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ARTICLE IN PRESSG ModelC-4233; No. of Pages 10

The International Journal of Biochemistry & Cell Biology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

jo ur nal home page: www.elsev ier .com/ locate /b ioce l

eview

unctional interaction of the cystic fibrosis transmembraneonductance regulator with members of the SLC26 family of anionransporters (SLC26A8 and SLC26A9): Physiological andathophysiological relevance�

lma El Khouria,b,c, Aminata Touréa,b,c,∗

INSERM, U1016, Institut Cochin, Paris, FranceCNRS, UMR8104, Paris, FranceUniversité Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France

r t i c l e i n f o

rticle history:eceived 4 December 2013eceived in revised form 29 January 2014ccepted 1 February 2014vailable online xxx

eywords:LC26ale infertility

ronchiectasisFTRystic fibrosis

a b s t r a c t

The solute carrier 26 (SLC26) proteins are transmembrane proteins located at the plasma membrane of thecells and transporting a variety of monovalent and divalent anions, including chloride, bicarbonate, sulfateand oxalate. In humans, 11 members have been identified (SLC26A1 to SLC26A11) and although partof them display a very restricted tissue expression pattern, altogether they are widely expressed in theepithelial cells of the body where they contribute to the composition and the pH regulation of the secretedfluids. Importantly, mutations in SLC26A2, A3, A4, and A5 have been associated with distinct humangenetic recessive disorders (i.e. diastrophic dysplasia, congenital chloride diarrhea, Pendred syndromeand deafness, respectively), demonstrating their essential and non-redundant functions in many tissues.During the last decade, physical and functional interactions of SLC26 members with the cystic fibrosistransmembrane conductance regulator (CFTR) have been highly documented, leading to the model of acrosstalk based on the binding of the SLC26 STAS domain to the CFTR regulatory domain. In this review,we will focus on the functional interaction of SLC26A8 and SLC26A9 with the CFTR channel. In particular
we will highlight the newly published studies indicating that mutations in SLC26A8 and SLC26A9 proteinsare associated with a deregulation of the CFTR anion transport activity in the pathophysiological contextof the sperm and the pulmonary cells. These studies confirm the physiological relevance of SLC26 andCFTR cross-regulation, opening new gates for the treatment of cystic fibrosis.
This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology,and therapeutic advances.

© 2014 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001.1. The SLC26 family of anion transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001.2. Interaction of the SLC26 family members with CFTR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: El Khouri E, Touré A. Functional interawith members of the SLC26 family of anion transporters (SLC26A8 andBiochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.02.0

2. SLC26A8 (TAT1; testis anion transporter 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.1. SLC26A8 presentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2. SLC26A8 physiological functions: control of sperm motility and c

Abbreviations: cAMP, cyclic adenosine monophosphate; CF, cystic fibrosis; CFBE4Io-onductance regulator; CHO-K1, Chinese hamster ovary-K1; DHPLC, denaturing high perfopithelial; HEK, human embryonic kidney; IRBIT, IP3 receptor binding protein released warrier 26; STAS, sulfate transporter and anti-sigma factor antagonist.� This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell bio∗ Corresponding author at: Department of Genetics, Development and Reproduction, Iu faubourg Saint Jacques, 75014 Paris, France. Tel.: +33 1 44 41 24 61.

E-mail address: [email protected] (A. Touré).

357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.biocel.2014.02.001

ction of the cystic fibrosis transmembrane conductance regulator SLC26A9): Physiological and pathophysiological relevance. Int J

01

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00apacitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

, cystic fibrosis bronchial epithelial cell line; CFTR, cystic fibrosis transmembranermance liquid chromatography; ENac, epithelial Na channel; HBE, human bronchialith IP3; PKA, protein kinase A; sAC, soluble adenylate cyclase; SLC26, solute-linked

logy, physiology, and therapeutic advances.nstitut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, 24 rue

dx.doi.org/10.1016/j.biocel.2014.02.001


http://www.sciencedirect.com/science/journal/13572725

http://www.elsevier.com/locate/biocel

mailto:[email protected]


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2 E. El Khouri, A. Touré / The International Journal of Biochemistry & Cell Biology xxx (2014) xxx–xxx

2.3. CFTR functions in sperm motility and capacitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Physical interaction and co-localization of SLC26A8 and CFTR in the sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5. Physical interaction of SLC26A8 with CFTR results in CFTR stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.6. Identification of SLC26A8 mutations destabilizing the SLC26A8–CFTR complex in human asthenozoospermia. . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. SLC26A9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. SLC26A9 presentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. SLC26A9 physiological functions in the stomach, lung and kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. SLC26A9 physical interaction with CFTR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.4. SLC26A9 interaction with CFTR stimulates CFTR activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.5. SLC26A9 interaction with CFTR modulates SLC26A9 function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.6. Identification of SLC26A9 mutations destabilizing the SLC26A9–CFTR complex in diffuse idiopathic bronchiectasis . . . . . . . . . . . . . . . . . . . . . . 00

4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

.1. The SLC26 family of anion transporters

The solute carrier 26 (SLC26) proteins are multifunctional trans-embrane proteins mediating the transport of various monovalent

nd divalent anions including Cl− (chloride), HCO3− (bicarbonate),

O42− (sulfate), iodide (I−) and C2O4

2− (oxalate), mainly across thelasma membrane of epithelial cells and contributing to the com-osition and the pH of secreted fluids in the body. SLC26 belongo the highly conserved superfamily of amino acid-polyamine-rganocation (APC) transporters and SLC26-related proteins haveeen found in various organisms among which, bacteria, yeast,lgae and plants (SulP/Sultr proteins). In mammals, 11 membersave been identified and designated as SLC26A1 to SLC26A11,LC26A10 most probably corresponding to a pseudogene (Table 1).

SLC26 proteins share a common structure including (i) a highlyonserved transmembrane region involved in the anion transportctivity, which comprises 10–14 hydrophobic spans and (ii) a lessonserved cytoplasmic region which comprises the STAS domainsulfate transporter and anti-sigma factor antagonist), involvedn protein–protein interaction and regulation. In addition, several

embers carry a PDZ binding domain at their carboxy-terminalxtremity (for reviews see Ohana et al., 2009; Alper and Sharma,013). In the last three years, significant progress in the field ofLC26s structure has been completed as X-ray crystallography andMR studies achieved the modeling of the STAS domain for twoacterial and one mammalian members of the SLC26/SulP familyEscherichia coli YchM, Mycobacterium tuberculosis Rv1739c SO4

2−

ransporter, Yersinia enterocolitica SLC26A2, rat SLC26A5/Prestin)Babu et al., 2010; Pasqualetto et al., 2010; Compton et al., 2011;harma et al., 2011). However, no structure of the transmembraneegion has been reported so far. One additional structural feature ofhe SLC26s that has emerged during the last years is their capacity toorm homo-dimers and -oligomers, most probably via their trans-

embrane regions; this property was in particular documented forLC26A5/Prestin in mammals (Detro-Dassen et al., 2008; Comptont al., 2011).

SLC26 members mediate the transport of various anions and theechanisms associated are variable between the different mem-

ers. Surprisingly, they can be multiple for a single transporter.he current classification of the SLC26 family, based on their modef transport, comprises three main groups: the SO4

2− transportersSLC26A1 and A2], the Cl−/HCO3

− exchangers [SLC26A3, A4 and6] and the ion channels [SLC26A7 and A9] (for reviews see Ohanat al., 2009; Alper and Sharma, 2013). So far SLC26A5 anion trans-


ort activity has not been reported in mammals but only in chicken,ebra fish and insects (Schaechinger and Oliver, 2007; Hirata et al.,012); however SLC26A5 activity as a motor protein was showno be anion-dependent (Rybalchenko and Santos-Sacchi, 2008).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Regarding SLC26A8 and SLC26A11, they have been poorly charac-terized and their mode of transport is ill defined. In total, althoughuseful, the above current classification is complex because of thelack of correlation with sequence similarities and most importantlythe capacity for some members to behave as both channels andanion exchangers depending on the substrates available and prob-ably their cellular and sub-cellular localization, as illustrated forSLC26A3 and A6.

SLC26 family members are undoubtedly essential to variousphysiological functions and differentiation processes; in human,pathogenic “loss of function” mutations in SLC26s have been asso-ciated with four hereditary genetic diseases: diastrophic dysplasia(SLC26A2), congenital chloride diarrhea (SLC26A3), Pendred syn-drome (SLC26A4) and deafness (SLC26A5) (Everett and Green,1999; Dawson and Markovich, 2005), which are all transmittedfollowing an autosomal recessive mode of inheritance. These phen-otypes are in line with the restricted tissue expression profilesobserved for most of the SLC26 genes. To date knock out and knockin mouse models have been generated for all SLC26 membersexcept SLC26A11. These models did reproduce most of the clini-cal features of the SLC26 human-related diseases when applicable(Liberman et al., 2002; Forlino et al., 2005; Schweinfest et al., 2006;Dallos et al., 2008; Dror et al., 2010; Lu et al., 2011). Interestingly,inactivation of the genes not related so far to a human disease alsogenerated a specific phenotype in the tissues where the concernedSLC26s were to be expressed (i.e. kidney, liver, stomach, testis, etc.)(Jiang et al., 2006; Touré et al., 2007; Xu et al., 2008, 2009; Dawsonet al., 2010), suggesting that in humans, mutations in other SLC26genes could be pathogenic.

1.2. Interaction of the SLC26 family members with CFTR

SLC26 proteins are expressed throughout the entire body andmost interestingly they were shown to be co-expressed with thecystic fibrosis transmembrane conductance regulator (CFTR) in var-ious epithelia. The existence of physical and functional interactionsbetween the cystic fibrosis transmembrane conductance regulator(CFTR) Cl− channel and several members of the SLC26 family ofanion transporters is now well established. Pioneer work describeda mutual regulation between CFTR and two members of the SLC26family: SLC26A3 and SLC26A6 (Ko et al., 2002, 2004; Chernovaet al., 2003). In the proposed model, physical interaction betweenCFTR and SLC26s first involves the binding of their PDZ domainswith a common scaffold protein bringing them to close proximityand allowing subsequent direct interaction of the STAS domain ofthe SLC26s with the R domain of CFTR (Fig. 1). Interestingly, this


01

interaction was shown to be enhanced by PKA-mediated phos-phorylation of the R domain (Ko et al., 2002, 2004; Shcheynikovet al., 2006). Following this discovery, physical and/or functionalinteraction of CFTR were further documented for SLC26A3, A6



E. El Khouri, A. Touré / The International Journal of Biochemistry & Cell Biology xxx (2014) xxx–xxx 3

Table 1Main characteristics of SLC26 family members. The table displays the principal characteristics of the SLC26 proteins, in particular the tissues were they are mainly expressedtogether with their anion specificity and their mode of transport when defined. The clinical signs of the human genetic disorders associated with SLC26 mutations and thephenotype of the corresponding mutant mouse model are also mentioned.

Protein Major tissue distribution Function Interaction withCFTR

Associated humanpathology

Phenotype of the mousemodelsa

SLC26A1 (SAT1) Liver–Pancreas–Brain–Kidney SO42− and oxalate

transporterND Deregulation of sulfate and

oxalate homeostasisSLC26A2 (DTDST) Ubiquitous SO4

2−/2Cl− ,SO4

2−/2OH− ,SO4

2−/OH−/Cl−

exchanger

ND Diastrophicdysplasia

KI: non-lethalchondrodysplasia

SLC26A3(DRA/CLD)

Gastrointestinal and Reproductivesystems–Sweat Gland

2Cl−/HCO3− ,

Cl−/OH− exchangerNO3

− and SCN−

channel

+ Congenital chloridediarrhea

Chloride-losing diarrheaEnhanced colonicproliferation

SLC26A4(PDS/Pendrin)

Thyroid–Inner ear–Kidney–Salivarygland duct

Cl−/HCO3-, Cl−/I− ,I−/HCO3

−

exchanger

+ Pendred syndrome DeafnessEnlargement of themembranous labyrinthEnlargement of thevestibular aqueduct

SLC25A5 (Prestin) Cochlea Electromotility + Deafness Loss of outer hair cellelectromotilityLoss of cochlear sensitivity

SLC26A6(CFEX/PAT1)

Kidney–Pancreas–Duodenum–Skeletalmuscle

Cl−/2HCO3− ,

Cl−/oxalate,Cl−/formateexchanger, NO3

− &SCN− channel

+ Alteration of Cl−/HCO3−

exchange in nativepancreatic ductCalcium oxalateurolithiasis

SLC26A67 (SUT2) Kidney–Stomach–Testis–Epididymis Cl−/HCO3−

exchanger Cl−

channel

ND Distal renal tubularacidosisImpaired gastricacidification

SLC26A8 (TAT1) Testis (male germ cells) Cl− , SO42− , oxalate

transporter+ Male infertility

(asthenozoosper-mia)

Male sterility(asthenozoospermia andflagellum structuraldefects)

SLC26A9 Lung–Stomach Cl−/HCO3− ,

Na+/anionexchanger Cl−

channel

+ Diffuse idiopathicbrochiesctasis

Gastric hypochlorhydriaAirway mucus obstructionin inflammatory conditionReduction of the renalchloride excretionElevated systemic arterialpressure

SLC26A10(Pseudogene)

– – – – –

2− orter

(eAc2e

nutSooeomeweeu

o

SLC26A11(SUT1/KBAT)

Ubiquitous SO4 transpCl− channel

a Knock-out unless otherwise specified.

Shcheynikov et al., 2008; Stewart et al., 2009) and reported for sev-ral other members of the SLC26 family (i.e. SLC26A4, A5, A8 and9), suggesting that cross-regulation between CFTR and SLC26 is aommon mechanism and is essential to their functions (Ko et al.,002, 2004; Rakonczay et al., 2008; Bertrand et al., 2009; Changt al., 2009a; Homma et al., 2010; Rode et al., 2012).

The functional consequences of SLC26 and CFTR binding haveot been established for all SLC26s but both reciprocal andnidirectional mechanisms of regulation (stimulation or inhibi-ion) were reported. Functional interaction between SLC26A3 andLC26A6 with CFTR was shown to be reciprocal as co-expressionf the STAS domain of both SLC26s resulted in increasing thepen probability of the CFTR channel and reciprocally, CFTR co-xpression resulted in increasing the HCO3

−/Cl− exchange activityf the SLC26s (Ko et al., 2004). In contrast, a “one-way” activityodulation was described for SLC26A4 and SLC26A5 as (i) over-

xpression of CFTR resulted in the activation of SLC26A4 (Pendrin)hereas deletion of SLC26A4 did not impact on CFTR activity (Ko

t al., 2002), (ii) the nonlinear capacitance of SLC26A5 (Prestin) was


nhanced upon CFTR interaction while CFTR conductance remainednchanged upon its association with SLC26A5 (Homma et al., 2010).

From these observations we assume that in vivo the selectivityf the anion transport mediated by the SLC26s is provided by their

ND ND

intrinsic properties and the composition of the complexes theymight form with CFTR and/or other associated proteins. In supportof this hypothesis, in vivo studies conducted in the mouse parotidgland duct clearly demonstrated the selective functions of Slc26a4and Slc26a6 in this same tissue: Slc26a4 mediates the active secre-tion of I− in a CFTR-independent mode whereas Slc26a6 mediatesHCO3

− transport under the regulation of CFTR (Shcheynikov et al.,2008).

Very recently, the IRBIT protein (IP3 receptor binding proteinreleased with IP3) has emerged as a novel actor in coupling theactivities of CFTR and SLC26 anion exchangers; hence IRBIT wasshown to bind to and to activate both CFTR and Slc26a6 upon Ca2+

and cAMP stimulation (Hong et al., 2013; Park et al., 2013). By inte-grating Ca2+ and cAMP signalings, IRBIT antagonizes the effects ofthe WNK/SPAK (with no Lysine/Ste20-related Proline Alanine-richKinase) pathway, and is involved in the synergic regulation of sev-eral transporters resulting in the stimulation and the coordinationof epithelial fluid secretion (Yang et al., 2009, 2011; Mikoshiba,2012).


01

During the last decade, the crosstalks existing between SLC26proteins and CFTR have been extremely documented. Recently,mutations in SLC26A8 and SLC26A9 abrogating the stimulationof CFTR anion transport activity have been reported in patients




Fig. 1. Model of the physical interaction between SLC26 family proteins and the CFTR channel. SLC26 family members share a common structure comprising a conservedtransmembrane region of 10–14 hydrohobic spans, associated with their anion transport activity, and a less conserved carboxy-terminal region including the STAS domain(sulfate transporter and anti-sigma factor antagonist), involved in protein–protein interaction and regulation. Several SLC26 members also contain a PDZ binding motif attheir carboxy-terminal extremity. The formation of the SLC26–CFTR complex relies on the direct interaction of the SLC26s’ STAS domain with the regulatory (R) domain ofCFTR, interaction, which is enhanced by PKA-dependent phosphorylation of the R domain. In addition indirect interaction of the proteins is mediated by the binding of bothSLC26s and CFTR to common PDZ motif-containing scaffold proteins.

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resenting functional defects in the sperm and in the lung wherehese two SLC26s are respectively co-expressed with CFTR. In thiseview, we will summarize data concerning the functional interac-ions of SLC26A8 and A9 with the CFTR channel in the physiologicalnd pathophysiological context.

. SLC26A8 (TAT1; testis anion transporter 1)

.1. SLC26A8 presentation

SLC26A8 also called TAT1 (testis anion transporter 1) was iso-ated through a two-hybrid screening as an interacting partner of

gcRacGAP, a male germ specific regulator of the Rho GTPases andharacterized as a novel member of the SLC26 family displaying anxclusive expression in the human adult testis and in the male germells at the spermatocyte and spermatid stages (Toure et al., 2001).urther studies indicated that expression of SLC26A8 is maintainedn the mature sperm as SLC26A8 protein was detected at the annu-us, a ring-shaped structure located at the junction of the midpiecend the principal piece of the sperm flagellum (Touré et al., 2007)nd at the equatorial segment of the sperm head (Rode et al., 2012).n additional study using a homology-based cloning strategy iden-

ified SLC26A8 as a member of the SLC26 family presenting the bestcore of similarity with SLC26A3 and SLC26A6 (Lohi et al., 2002) andonfirmed the testis specific expression pattern of the gene.

Altogether the functional studies performed so far indicatehat SLC26A8 displays a SO4

2− transport activity at least partlyhrough a SO4

2−/Cl− exchange mechanism. SLC26A8 transportctivity toward the C2O4

2− but not the I− was also demonstrated


Toure et al., 2001; Lohi et al., 2002). So far, SLC26A8 remains onef the less characterized members of the SLC26 family as its physi-logical substrates in the male germline together with the mode ofransport possibly involved are not known.

2.2. SLC26A8 physiological functions: control of sperm motilityand capacitation

Ion fluxes, more precisely, Ca2+, Cl− and HCO3− influxes trig-

ger sperm motility and capacitation events that are required foroocyte fertilization (i.e. flagellum hyperactivation and sperm headacrosomal reaction) (Fig. 2A). During these processes substan-tial changes are occurring both at the plasma membrane and thecytoplasmic levels; hence cholesterol depletion induces the reorga-nization of membrane phospholipids, and complex ion movementslead to hyperpolarization of the sperm plasma membrane poten-tial, intracellular alkalinization and protein hyperphosphorylation.Interestingly, Ca2+, Cl− and HCO3

− influxes are responsible forincreasing the intracellular cAMP concentration and the subse-quent activation of PKA and phosphorylation cascades essentialfor sperm capacitation. Part of these intracellular responses of thesperm are mediated by the soluble adenylate cyclase (sAC), whichis highly expressed in the sperm and is activated upon capacitationby direct binding of Ca2+ and HCO3

− ions (Chen et al., 2000; Jaiswaland Conti, 2003; Xie et al., 2006).

Deletion of Slc26a8 in the mouse was shown to induce a malesterility phenotype due to the lack of sperm motility (i.e. astheno-zoospermia) and impaired capacitation but also to severe structuraldefects of the flagellum (Touré et al., 2007). In particular the lackof PKA-dependent hyperphosphorylation and reduced acrosomalreaction were observed upon sperm incubation in a capacitatingmedium containing Ca2+, Cl− and HCO3

− and albumin, the lat-ter facilitating cholesterol depletion, suggesting that (i) Slc26a8anion transport activity was essential for these processes and (ii)in human, SLC26A8 mutations could lead to male infertility. In con-sistence with the flagellum defects also observed in Slc26a8-null


01

sperm, the absence of the annulus, the structure of which SLC26A8is a component, was correlated with a human asthenozoosper-mia phenotype by analysis of a Caucasian and a Japanese cohorts(Sugino et al., 2008; Lhuillier et al., 2009).



E. El Khouri, A. Touré / The International Journal of Biochemistry & Cell Biology xxx (2014) xxx–xxx 5

Fig. 2. Major ions movements occurring at the plasma membrane of the sperm cell upon capacitation and of the epithelial cells in the airways. (A) Changes in ion permeabilityoccurring at the plasma membrane of the sperm cell lead to membrane hyperpolarization, intracellular alkalinization and protein hyperphosphorylation, which togetherare responsible for the hyperactivation (i.e. increased beating frequency and amplitude of the sperm flagelum) and the capacitation of the sperm. ENaC: epithelial sodiumchannel; CFTR: cystic fibrosis transmembrane conductance regulator; SLC26A8: solute carrier 26 A8; Hv1: voltage-gated hydrogen channel 1; NHE: sodium/hydrogene nnel so transr g pot

2

ag(gtodswrith2

2i

mtmam

scCS

xchanger; CatSper: cation channel sperm-associated protein; Slo3: potassium chaf the airways epithelial cells. ENaC: epithelial sodium channel CFTR: cystic fibrosisectifying Cl− channels; CaCC: Ca2+-dependent Cl− channels; Kir: inwardly rectifyin

.3. CFTR functions in sperm motility and capacitation

Recently, the expression of CFTR was reported in the mousend human sperm and shown to be restricted to the midpiece fla-ellum compartment and to the equatorial segment of the headHernández-González et al., 2007; Xu et al., 2007); expression inuinea pig was also documented but shown to be restricted tohe acrosome of the sperm head. Importantly, the requirementf CFTR for sperm motility and capacitation processes was alsoemonstrated, in particular by using genistein and CFTR inh-172 topecifically activate or inhibit the CFTR channel, respectively. CFTRas shown to be required for the cAMP and the intracellular pH

ise upon sperm incubation in a capacitating medium. Besides, Cl−

nfluxes mediated by CFTR were shown to be required for inhibi-ion of the Epithelial sodium channel (ENac) and sperm membraneyperpolarization upon capacitation (Hernández-González et al.,007; Xu et al., 2007).

.4. Physical interaction and co-localization of SLC26A8 and CFTRn the sperm

The discovery of CFTR expression in the mature sperm andore importantly of its requirement for correct sperm capacitation

ogether with the multiple evidence of interaction between SLC26embers and CFTR led to the investigation of a possible physical

nd functional interaction between CFTR and the sperm-specificember of the SLC26 family, SLC26A8.A physical interaction between SLC26A8 and CFTR was demon-


trated by co-immunoprecipitation experiments in heterologousells transiently transfected with plasmids encoding SLC26A8 andFTR proteins; this interaction was shown to be dependent on theTAS domain. Moreover, this interaction was verified in vivo as the

ubfamily U member 1. (B) Ion transports occurring at the apical plasma membranemembrane conductance regulator; SLC26A9: solute carrier 26 A9; ORCC: outwardassium channels.

chimeric Flag-Slc26A8 protein produced in transgenic mice testeswas able to co-precipitate Cftr (Rode et al., 2012).

Similarities in the intracellular distribution of SLC26A8 and CFTRin mature sperm are compatible with such an interaction betweenthe two proteins: SLC26A8 is present at both the annulus and theequatorial segment of the sperm head and as mentioned above,CFTR is detected at the equatorial segment of the head and at themidpiece of mouse and human sperm (Hernández-González et al.,2007; Xu et al., 2007). Interestingly, in some spermatozoa a bulk ofCFTR protein was also detected at the proximal border of the annu-lus (Rode et al., 2012), suggesting that CFTR might be subject tosubcellular relocation in response to specific environmental condi-tions and/or interactions. Altogether these data suggest that CFTRand SLC26A8 might cooperate in both the sperm head and flagellumto regulate ion fluxes upon capacitation, which are required for theacrosomal reaction and the flagellum hyperactivation, respectively.

To note, two other SLC26 members, SLC26A3 and SLC26A6, havebeen shown to co-localize with CFTR in the midpiece compartmentof the sperm flagellum and are likely to contribute to the complexregulation of anion exchanges occurring during sperm motility andcapacitation processes (Chávez et al., 2012); the exact contributionof each transporter is so far unknown.

2.5. Physical interaction of SLC26A8 with CFTR results in CFTRstimulation

Voltage clamp experiments conducted in Xenopus laevis oocytesinjected with CFTR and/or SLC26A8 RNAs showed that in the


01

oocytes expressing CFTR in combination with SLC26A8, PKA-stimulated currents were significantly larger than those observedin the oocytes expressing CFTR alone, demonstrating the stimu-lation of CFTR activity by SLC26A8 (Rode et al., 2012). Moreover,


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easurement of I− efflux mediated by CFTR in CHO-K1 cells co-xpressing CFTR and SLC26A8, confirmed the stimulation exertedy SLC26A8 on CFTR.

In order to elucidate the cellular mechanisms associated withFTR and SLC26A8 cooperation in the sperm, further studiesere performed in the Slc26a8 knock out mouse model. Slc26a8-ull sperm were unable to induce the intracellular rise of cAMPnd the PKA-dependent downstream phosphorylation cascades, inesponse to their incubation in a capacitating medium. Interest-ngly, supplementation of the capacitating medium with permeantnalogs of the cAMP partially rescued the sperm motility and theapacitation defects in a PKA-dependent manner (Rode et al., 2012).ltogether, these results strongly suggested that in the mouse,

he Slc26A8–CFTR complex is involved in the regulation of thel− and/or HCO3

− fluxes that are required to switch on the PKA-ependent downstream phosphorylation cascades during spermotility and capacitation (Fig. 2A).

.6. Identification of SLC26A8 mutations destabilizing theLC26A8–CFTR complex in human asthenozoospermia

The identification and the characterization of SLC26A8 as aale germ cell specific member of the SLC26 anion transporter

amily rationally suggested that mutations of the gene could bessociated with human male infertility. A primary study was per-ormed by screening of the SLC26A8 gene in a cohort of Finishatients presenting with a spermatogenic arrest (oligozoospermiar azoospermia) but no mutations were identified (Mäkelä et al.,005). Retrospectively, these negative results are in consistenceith the Slc26A8 mutant mice phenotype, which consists in spermotility and maturation defects but normal sperm production.Very recently, based on the phenotype of the Slc26a8 mutant

ice, the screening of SLC26A8 was performed in a cohort ofatients presenting with asthenozoospermia (Dirami et al., 2013).he analysis of SLC26A8 coding regions in a cohort of 146 meneing treated for infertility and presenting asthenozoospermiaaccording to the World Health Organization values) allowed thedentification of seven asthenozoospermic individuals carryingeterozygous sequence variations that were not found by directequencing of the gene in an ethnicity-matched control popula-ion of 121 individuals. The frequency of these variants in a largeropulation control of 8600 individuals available in the databases1001 genomes, dbSNP) and their functional consequences wereurther investigated for three of them. The sequence variations.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys) and c.2860C>Tp.Arg954Cys) were shown to be associated with asthenozoosper-

ia with a power >95%: their frequency in the control populationsf the databases was about 5/8600, 0/8600 and 0/8600, respec-ively; the power of this association being even stronger as the sexnd the fertility status of the individuals recorded in the databasesre unknown. A first assessment of the pathogenicity of the variantsas performed in silico and predicted the variation p.Arg87Gln to beossibly damaging and the variations p.Glu812Lys and p.Arg954Cyso be probably damaging (PolyPhen software: scores 0.916, 0.985nd 0.993, respectively).

Interestingly, all three variants were deleterious in terms of theirunctional cooperation with the CFTR channel. Recording of CFTR

ediated I− efflux in CHO-K1 cells co-expressing CFTR with eachf the SLC26A8 variants or the wild type SLC26A8 indicated that allhree variants were unable to stimulate CFTR activity in contrast tohe wild type protein (Dirami et al., 2013). The abrogation of CFTRtimulation was associated in vitro with reduced amount of the vari-


nts and surprisingly of CFTR protein amount as well. The reducedmounts of the SLC26A8 variants were shown to result from pro-easomal degradation as normal protein amounts could be restoredpon proteasome inhibition with MG132. In consistence with these

PRESSiochemistry & Cell Biology xxx (2014) xxx–xxx

findings, semen analysis of the patients carrying the sequence vari-ations p.Glu812Lys and p.Arg954Cys showed an abnormal diffusestaining of SLC26A8 in the flagellum and its absence from the equa-torial segment of the head where it would normally interact withCFTR channel (Rode et al., 2012; Dirami et al., 2013). Altogether,these results indicate that mutations in SLC26A8, by compromis-ing the formation and the stability of the SLC26A8–CFTR complex,abrogate normal stimulation of the CFTR channel and impact on ionfluxes regulation in the sperm.

Remarkably, all three mutations identified in SLC26A8 werefound at the heterozygous state, in contrast to the mutations pre-viously identified at the homozygous state in SLC26A2, A3, A4 andA5, which follow an autosomal recessive mode of inheritance.This difference could be related to the haploid state of the germcells and their permanent connection through the intercellularbridges allowing limited sharing between the cells. These specificproperties of the sperm cell are likely to reinforce any mild phe-notype resulting from monogenic heterozygous alterations andto contribute to the heterogeneity of the sperm phenotype in agiven individual. Consistent with this, all three individuals carryingheterozygous mutations in SLC26A8 presented with mild astheno-zoospermia and heterogeneous sperm parameters in terms of theirmotility and morphology.

Finally, as less CFTR protein accumulate in the presence of theSLC26A8 variants in comparison to the SLC26A8 wild type protein,CFTR stability is likely to be hindered as well. It is conceivable thatSLC26A8 and CFTR interaction occurs in the early steps of the bio-genesis of both proteins. One could therefore speculate that theformation of the SLC26A8–CFTR complex protects each transporterfrom degradation by preventing their recognition by componentsof the endoplasmic reticulum-associated degradation (ERAD) path-way, well known for its role in the degradation of the CFTR channel(Ward et al., 1995).

3. SLC26A9

3.1. SLC26A9 presentation

SLC26A9 was cloned along with SLC26A7 and SLC26A8 byhomology searches on genomic and expressed sequences databases(Lohi et al., 2002). SLC26A9 is predominantly expressed in the lungalveolar and tracheal epithelial cells (Fig. 2B) and in the stomach, inthe gastric surface epithelial and parietal cells where it is involvedin acid secretion (Lohi et al., 2002; Xu et al., 2005; Chang et al.,2009b); lower expression of SLC26A9 was also detected in the kid-ney (Amlal et al., 2013).

SLC26A9 anion transport activity has been highly documentedbut is still controversial as it was shown to exhibit distinct modesof transport in the different studies performed so far. SLC26A9 wasinitially described as an electrogenic nCl−/HCO3

− exchanger likeSLC26A3 and SLC26A6 (Xu et al., 2005) but further studies pointedout a Cl− channel activity with minimal permeability to OH− orHCO3

−, which led to classify SLC26A9 along with SLC26A7 as properchannels in the SLC26 family (Dorwart et al., 2007; Bertrand et al.,2009). More recent studies indicate that HCO3

− might indeed stim-ulate the Cl− channel activity of SLC26A9 (Loriol et al., 2008; Xuet al., 2008; Chang et al., 2009b; Chen et al., 2012). Lastly andmore intriguingly, SLC26A9 was also reported to exhibit a uniquefunction among the SLC26 members, as its anion transport activitywas shown to be coupled to cation transport (Na+/anion trans-


01

port) (Chang et al., 2009b). In a model proposed by Romero et al.for SLC26A9 function in oocytes, this protein would behave asboth a Cl− channel and a nCl−/HCO3

− exchanger (Romero et al.,2006).


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.2. SLC26A9 physiological functions in the stomach, lung andidney

Inactivation of Slc26a9 in the mouse did not affect growth orealth but led to the loss of the gastric acid secretion, the absencef tubulovesicles in the gastric parietal cells and a reduced num-er of parietal and zymogen cells were also reported (Xu et al.,008). As Slc26a9 protein could be detected in the tubulovesiclesf wild type mice, it was suggested to be required for the viabilityf these structures and the Cl− secretion of the parietal cells. Usinghe Slc26A9-null model, Demitrack et al. also demonstrated thathe pH increase physiologically occurring at the gastric surface inesponse to focal epithelial damage or to prostaglandin expositions predominantly mediated by cellular HCO3

− secretion throughhe SLC26A9 anion transporter (Demitrack et al., 2010).

In the lung, studies conducted on Slc26a9-null mice identified anssential role of Slc26a9-mediated Cl− secretion in response to air-ay inflammation induced by IL-13 treatment (Anagnostopoulou

t al., 2012). In consistence with this, polymorphisms located inhe 3′UTR of SLC26A9 resulting in reduced amount of SLC26A9 pro-ein were found to be associated with human asthma conditions,uggesting that SLC26A9 function might be required for preventingirway obstruction in allergic airway disease.

In the kidney, Slc26a9 was shown to localize at the apical mem-rane of the principal cells in the medullary collecting duct. Thebsence of Slc26a9 in the mouse led to a reduction of the renall− excretion under a salt-rich diet or water deprivation togetherith elevated systemic arterial pressure, suggesting that Slc26a9 is

equired for the regulation of both renal salt excretion and bloodressure (Amlal et al., 2013).

.3. SLC26A9 physical interaction with CFTR

The overlapping expression pattern of CFTR and SLC26A9 inhe gastric and the airway epithelial cells led to the investiga-ion of a potential physical and functional interaction betweenhe two proteins. Evidence for this physical interaction is docu-

ented in multiple studies. Co-immunoprecipitation experimentsonducted in HEK 293 (Human Embryonic Kidney 293) cells tran-iently expressing SLC26A9 and CFTR proteins demonstrated thexistence of the SLC26A9–CFTR complex (Bertrand et al., 2009).his complex was also confirmed by co-immunoprecipitation inhe cystic fibrosis bronchial epithelial cell line (CFBE4Io-) sta-ly expressing the wild type or p.Phe508del CFTR proteins andransduced with SLC26A9 (Avella et al., 2011). Demonstration ofhe direct interaction between the two proteins was obtained byo-immunoprecipitation experiments using the purified SLC26A9TAS domain and the purified CFTR R domain (Chang et al., 2009a).hereas most studies are in agreement with the existence of

physical interaction between SLC26A9 and CFTR proteins, theonsequences of this interaction, as described below, are still con-roversial.

.4. SLC26A9 interaction with CFTR stimulates CFTR activity

The physical interaction of SLC26A9 with CFTR was shown toodulate the CFTR channel activity. Bertrand et al. showed that

o-expression of CFTR with SLC26A9 in HEK cells enhances CFTRctivity in a STAS domain- and R domain-dependent manner butad no effect on the p.Phe508del CFTR mutant activity (Bertrandt al., 2009). Avella et al. also showed that the co-expression ofLC26A9 stimulates CFTR activity in both the CFBE41o-cell line


nd Xenopus laevis oocytes; interestingly they observed that CFTRrotein amounts were significantly increased in the presence ofLC26A9, suggesting that SLC26A9 might impact on the biogen-sis and/or the stabilization of CFTR (Avella et al., 2011); further

PRESSochemistry & Cell Biology xxx (2014) xxx–xxx 7

experiments with inhibitors of the proteasome should be per-formed in order to distinguish between enhanced proteinbiogenesis or reduced stability due to excessive degradation. Tonote, although SLC26A9 co-expression similarly increased theprotein amount of the p.Phe508del CFTR mutant, it could notstimulate the forskolin-activated Cl− secretion of CFBE4Io-cellsexpressing the p.Phe508del CFTR mutant protein, suggesting thatSLC26A9 overexpression cannot circumvent the lack of function ofp.Phe508del CFTR (Avella et al., 2011).

3.5. SLC26A9 interaction with CFTR modulates SLC26A9 function

Bertrand et al. reported a reciprocal regulatory interactionbetween SLC26A9 and CFTR by showing that functional CFTR isa prerequisite for the activation of SLC26A9 in HEK and HBE(human bronchial epithelial) cells; in those cells the expression ofp.Phe508del CFTR or the pharmacological inhibition of CFTR pre-vented the SLC26A9-dependent constitutive Cl− currents (Bertrandet al., 2009). This alteration does not result from decreased avail-ability of SLC26A9 in the cells as SLC26A9 transcripts are equallypresent in normal cells and cystic fibrosis cells (Bertrand et al.,2009; Braun et al., 2010). However it could result from the absenceof p.Phe508del CFTR at the apical membrane of the epithelialcells (Cheng et al., 1990). Interestingly, Avella et al. also reportedSLC26A9 protein amounts to be increased in response to the co-expression with the CFTR channel.

In opposition to the above observations, a study conductedwith purified protein domains of both SLC26A9 and CFTR indicatedthat SLC26A9 mediated nCl−/HCO3

− exchange and Cl− currents arealmost fully inhibited by the R domain of CFTR in a STAS domain-dependent manner; this possibly by interfering with the formationof SLC26A9 functional homodimers (Chang et al., 2009a). The use ofchimeric constructs in which part of the STAS domain of SLC26A9was replaced with that of SLC26A6, showed that it is specifically theSTAS domain of SLC26A9 that supports SLC26A9 inhibition by the Rdomain of CFTR, demonstrating that although all SLC26s are likelyto interact with the CFTR channel, the outcome of this interactionon their activity is SLC26-dependent (Chang et al., 2009a).

Finally, a recent study performed on different cell types reporteda positive cross-regulation of SLC26A9 and CFTR in polarized airwayepithelial cells whereas SLC26A9 was shown to be inhibited by CFTRin non-polarized HEK293 cells (Ousingsawat et al., 2012), suggest-ing that the cellular localization might contribute to the functionalversatility of the SLC26s and of the SLC26s–CFTR complexes.

3.6. Identification of SLC26A9 mutations destabilizing theSLC26A9–CFTR complex in diffuse idiopathic bronchiectasis

Taking into account the high expression of SLC26A9 in the lungand the implication of Slc26a9 in airway inflammation, in themouse, it was hypothesized that SLC26A9-mediated Cl− secretionwas required to prevent airway obstruction in allergic conditionand that mutations in SLC26A9 might be in cause in human mucocil-iary disorders. A primary study performed by Anagnostopoulouet al. (2012), using chip genotyping in a cohort of 661 childrenpresenting with asthma and 658 healthy individuals led to theidentification of several polymorphisms in the 3′ UTR of SLC26A9(rs12031234, rs2282429, rs2282430) that were significantly asso-ciated with asthma in this population. Among them rs2282430was shown to result in reduced protein expression in vitro indicat-ing that SLC26A9 mutations could constitute modifiers in humanmucociliary diseases (Anagnostopoulou et al., 2012).


01

More recently Bakouh et al. have studied a cohort of 147 patientspresenting with diffuse idiopathic bronchiectasis, which corre-sponds to a common lung disease primarily induced in CysticFibrosis (CF) patients. The entire coding regions and the flanking




Fig. 3. Major sites of the physical and functional interaction of SLC26 proteins with the CFTR channel. Taken together, the tissue expression patterns of SLC26 family memberscover most of the epithelial cells of the body where they are co-expressed with the CFTR channel. Physical and functional interaction with CFTR has been described for severalS whend

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LC26 proteins namely SLC26A3, A4, A5, A6, A8 and A9. The asterisk (*) indicates

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ntron sequences of SLC26A9 were analyzed by DHPLC and directequencing and resulted in the identification of the followingwo sequence variations: (c.1723C>T) p.Arg.575Trp and (c.1456G>A).Val 486Ile, which were found at the heterozygote state in twoistinct patients. Interestingly, the patient carrying the sequenceariation (c.1723C>T) p.Arg.575Trp, also carried the p.Phe508delFTR mutation at the heterozygous state. Both SLC26A9 sequenceariations were not found by direct sequencing in an ethnicity-atched control population including 50 unaffected individuals

nd 78 CF patients carrying two CF-causing mutations. Furthermoren silico analysis of their pathogenicity predicted the variations.Arg.575Trp and p.Val 486Ile to be possibly and probably dam-ging, respectively (PolyPhen software: scores 0.99 and 0.802,espectively).

When variants p.Arg.575Trp and p.Val 486Ile were expressedn Xenopus laevis oocytes to assess their impact on SLC26A9 func-ion, SLC26A9-mediated Cl− currents were fully abolished withoutffecting protein membrane expression. In addition, co-expressionf the variant p.Arg.575Trp with CFTR (but not of variant p.Val86Ile) inhibited the SLC26A9-dependent activation of CFTR uponKA stimulation. This inhibition is likely due to the impairmentf a functional SLC26A9–CFTR interaction as the residue Arg.575ocates in the STAS domain and the use of a 20 amino-acids pep-ide overlapping the Arg.575 residue could trigger the stimulationf CFTR as observed with the full length and wild type SLC26A9rotein (Bakouh et al., 2013).

Here again the mutations were found at the heterozygous staten both patients suggesting the existence of a residual activity ofhe SLC26A9–CFTR complex and/or a non-recessive mode of in her-tance in contrast to the mutations identified in SLC26 A2, A3, A4nd A5.

Altogether these data indicate that SLC26A9 has a physiolog-cal relevance in the regulation of bronchic transepithelial anionuxes and partly through its functional cooperation with the CFTR


hannel (Fig. 2B). SLC26A9 could therefore be in cause in humanucociliary diseases but also constitute a modifier gene in the

ontext of the cystic fibrosis. In consistence with this, a com-rehensive genome-wide association study previously identified

mutations in SLC26 have been identified in human and associated with a genetic

polymorphisms in SLC26A9 that were associated with the presenceof meconium ileus in CF patients (Sun et al., 2012).

4. Conclusion

Since the recent discovery of the SLC26 family, tremendous workhas been performed in order to better define their specific func-tions and their mode of transport in the various epithelia wherethey are most often specifically or quasi-specifically expressed. Inparticular the development of mouse models for all SLC26 genes(except SLC26A11) constitutes a powerful tool for the understand-ing of their physiological functions and the anticipation of thepotential pathophysiological consequences of their mutations inhuman. Future work should allow a better characterization of theirstructural properties but also the identification of their interactingproteins in order to progress on the molecular pathways regulatingtheir activity, in a most likely tissue- and cell-dependent manner.

One of the most interesting features of the SLC26 proteins high-lighted during the last decade is their physical and functionalinteraction with the CFTR channel. Several SLC26 members areco-expressed with CFTR in various epithelia throughout the entirebody and most interestingly in the tissues affected in CF patients(Fig. 3). The increasing number of studies reporting the regulationof CFTR by SLC26 proteins in vitro suggests a physiological rele-vance for this crosstalk. As described in this review, recent studiesin the field demonstrated that SLC26s can induce CFTR-related dis-ease when mutated but also behave as modifier genes in the contextof the cystic fibrosis. The latter is of considerable importance as thediscovery of novel ways to modulate SLC26 activity and/or expres-sion could circumvent the defects of CFTR and therefore representnovel therapeutic strategies for the cystic fibrosis.

Acknowledgements


01

We are grateful to Baptiste Rode (Faculty of Biological Sciences,Leeds, UK) for critical reading of the manuscript. We also wouldlike to thank the scientific community working on CFTR for inter-action and collaboration. Our work is supported by the Institut


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ational de la Santé et de la Recherche Médicale, Centre Nationale la Recherche Scientifique, Université Paris Descartes and Agenceationale de la Recherche (ANR-07-JCJC-0099; ANR-12-BSV1-011-01 MUCOFERTIL) and Association Vaincre la MucoviscidoseRF20110600465).

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