ***P , 0.001. ns, no significant difference.

Antigny, Norez, Dannhoffer, et al.: TRPC6 Links Ca21 Mishandling to CFTR Dysfunction 85

experimental conditions to Ca21 entry via TRPC6 (Figure 3C,right traces). Under these different experimental conditions, weobtained similar unitary conductance for TRPC6 channels,calculated by a linear regression of the corresponding current/voltage (I/V) curve, in CF-KM4 cells (5.1 6 0.1 pS, n 5 12),MM39 cells (4.3 6 0.1 pS, n 5 10), corrected CF-KM4 cells (4.5 6

0.1 pS, n 5 7), and reverted CF-KM4 cells (4.9 6 0.1 pS, n 5 4).In contrast, the number of channels 3 open probability (NPo)was abnormally increased in CF-KM4 cells (0.72 6 0.09, n 5

11), compared with MM39 cells (0.33 6 0.06, n 5 9), correctedCF-KM4 cells (0.33 6 0.07, n 5 9), and reverted CF-KM4 cells(0.23 6 0.04, n 5 7) (Figure 3D). The pharmacologic rescue ofF508 del-CFTR also normalized NPo to a level similar to thatof non-CF cells (Figure 3D). These results highlight the role ofTRPC6 as a receptor-operated Ca21 influx channel, in theabnormal Ca21 entry into human airway epithelial CF cells.

Silencing TRPC6 Reduced the Abnormal Ca21 Influx

into Airway Epithelial CF Cells

To confirm the role of TRPC6 in CF cells, we used an siRNAstrategy of knockdown TRPC6 in CF-KM4 and MM39 cells(Figure 4). As shown in Figures 4A and 4C, the OAG-inducedCa21 influx was reduced in CF-KM4 with siRNA–TRPC6, froman AUC of 385 6 20 nM 3 10 minutes (n 5 160) to an AUC of129 6 8 nM 3 10 minutes (n 5 92). In non-CF cells, TRPC6silencing also induced a decrease of Ca21 influx in response to100 mM OAG, from an AUC of 204 6 7 nM 3 10 minutes (n 5

228) to an AUC of 147 6 8 nM 3 10 minutes (n 5 70) (Figures4B and 4C). These data confirm that the OAG-induced Ca21

influx is attributable to TRPC6 in human tracheal-gland serousnon-CF cells, and that TRPC6 activity is exacerbated in CFcells.

Next we explored the effect of siRNA–TRPC6 treatment onCFTR activity in MM39 and CF-KM4 cells (Figure 5). To thisend, we measured CFTR-dependent ionic transport by single-cell fluorescence imaging, using the membrane potential-sensitiveprobe DiSBAC2(3). As shown in Figure 5A, a sharp increase offluorescence, corresponding to membrane depolarization, was

recorded after the addition of the CFTR activators forskolin(Fsk) and genistein (Gst) on MM39 cells. Moreover, the de-crease in fluorescence level observed after the addition ofCFTRinh-172 further confirmed the identity of CFTR. Theaddition of Fsk (10 mM) plus Gst (30 mM) stimulated CFTR-dependent fluorescence (Fx) in MM39 cells (Fx 5 25.16 6 1.62,n 5 30), whereas no variation of fluorescence was evident inCF-KM4 cells (Fx 5 0.66 6 0.42, n 5 22) (Figure 5B). We usedscrambled siRNA as a negative control (denoted as siRNA–Ctr). No significant differences for AUC or Fx were measuredin MM39 and CF-KM4, with or without siRNA–Ctr (Figures 4Cand 5B). On the contrary, when we knocked down TRPC6 withsiRNA in MM39 cells, we observed a twofold decrease inCFTR-dependent fluorescence (Fx 5 10.69 6 0.21, n 5 30).No effect of siRNA–TRPC6 was observed in the F508 del-CFTR activity of CF-KM4 cells (Figure 5B), as expected, be-cause F508 del is a misfolded protein with altered trafficking.

Functional and Reciprocal Coupling between CFTR

and TRPC6

TRPC isoforms are able to form a heteromultimeric complex inseveral cell types (18–20). In co-immunoprecipitation experi-ments, we found no evidence for TRPC1/TRPC6, TRPC1/CFTR, and TRPC1/F508 del-CFTR interactions in CF or non-CF cells (Figure 6A). By contrast, to explore the molecularmechanism linking TRPC6 to CFTR, we immunoprecipitatedCFTR onto TRPC6 Western blots, and found that wildtype-CFTR and F508 del-CFTR are both able to interact with TRPC6in CF-KM4 and MM39 cells (Figure 6B).

We also explored the effects of siRNA–CFTR on OAG-induced Ca21 influx in CF and non-CF cells (Figure 7). First weevaluated the activity of CFTR channels by single-cell fluores-cence imaging in non-CF (Figure 7A) and CF (Figure 7B) cellstreated or not treated using siRNA–CFTR for 48 hours. Noeffect of scrambled siRNA was evident in MM39 and CF-KM4cells (Figure 5B). As shown in Figure 7A, membrane depolar-ization was recorded after the addition of the CFTR activators,Fsk plus Gst, to MM39 cells. When MM39 cells were treated

Figure 3. Activation of TRPC6 by 1-oleoly-2-

acetyl-sn-glycerol (OAG) in human tracheal-gland serous cells. (A) Typical traces of Ca21

mobilization induced by 100 mM OAG in

MM39 and CF-KM4 cells for 10 minutes inpresence of 2 mM extracellular Ca21. (B) Corre-

sponding histogram shows normalized AUC

corresponding to Ca21 mobilization induced by

100 mM OAG for 10 minutes, in the presence orabsence of 10 mM SKF96365 (SKF). Results are

presented as mean 6 SEM. ***P , 0.001. ns, no

significant difference. (C) Single-channel TRPC6

currents in human tracheal-gland serous cells.Original traces in cell-attached configuration

show single-channel inward currents (at 260 mV)

for 1 minute before (Basal) and after stimulationby 10 mM OAG. As indicated at left, recordings

are from CF-KM4, MM39, corrected CF-KM4

(2 hours, with 100 mM miglustat), and reverted

CF-KM4 cells. The closed (c) and open (o)states of the channel are marked to the right

of each tracing. (D) Histogram shows mean

channel NPo, as indicated. Results are presented

as mean 6 SEM. **P , 0.01. ***P , 0.001. ns, nosignificant difference.


with siRNA–CFTR for 48 hours, we measured a lower fluores-cence level (Fx 5 19.61 6 4.29, n 5 22) than in its absence (Fx 5

40.10 6 3.31, n 5 28) (Figures 7A and 7C). The knockdown ofCFTR had no effect on CF-KM4 cells (Figure 7C). On the otherhand, when we rescued F508 del-CFTR on CF-KM4 using 100 mMmiglustat for 2 hours, we observed a significant stimulation ofF508 del-CFTR–dependent fluorescence (Fx 5 49.08 6 2.02, n 5

24), which was significantly decreased when miglustat-correctedCF-KM4 cells were treated with siRNA–CFTR (Figures 7B and7C).

To validate the functional coupling between TRPC6 andCFTR channels in CF and non-CF cells, we also measured theeffect of siRNA–CFTR on OAG-dependent Ca21 mobilization(Figure 8). We observed a significant increase of Ca21 response,from AUC 5 214.40 6 7.16 nM 3 10 minutes (n 5 228) inMM39 cells, to AUC 5 336.10 6 39.81 nM 3 10 minutes (n 5

25) in siRNA–CFTR-treated MM39 cells (Figures 8A and 8B).Conversely, no significant variation of Ca21 mobilization wasobserved between CF-KM4 and siRNA–CFTR CF-KM4 cells(Figure 8B). These results suggest that the knockdown of CFTRcan induce an abnormal Ca21 response in non-CF cells. Thus, ourresults suggest the existence of functional and reciprocal couplingbetween CFTR (or F508 del-CFTR rescued at the plasma mem-brane) and TRPC6 channels in human epithelial cells.

We repeated some of our key experiments in freshly ciliatedhuman bronchial epithelial cells (Figure 9). The expression

Figure 4. Effect of silenc-

ing TRPC6 on OAG–Ca21

entry. (A) Typical traces of

Ca21 mobilization induced

by 100 mM OAG in CF-

KM4 and small interfering(si) RNA TRPC6 CF-KM4

cells for 10 minutes in the

presence of 2 mM extra-

cellular Ca21. (B) Typicaltraces of Ca21 mobilization

induced by 100 mM OAG

in MM39 and siRNA–

TRPC6 MM39 cells for10 minutes in the presence

of 2 mM extracellular

Ca21. (C) Correspondinghistograms summarize

normalized AUC after stim-

ulation by 100 mM OAG.

Results are presented asmean 6 SEM. ***P ,

0.001. ns, no significant

difference.


ing TRPC6 on CFTR activ-ity. (A) Example of typical

time courses obtained

with MM39 cells in the

presence or absence ofsiRNA–TRPC6 treatment.

Fsk, forskolin; Gst, genis-

tein; CFTR, cystic fibrosis

transmembrane conduc-tance regulator. (B) Histo-

grams of mean relative

fluorescence collectedfrom separate experi-

ments, with a total num-

ber of cells from 22–30.

Results are presented asmean 6 SEM. ***P ,


difference.

Figure 6. Co-immunoprecipitation of wild-type or F508 deletion

(del)-CFTR and TRPC6 in human tracheal-gland serous cells. (A) Co-

immunoprecipitation of CFTR with TRPC1 in CF-KM4 and MM39 cells.

Protein sepharose (Prot. G and Prot. A) and TRPC1 immunoprecipitationwere used as negative and positive controls, respectively. (B) Co-

immunoprecipitation of CFTR with TRPC6 in CF-KM4 and MM39 cells.

Prot. G, Prot. A, and TRPC6 immunoprecipitation were used as neg-ative and positive controls.


profile of the TRPC1–TRPC7 isoforms was analyzed by theRT-PCR technique in human bronchial epithelial cell cultures,and only TRPC6 mRNA was detected (Figure 9A). In co-immunoprecipitation experiments, we confirmed that CFTRcan interact with TRPC6 in human bronchial primary cellcultures (Figure 9B). We evaluated the activity of CFTRchannels via single-cell fluorescence imaging in isolated, ciliatedhuman epithelial cells treated or not treated with siRNA–CFTRor siRNA–TRPC6 (Figures 9C and 9D). No effect of scrambledsiRNA (siRNA–Ctr) was evident, compared with untreated cells(Figure 9D). Figures 9C and 9D show that CFTR activity wasreduced by 60% in isolated, ciliated human epithelial cells withsiRNA–CFTR. As shown in Figures 9E and 9F, the OAG-inducedCa21 influx was reduced by 66% in isolated, ciliated humanepithelial cells with siRNA–TRPC6, and was increased by 63%in cells treated with siRNA–CFTR. These results confirm that theknockdown of CFTR is able to induce an abnormal Ca21 responsein non-CF human polarized epithelial cells, and also confirm theexistence of functional and reciprocal coupling between CFTR andTRPC6 in primary human ciliated epithelial cells.

DISCUSSION

We showed here that TRPC6 is endogenously expressed in non-CF and CF airway epithelial human cells. TRPC6 played a rolein cytosolic Ca21 handling within a macromolecular proteincomplex including CFTR (or F508 del-CFTR in CF cells).Compared with non-CF airway epithelial cells, the TRPC6-mediated Ca21 influx was increased twofold in CF cells. The useof siRNA–TRPC6 and scrambled siRNA implicated TRPC6 inthe abnormal OAG–Ca21 influx into CF cells. The pharmaco-logic corrector miglustat (17), or a protocol of low-temperatureincubation of CF cells allowing the rescue of abnormal traffick-ing of F508 del-CFTR (21), both succeeded in reversing the up-regulated TRPC6-dependent Ca21 influx in CF cells. On theother hand, using an siRNA strategy after CFTR knockdown innon-CF cells, the OAG–Ca21 influx was up-regulated to a levelsimilar to that in CF cells. Based on these observations, we proposea model in which CFTR down-regulates TRPC6-dependent Ca21

influx, and in which TRPC6 up-regulates CFTR-dependent Cl2

transport in airway epithelial cells. This reciprocal functionalcoupling is favored by the fact that CFTR and TRPC6 could beassociated, probably within a multiprotein complex. In CFairway epithelial cells, this functional coupling is lost becauseof abnormal trafficking of F508 del-CFTR, leading to abnormalcontrol of the OAG–Ca21 influx.

The misregulation of TRPC channels has been increasinglyimplicated in the physiopathology of many pulmonary humandiseases. TRPC1 plays a role in asthma and chronic obstructivepulmonary disorder (COPD). In proliferating airway smoothmuscle cells, TRPC1 is up-regulated (22). Interestingly, amongthe TRP isoforms, TRPC6 is highly expressed in the human lung,and especially in smooth muscle and epithelial cells. TRPC6 up-regulation in response to platelet-derived growth factor wasassociated with pulmonary artery smooth muscle proliferation(23), and with idiopathic pulmonary arterial hypertension (24).TRPC6 may also contribute to the hypersecretion of mucus,


ing CFTR on CFTR activity.

(A) Functional evaluation ofCFTR and F508 del-CFTR by

bis-(1,3-diethylthiobarbituric

acid)-trimethine oxonol

assay. Example of typicaltime courses obtained

with MM39 cells in the

presence or absence of

siRNA-CFTR treatment. (B)Example of typical time

courses obtained with CF-

KM4 cells treated for2 hours with 100 mM

miglustat in the presence

or absence of siRNA–CFTR.

Data represent the mean 6

SEM of relative fluorescence

collected from all cells of

a field. (C) Histograms of

mean relative fluorescencecollected from separate ex-

periments, with a total

number of cells from22–44. Results are pre-

sented as mean 6 SEM.

***P , 0.001. ns, no signif-

icant difference.

Figure 8. Effect of si-lencing CFTR on OAG–

Ca21 entry (A) Typical

traces of Ca21 mobiliza-

tion induced by 100 mMOAG in MM39 and in

siRNA–CFTR MM39

cells. (B) Histograms

summarize normalizedAUC after stimulation

by 100 mM OAG in

MM39 and CF-KM4cells in the presence or

absence of siRNA–CFTR.

Results are presented as

mean 6 SEM. ***P ,


difference.


another characteristic feature of COPD (25). Recently, a unique,nonredundant role for TRPC6 was postulated in allergic airwayinflammation, making this cation channel a potential new drugtarget in asthma and COPD (26). In cystic fibrosis, persistentairway infection and inflammation are predominant clinical phe-notypic characteristics, relevant to the impaired clearance ofmucus (27). Thus, we may reasonably propose that the up-regulation of TRPC6 activity in CF cells reflects a responsecharacterized by persistent epithelial exposure to an inflamma-tory environment. These observations may prove important indesigning strategies for therapeutic agents aimed at restoringnormal TRPC6 activity in patients with chronic infectious lungdisease (e.g., CF).

The down-regulation of TRPC6-dependent Ca21 influx byCFTR is reminiscent of the picture already observed for theepithelial Na1 channel (ENaC), and for CFTR in airwayepithelia (28). Indeed, a hallmark of CF is the hyperabsorptionof Na1 by epithelial cells from the lumen of the tracheobron-chial tree to the blood compartment, via pores of the cationchannel ENaC (29). Among multiple local signals that controlthe activity of ENaC, CFTR plays a particular role (althoughnot fully understood), because both channels may interact viaintermolecular links, such that CFTR down-regulates ENaC(29, 30). We recently showed that the pharmacologic correction

of the abnormal trafficking of F508 del-CFTR induced a nor-malization of ENaC activity (31). Thus, we hypothesize thatENaC and TRPC6 are two channels under the control of CFTRin airway epithelial cells. In CF cells, where the biogenesis ofmutated CFTR proteins is abnormal, the absence of CFTR atthe plasma membrane precludes interactions with ENaC orTRPC6, and thus leads to enhanced Na1 and Ca21 influx bythese cation channels. In non-CF or in corrected CF cells, wherethe CFTR protein is relocated and functional at the plasmamembrane, both enhanced Na1 and Ca21 influx are down-regulated, and normalized because of recovered channel interac-tions. Thus, modulating protein–protein interactions providesa complex but efficient mechanism for airway epithelial cells toregulate the transepithelial transport of cations with CFTR as anintermediary.

In the airways, TRPC6, already known as an essentialcomponent of the receptor-activated Ca21 influx machinery insmooth muscle cells, is proposed to be the channel responsiblefor abnormal Ca21 influx in human CF epithelial cells, linkingcalcium signaling to CFTR pathobiology. A model couldemerge in which CFTR and TRPC6 are present within a multi-protein complex, each channel regulating the other, i.e., CFTRdown-regulates TRPC6-dependent Ca21 influx, and TRPC6 up-regulates CFTR-dependent Cl2 transport. In CF airway epithe-

Figure 9. Functional coupling between CFTR and TRPC6 in

freshly isolated ciliated human epithelial cells. (A) Expression

of TRPC6 mRNA was assessed by RT-PCR in humanbronchial epithelial cell culture. (B) Co-immunoprecipitation

of CFTR with TRPC6 in primary human epithelial cell

culture. (C ) Effects of silencing TRPC6 and CFTR on CFTRactivity. Example of typical time courses obtained with

isolated ciliated epithelial human cells, in the absence

(untreated) or presence of siRNA–Ctr, siRNA–TRPC6. or

siRNA–CFTR treatment. (D) Histograms of mean relativefluorescence collected from separate experiments. Results

are presented as mean 6 SEM. ***P , 0.001. ns, no

significant difference. (E ) Effects of silencing TRPC6 or

CFTR on OAG–Ca21 entry. Typical traces of Ca21 mobi-lization induced by 100 mM OAG in isolated, ciliated

human epithelial cells in the absence (untreated ) or

presence of siRNA–Ctr, siRNA–TRPC6, or siRNA–CFTR

treatment. (F ) Corresponding histograms summarize nor-malized AUC after stimulation by 100 mM OAG. Results

are presented as mean 6 SEM. ***P , 0.001. ns, no

significant difference.


lial cells, the macromolecular edifice is disrupted and functionalcoupling is lost because of the abnormal trafficking of F508 del-CFTR, leading to abnormal control of OAG–Ca21 influx. Theseobservations and hypotheses open up a novel area of research,i.e., to understand how epithelial Ca21 and Cl2 transports areregulated. TRPC6 may represent a novel drug target in the fieldof CF for pharmacologic therapeutic interventions.

Author Disclosure: None of the authors has a financial relationship with acommercial entity that has an interest in the subject of this manuscript.

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