Pediatric Pulmonology 39:289–291 (2005)

Introduction

Beyond Chloride Transport: CFTR in the21st Century—Introductory Remarks

to a New State of the Art Series

Anil Mehta, MB, BS, MSc, FRCP (Edin), FRCPCH1* and Andrew Bush, MB, BS (Hons), MA, MD, FRCP, FRCPCH

2

Summary. This new series of articles on cystic fibrosis provides an overview of the confusing

plethora of problems that arise from the loss of function in a low abundance protein, the cystic

fibrosis membrane conductance regulator CFTR. The references are designed to take the clinical

reader into areas and journals that they might not normally read. In particular we have concentrated

on recent advances that suggest CFTR has functions that do not relate to chloride transport alone.

In forthcoming issues of the journal the topics covered range from prospects and difficulties in the

translation of new therapies into clinical practice, the regulation of the defective gene (promoters,

enhancers, silencers, etc.), regulation and interaction of the CFTR protein product with other

proteins in the cell, to functional approaches using developmental and secretory paradigms. These

themes have been chosen to bring controversies at the cutting edge of cystic fibrosis research to

the practicing pulmonologist in order to stimulate lateral thinking, which we hope will ultimately

benefit our patients. Pediatr Pulmonol. 2005; 39:289–291. � 2004 Wiley-Liss, Inc.

Life used to be so simple.We thought that cystic fibrosis(CF) was caused by mutations in the CF transmembraneconductance regulator protein (CFTR),1 which encoded achloride channel. It followed that mutation of the genewould disrupt the movement of this anion; however,the first discordant note sounded was that the steps betw-een chloride channel dysfunction and CF disease wereunclear, with many conflicting hypotheses. Unlike thechloride ‘‘channelopathy’’ causing idiopathic generalizedepilepsy,2 where it is much easier to explain how disruptedchloride movement could cause neuronal misfiring whenmembrane voltage changes abruptly following an actionpotential, the apparently simple CF chloride channel storyhas now suffered a series of severe setbacks. Even theelementary mantra that genomic DNAmakes RNAmakesprotein is no longer so simple at the RNA level,3 with extracomplexity at the CFTR protein level introduced by thenotion that systems of proteins may operate together toproduce the final ‘‘microanatomical’’ phenotype.4,5 Fur-thermore, we realize that there are newly recognizedsignaling proteins (colorfully known as traffic COPS)involved in the complex intracellular trafficking that isneeded to produce final, mature, apical CFTR.6,7 Themature CFTR is a complexly glycosylated protein that hasfar more functions than a mere chloride channel,8 andrecent evidence suggests that it may even be a functionaldimer,9 thus adding to our inability to grasp how four

potential nucleotide binding folds might interact. Suchdata also beg the question as towhat exactly constitutes theCFTR(s) pore. So are all CFTRs the same? All we can sayis that CFTR(s) can interact with many adjacent proteinsin the cytoskeleton, conducting a complex molecularorchestra—but what is the tune?Although the abnormalities in chloride transport re-

sulted in the development of the most widely useddiagnostic test for CF, albeit in a very unusual double-layered gland duct that forms a syncitium with CFTRexpressed in both apical and basolateral cell membranes, aconsiderable body of evidence suggests that much of CF

1UK Cystic Fibrosis Database, Ninewells Hospital Medical School,

Dundee, Scotland, UK.

2Department of Paediatric Respirology, Imperial School of Medicine at

National Heart and Lung Institute, Royal Brompton Hospital, London UK.

*Correspondence to: Anil Mehta, M.B., B.S., M.Sc., F.R.C.P. (Edin),

F.R.C.P.C.H., Division of Maternal and Child Health Sciences, Ninewells

Hospital Medical School, Dundee DD1 9SY, Scotland, UK.

E-mail: a.mehta@dundee.ac.uk

Received 28 September 2004; Accepted 28 September 2004.

DOI 10.1002/ppul.20146

Published online 30 November 2004 in Wiley InterScience

(www.interscience.wiley.com).

� 2004 Wiley-Liss, Inc.

disease cannot be readily explained by chloride transportalone.10 Some now suggest that CF disease segregatesbetterwith bicarbonate transport.11A recent paper showedthat non-CF mice with overexpressed mouse ENaC havea lung disease that is very similar to human CF lungpathology, suggesting that lung disease may have nothingto do with chloride, which would be supported by theobservation that lung disease does not correlate with resi-dual chloride channel function.11 The conventional viewis that in the CF airway epithelium, both apical Cl� sec-retion and Naþ absorption are disordered.12–14 It remainsproblematic how defective chloride transport results notonly from abnormal CFTR function, but also frommalfunctioning Cl� channels distinct from CFTR. Theseabnormalities may also differ in different tissues. Forexample, in the lung, sodiumabsorption isupregulated, butit is presently controversial whether this occursvia protein-protein interactions between CFTR, apicalsodium channels, and cytoskeletal proteins.12–14 Somedisagree with that notion.15 Further, the cellular ab-normalities in CF cannot be wholly explained by channeldysfunction alone, because when the most commondisease-inducing mutant CFTR (DF508) is reconstitutedinto lipid bilayers, its channel function is no different fromwild-type.16 Once again, in the cell a different result isobtained,17 thus alluding to the above cell-dependentregulation of the channel itself. A complete understandingof the disease must also explain the complex relation-ship between infection and inflammation,18–20 or in cellmodels, how defective CFTR causes abnormalities inendocytosis21 and excessive turnover of phosphatidylcho-line.22 The diversity of defects suggests that either CFTRcontrols each of the above processes directly by a CFTRinteraction, or that the ‘‘R’’ in CFTR indirectly regulates aplethora of functions by unrecognized cellular pathways.A further complexity is that an increasing number of othergenesmodify themanifestations of CF disease. Rare casesof phenotypic CF have been described in patients with acompletely normal CF gene!23 The caveat here is that onlythe coding exons are reported, and studies are beingplanned to examine the large intronic elements and regu-lators of expression to complete the picture. It remainspossible that such cases are due to mutations in genesencoding proteins responsible for intracellular processingof and/or final function of mature CFTR.These issues have real clinical consequences because

the hardest task ahead, as survival improves to the fourthdecade of life, is to find optimum therapies. Finding thecorrect explanations for each patient will also be vital forthe nascent screened CF populations, as newborn screen-ing becomes commonplace. This requires translation ofcurrent research into the elucidation of the mechanismsthat must explain how likely it is that a particular mutatedCFTR will cause the premature death of that CF patient.This in turn will inform the choice of biologically relevant

endpoints for novel treatments such as gene or long-termpharmacological therapy by weighing risks and benefits.Indeed, if CF lung disease has nothing whatever to dowith chloride channel dysfunction, replacing the chloridechannel by gene therapy, which never makes any dif-ference to control of ENaC, may be a fruitless strategy! Itis therefore timely to review our present knowledge ofCFTR in this second series of ‘‘State of the Art’’ articles inthis Journal. The series will provide an overview ofattempts to understand the basic physiology of CFTR andits molecular interactions. It is offered in particular as anupdate for the clinical pediatric pulmonologist. We havecarefully chosen a blend of youth and experience in theauthors, with the aim of stimulating lateral thinking andthe creation of hypotheses that could link the defectiveCFTR protein to clinical disease. If you disagree, we hopeyou will write to us. To paraphrase George Bernard Shaw,progress is dependent on those who adopt unreasonableand not reasonable positions.

REFERENCES

1. Kerem BS, Rommens JM, Buchanan JA, Markiewicz D, Cox

TK, Chakravarti A, Buchwald M, Tsui LC. Identification of the

cystic fibrosis gene: genetic analysis. Science 1989;245:1073–

1080.

2. Haug K, Warnstedt M, Alekov AK, Sander T, Ramirez A, Poser

B, Maljevic S, Hebeisen S, Kubisch C, Rebstock J, Horvath S,

Hallmann K, Dullinger JS, Rau B, Haverkamp F, Beyenburg S,

Schulz H, Janz D, Giese B, Muller-Newen G, Propping P, Elger

CE, Fahlke C, Lerche H, Neils A. Mutations in CLCN2 encoding

a voltage-gated chloride channel are associated with idiopathic

generalized epilepsies. Nat Genet 2003;33:527–532.

3. Denli AM, Hannon GJ. RNAi: an ever-growing puzzle. Trends

Biochem Sci 2003;28:196–201.

4. Pollard HB. Anatomic genomics: systems of genes supporting the

biology of systems. Anat Rec 2000;259:1003–1009.

5. Papin JA, Price ND, Wiback SJ, Fell DA, Palsson BO. Metabolic

pathways in the post-genome era. Trends Biochem Sci 2003;28:

250–258.

6. Kim PS, Arvan P. Endocrinopathies in the family of endoplasmic

reticulum (ER) storage diseases: disorders of protein trafficking

and the role of ER molecular chaperones. Endocr Rev 1998;19:

173–202.

7. Arven P, Halban PA. Sorting ourselves out: seeking consensus on

trafficking in the beta-cell. Traffic 2004;5:53–61.

8. Widdicombe JH. Yet another function for the cystic fibrosis

transmembrane conductance regular. Am J Respir Cell Mol Biol

2000;22:11–14.

9. Li C, Roy K, Dandridge K, Naren AP. Molecular assembly of the

cystic fibrosis transmembrane conductance regulator in plasma

membrane. J Biol Chem 2004;279:24673–24684.

10. Freedman SD, Blanco PG, Zaman MM, Shea JC, Ollero M,

Hopper IK, Weed DA, Gelrud A, Regan MM, Laposata M,

Alvarez JG, O’Sullivan BP. Association of cystic fibrosis with

abnormalities in fatty acid metabolism. N Engl J Med 2004;350:

560–569.

11. Gray MA. Bicarbonate secretion: it takes two to tango. Nat Cell

Biol 2004;6:292–294.

12. Donaldson SH, Poligone EG, Stutts MJ. CFTR regulation of

ENaC. Methods Mol Med 2002;70:343–364.

290 Mehta and Bush

13. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier

BC, Boucher RC. CFTR as a cAMP-dependent regulator of

sodium channels. Science 1995;269:857–850.

14. Mall M, Grubb BR, Harkema JR, O’Neill WK, Boucher RC.

Increased airway epithelial sodium absorption Naþ produces

cystic fibrosis-like disease in mice. Nat Med 2004;10:487–

493.

15. Kunzelman K. ENaC is inhibited by an increase in the

intracellular Cl� concentration mediated through activation of

Cl� channels. Pflugers Arch 2003;445:504–512.

16. Li C, Ramjeesingh M, Reyes E, Jensen T, Chang X, Rommens

JM, Bear CE. The cystic fibrosis mutation (DF508) does not

influence the chloride channel activity of CFTR. Nat Genet 1993;

3:311–316.

17. Taddei A, Folli C, Zegarra-Moran O, Fanen P, Verkman AS,

Galietta LJV. Altered channel gating mechanism for CFTR

inhibition by a high-affinity thiazolidinone blocker. FEBS Lett

2004;558:52–56.

18. Becker MN, Sauer MS, Mulebach MS, Hirsch AJ, Wu Q,

Verghese MW, Randell SH. Cytokine secretion by cystic fibrosis

airway cells. Am J Respir Crit Care Med 2004;169:645–653.

19. Triouvanziam R, Khazaal I, Peault B. Primary inflammation in

human cystic fibrosis small airways. Am J Physiol Lung Cell Mol

Physiol 2002;283:445–451.

20. Khan TZ, Wagener JS, Bost T, Martinez J, Accurso F,

Riches DWH. Early pulmonary inflammation in infants with

CF. Am J Respir Crit Care Med 1995;151:1075–1082.

21. Schwiebert EM, Gesek F, Ercolani L, Wjasow C, Gruenert D,

Karlson K, Stanton B. Heterotrimeric G proteins, vesicle traffick-

ing and CFTR Cl� channels. Am J Physiol 1994;267:272–281.

22. Ulane MM, Buttler JD, Peri A, Miele L, Ulane RE, Hubbard V.

Cystic fibrosis and phosphatidylcholine biosynthesis. Clin Chim

Acta 1994;230:109–116.

23. Groman JD, Meyer ME, Wilmott RW, Zeitlin PL, Cutting GR.

Variant cystic fibrosis phenotypes in the absence of CFTR muta-

tions. N Engl J Med 2002;347:401–407.

Beyond Chloride Transport: 21st Century CFTR 291