cftr: more than just a chloride channel

Pediatric Pulmonology 39:292–298 (2005)

CFTR State of the Art Series

CFTR: More Than Just a Chloride Channel

Anil Mehta, MBBS, MSc, FRCP (Edin), FRCPCH*

Summary. This review examines the cystic fibrosis transmembrane conductance regulator

(CFTR) protein. After summarizing the ion channels regulated byCFTR, the review focuses on the

functions of CFTR that do not relate directly to a disease mechanism based on a channelopathy.

The key concept is that newly synthesized CFTR has to enter lipid vesicles which bud from the

endoplasmic reticulum. This is abnormally low in DF508 CFTR. Normal wild type vesicular CFTR

enters a recycling pool of lipid vesicles which transiently dock with the apical membrane only for

CFTR to be retrieved shortly after into a sub-apical recycling compartment. This retrieval is

abnormally fast in DF508 CFTR. The review discusses the relationship between this process and

thedifficult topic of fatmetabolismand thenexplores thepossible linksbetweenabnormal fattyacid

turnover and inflammatory cascades that are abnormal in cystic fibrosis. Finally the review

concentrates on the emerging functions of a protein kinase (AMP-activated kinase)which is bound

near the C terminus of the CFTR protein whose functions could intergrate some of the

abnormalities in lipid metabolism that result from mislocalization of CFTR in clinical disease.

Pediatr Pulmonol. 2005; 39:292–298. � 2004 Wiley-Liss, Inc.

Key words: endocytosis; lipids; clathrin; AMPK; lipoxin; arachidonic acid; CF mouse.

INTRODUCTION

Although the autosomal-recessive disease cystic fibro-sis (CF) is characterized by abnormalities in a small-conductance ion channel (the cystic fibrosis transmem-brane conductance regulator; CFTR1), the pathway fromdefective protein to clinical disease remains unclear.2,3 CFhas been thought of as a disease of chloride transport,because serendipitously, the measurement of chlorideconcentration in sweat is a useful diagnostic test for thecondition. However, linking chloride dysregulation tothe clinical disease has not been easy. Numerous otherfunctions of CFTR have been described. The purpose ofthis review is to stimulate thought as to whether some ofthe other functions of CFTR may be as important incausing the clinical disease. That the CFTR protein can bea chloride channel is beyond, doubt but this function alonecannot explain the entire pathology of the disease.4 Table 1illustrates some of the proposed defects consequent uponmutation of CFTR, as based on a recent review.5 Thesedefects cannot be easily related to one another, and thissuggests that the CFTR regulates multiple pathways.

CFTR: ION TRANSPORT-RELATED FUNCTIONS

CFTR-dependent movement of chloride can be repre-sented as a two-step process.6 Firstly, the open CFTR porerequires prior phosphorylation of the CFTR protein by

protein kinase A, and also protein kinase C. This phos-phorylation is countered bymultiple CFTR-bound proteinphosphatases, each regulated differently.7 Secondly,CFTR-induced hydrolysis of ATP is a required step, andyet there is no direct relationship between chloridetransport and ATP cleavage of the terminal phosphate.ATP cleavage and channel-gating involve a complexhead-to-tail (‘‘69’’) interaction (Fig. 1) between the twonucleotide-binding domains (NBDs).8 This opens thechannel pore, which cannot be accomplished by eitherNBD alone. In this scheme, NBD2 acts as the ATP-hydrolytic moiety,9 because it alone has the necessarycatalytic amino acids in the required spatial locations

Division of Maternal and Child Health Sciences, Ninewells Hospital

Medical School, Dundee, Scotland, UK.

Grant sponsor: Wellcome Trust; Grant sponsor: CF Trust; Grant sponsor:

Anonymous Trust.

*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: [email protected]

Received 20 July 2004; Accepted 5 October 2004.

DOI 10.1002/ppul.20147

Published online 30 November 2004 in Wiley InterScience

(www.interscience.wiley.com).

� 2004 Wiley-Liss, Inc.

(arrows in Fig. 1). This sequence of ATP hydrolysis andphosphorylation does not happen in reverse, since PKA-dependent phosphorylation must precede ATP-dependentchannel opening. The reason for this complexity duringcell chloride secretion is unclear, since the continuousoutward flux of potassium ions from the cell interior

creates a relatively negative cell interior that should spon-taneously drive negatively charged chloride out of the cell.It must be remembered, however, that although theintracellular chloride concentration is around 40 mM(compared with intracellular Kþ at 120 mM), periciliaryfluid chloride concentration is higher. In the absence ofactive transport, the net direction ofmovement of chlorideis dependent on the opposing gradients of electricallydriven exit and diffusion-driven entry. The latter is logorders less potent than the former. In the unstimulated cell,some consider the two opposing forces to be balancedagainst one another across the apical membrane.10 Otheranswers to this paradox may be gleaned from the ATP-binding cassette (ABC) family of membrane proteins towhich CFTR belongs, e.g., proteins that commonly useATP to transport organic molecules.11 The nature of theputative CFTR substrate(s) remains controversial, withdata showing that the antioxidant glutathione found inmMconcentrations inside cells is an alternative and plausiblephysiological substrate because of its antioxidant effects.However, the list is long and controversial, and might alsoinclude ATP and bicarbonate.11–13 This complexity isdifficult to reconcile in a single model. When Burnstockfirst suggested that ATP could act as a ligand at purinergicreceptors,14 the notion was greeted with disbelief. Todaynone can dispute the idea that ATP has a dual role inproviding energy for metabolism inside cells and in gatingextracellular purinergic receptors. This theme invokesCFTR as a regulator of transmembrane pH gradients(HCO3

�), intracellular energy (ATP), extracellular pur-inergic receptor function (UTP,ATP, and other breakdownproducts), and antioxidant transport (GSH). These ideasare still continuing to develop,15 and their clinicalconsequences, which remain controversial, are reviewedelsewhere.16 However, no molecular mechanism exists toexplain the pleotropic effects of CFTR with respect to theproteins listed in Table 1.

CFTR: FUNCTIONS UNRELATEDTO ION TRANSPORT

CFTR functions unrelated directly to ion transport arenow reviewed by first describing the dynamic nature ofCFTR, and then considering how such dynamism occursthrough molecular sorting proteins that transiently colo-calize with CFTR as it moves in intracellular vesicles.Since lipid vesicles are key players in that process, lipidsand inflammation will then be applied as another CFTRparadigm before considering the roles of a protein kinasebound toCFTR that regulates lipidmetabolism.17 The aimis to demonstrate that CFTR provides a platform to permitregulation of cellular metabolism extending far beyondion channel function.The birth and death of CFTR is a complicated process.

If it is assumed for the purposes of argument thatwild-type

TABLE 1—Regulatory Roles of CFTR1

ENaC (sodium channel) KVLQT1(K channel)

Electroneutral Naþ absorption AQP3,7 (water clearance)

HCO3�/Cl� exchanger (pancreas) Gap-junction channels

ICOR Cl� channels Mucus secretion

Ca2þ- and swelling-activated Cl� channels ATP-transport

ROMK2, Kir6.1 (K channel) Glutathione transport

KCNN4 (SK4, ik) (K channel)

1Epithelial sodium channel (ENaC) gates sodium entry from surface

epithelial fluid space; this is electrogenic due to apical entry of positively

charged sodium, but this process can also occur across basolateral

membrane by electroneutral cotransport of ions such as chloride. In

pancreas, a related process of apical electroneutral exchange in opposite

directions occurs for negatively charged chloride and bicarbonate. CFTR

also regulates other non-CFTR apical channels such as ICOR (a channel

that shows Outward Rectification), which become regulated when

subdomains such as NBD1 of CFTR are present. Other apical channels

include calcium-activated chloride channels regulated by UTP. At

basolateral membrane, potassium channels such as ROMK2/Kir 6.1 also

form part of CFTR-dependent regulatory family, similarly for the KCNN

family of Kþ channels. A different class of K channel, KvLQT1, which

causes long QT disease in heart, is part of this regulatory spectrum, as are

water channels in aquaporin family (AQP3A).GAP junctions aremadeof

proteins whose function is to tunnel communications between epithelial

cells. These junctions lie underneath tight junctions and are also regulated

by CFTR. This illustrates only some functions deranged by mutating

CFTR; see Kunzelmann.5

Fig. 1. The two nucleotide binding folds act cooperatively to

regulate the binding (NBD1) and hydrolysis (NBD2) of ATP in

CFTR. Note the head to tail arrangement and only the NBDs in

CFTRareshownfor clarity.Aminoacidsequenceanalysisshows

that the amino acids adjacent to the ATP molecules in NBD1 are

unable to catalyse the cleavage of ATP. In contrast the required

amino acids are present inNBD2at the sites bordering thepart of

the protein indicated by the two arrows.

CFTR: More Than Just a Chloride Channel 293

CFTR is synthesized, is correctly folded, and ends up inthe post-Golgi compartment in the vicinity of the apicalmembrane, then this CFTR compartment is relativelystable (typically on the order of 16–24 hr, depending oncell type).18,19 What then is the fate of this mature CFTR?Cellular models suggest that every few minutes, CFTRleaving the apical membrane exits into the cytosol inintracellular lipid-coated vesicles which continuously budfrom the inner lipid leaflet of the apical membrane to formearly endosomes (literally, inside-bodies). Most endocy-tosed vesicular CFTR evades fusion with lysosomes andthus destruction, and rapidly recycles back to the cellsurface, using late endosomes in a manner similar to thetransferrin and LDL receptors. These different types ofvesicle are distinguishable by their different proteincontents, including Rab family members which are smallproteins that hydrolyze GTP.18 This is clinically pertinentbecause the difference between the rate of appearance ofapical CFTR (new synthesis/recycling late endosomes)and its rate of disappearance (destruction, and subapicalreserve pool of endosomes) determines how much CFTRis in the apical membrane at any one time. This number isdifferent for unstimulated wild-type CFTR, stimulatedwild-type CFTR, and the mutants DF508/G551D CFTR(see below). Thus it is conceivable that different treatmentstrategies might be needed to increase apical mutantCFTR concentration. There is also a relationship betweenthe rapid rate of CFTR endocytosis and motifs on the C-terminus of CFTR that bind clathrin-coated endocytoticvesicles. These form from the invaginated lipid pocketsand are excised by a different GTPase called dynamin thatinduces acute membrane curvature to promote vesiclefission, thus forming nascent vesicles.18 These may belaunched, like cannonballs, into the cytosol by amolecular‘‘kick’’ from the microtubules in the cytoskeleton. Thisesoteric cell biology may be clinically relevant to CFpathophysiology, firstly because these pathways are oftenutilized by pathogens such as cholera toxin to gain entryinto epithelia, and secondly because CF-relevant pro-cesses such as osteoporosis, bone destruction, and increa-sed turnover occur when acid-laden vesicles fuse withbone to remodel it. The dissolved bone detritus is carriedaway as cargo by late endosomes.20

Thermodynamically, it is no easy task to move a trans-membrane protein from one apical membrane environ-ment via a water-filled space to another lipid environmentin a vesicle. Such apical CFTR turnover is dependent onprotein bridges, i.e., links between adapter proteins suchas AP2 which bind not only CFTR at specific motifs in itsC terminus but also to inositol phosphate lipids anchoredto AP2 in restricted sites within the membrane (not to beconfused with AP-1 proteins acting in gene transcription).Much research is directed at finding out how CFTRinitially sorts itself into spatially fixed, endosome-generating specialized apical microdomains rather than

localizing in the general apical membrane. It is excitingthat CFTR regulation may occur directly through inositollipids, which form a different class of lipids compared tothose found in the general apical membrane21 (see below).This idea of a dynamic cycling CFTR does not sit com-fortably with the rather static concept of a CFTR bound tothe cytoskeleton pumping substrates out to the lumen orvice versa. Thus if CFTR moves substrates across theapical membrane, it does so only during its (regulated)apical transit. Therapeutically, increasing the amount ofCFTR in the apical membrane could be induced bystopping clathrin-/dynamin-mediated endocytosis to re-duce apical losses, by enhancing late endosome fusionwith the apical membrane to increase CFTR arrival or by acombined strategy to cause both to happen simultane-ously. For some epithelia, cAMP-like molecules wereproposed as such agents.22 However, pharmacologicalattempts to increaseDF508 CFTR at the apical membranemay be futile, because the apical residence time of thiscommon mutant CFTR is already much shorter than forwild-type.18 Typically in cell culture, DF508 CFTR exitsfour times faster than a wild-type in the intracellularvesicles that each carry between 1–10 (wild-type) CFTRmolecules.22 The content of DF508 CFTR molecules pervesicle is unknown. Other hypotheses about the genesis ofCFTR raise more fundamental problems. Quantitativeanalysis suggests that up to 75% of wild-type CFTR isdegraded during synthesis.22 The reason for such seemingreckless profligacy is unclear. These static-dynamic con-siderations have profound implications for therapy. Forexample, one of the proteins that binds CFTR only at itsN-terminus belongs to the aptly named t-SNARE familyof proteins23 that trap CFTR and turn off its ion channelfunction. It is possible that this binding could be a preludeto the intracellular transport of switched-off CFTR. Thisparticular t-SNARE is called syntaxin 1A. Its cousin,syntaxin 8, binds to the R domain, providing a relatedregulatory role in different endosome classes.23 Thiscomplexity of CFTR-linked protein binding extendsfurther toward the C terminus of CFTR, where a differenttype of interaction occurs through an adapter complexusing an AP2 protein whose role is to facilitate endo-cytosis of CFTR using a different mechanism, but cruci-ally, one that also involves specialized inositol lipids. Suchinteractions between lipids and ion channels also occur forthe ENaC sodium channel, whose function is upregulat-ed in CF airways.24,25 Recently, regulation of CFTR andENaC by specialized inositol phosphate-containing celllipidswas proposed. Thismay also provide ameans for thespatial colocalization of ENaC and CFTR. It is importantto remember that phosphoinositol lipids make up less than0.01% of general membrane phospholipids, and yet theyhave potent effects on cell signaling. The links betweenlipids and inflammation are now discussed, in order to tryto understand the behavior of such lipids in cystic fibrosis.

294 Mehta

SPECIAL LIPIDS AND MEMBRANE FUNCTION:TOO MUCH INFLAMMATION?

‘‘An exaggerated, sustained and extended inflammatoryresponse to bacterial and viral pathogens—characterisedby neutrophil dominated airway inflammation—is anaccepted feature of lung disease in cystic fibrosis.’’2 Evid-ence in support of this hypothesis comes from transgenicCF mouse models,26 and recently, a human xenograftmodel system was developed. In experiments engraftinghuman fetal CF lung explants under the skins of immuno-compromised ‘‘nude’’ mice (so called because they arehairless), murine neutrophils migrate into CF explants innumbers that significantly exceed those found in non-CFcontrols.27 The key point is that this CF-specific, excessneutrophil-dominated state of lungmigration is induced inthe absence of infection.2 Although the evidence for anexcessive inflammatory response to infective stimuli isindubitable, whether the CF epithelium is proinflam-matory in the absence of infection is still disputed. Onehypothesis to account for the characteristic excessiveinflammation invokes essential fatty-acid (EFA) defi-ciency in CF patients in the absence of protein-energymalnutrition.2,28,29 The complexities of EFA metabolismwill briefly be summarized. Fatty acids containing doublebonds can be defined using a D-number combinationwhich refers to the carbon atom counting from (andincluding) the terminal carboxylicmoiety of the fatty acid.This notation is applied when defining enzymes targetingspecific carbon-carbon links in fatty acids. Two suchenzyme classes provide this role atD5 andD6 sites duringAA and DHA synthesis, as described below. Alternativelywhen describing fatty acids themselves, two differentletter/number combinations are used by counting carbonatoms from the opposite (o) end of the fatty acid. Briefly,the essential dietary fatty acid 18:2o6 (also known aslinoleic acid, has 18 carbons, and two double bonds, thefirst of which lies at the sixth carbon from the noncarboxyl(o) end of the molecule). Linoleic acid is a precursor of abent, C-shaped, cell-synthesized 20-carbon arachidonicacid (20:4o6, AA) that is commonly inserted into themiddle glycerol carbon during membrane glycerol-phos-pholipid biosynthesis. Release of this semicircular fattyacid following cell stimulation and activation of cytosolicphospholipase A2 is commonly assumed to upregulatethe inflammatory state. However, this model notion maybe altered by the presence of anti-inflammatory drugs(see below). In contrast, dietary 18:3o3 (common name,linolenic acid) eventually generates a 22-carbon docosa-hexaenoic acid fatty acid (22:6o3, DHA) by a morecomplex route of carbon chain elongation and desatura-tion involving bidirectional mitochondrial membranetransport (and b-oxidation while therein). The keyenzyme mediating the synthesis of a metabolite that gatesthe mitochondrial entry step is regulated by a protein

kinase bound to CFTR, and will be discussed later. Onecurrent model suggests that abnormal EFA metabolismcould contribute to the excess inflammation observedwhenCFTR ismutated. It should be noted that CF patientsand CF mice do not always have precisely the same fatty-acid abnormality that is present in CF cell lines used tomodel the disease.30–32 Nevertheless, the combined dataaccumulated from in vivo, in vitro, and ex vivo32 studiespoint to one or more defects in fatty-acid metabolisminduced by mutant CFTR.2,29 Unfortunately, these path-ways are not easy to study due to complex feedback loopsbetween precursors and products. Further, reports in theCF literature do not measure fluxes across the enzymaticactivities needed to generate these complex lipids. Neitherare specific inhibitors available for the enzymes inquestion, which themselves remain poorly characterized.These difficulties should be remembered when con-sidering that the human CF phenotype has an increase inAA content in cellular phospholipids,32 noting that someconsider that the decrease in the DHA lipid class is not aconsistent feature.28,29 Freedman et al.26,32 claimed thatoral administration of DHA both corrects the AA/DHAratio and reverses the pathological manifestations of CF inmice. It also remains possible that the altered membranelipid composition is part of the problem whereby abnor-mal mutant CFTR function also results from the abnormallipid environment. Related effects of different lipids onother ion channels exist in the literature.33,34 The hypo-thesis relating lipids to channel function in general is thatall ion channels are surrounded by a collar of fatty acids,which may be different from general membrane lipids,some of which interact with complementary domains onthe channel protein. Thus ion channel dysfunction couldtheoretically occur by altering the type of fatty acid foundin close proximity to cellular proteins.34 As far as theauthor is aware, no one has explored this possibility incystic fibrosis, despite the existence of a precedent as inthe case of AA for a different ion channel.33 Overall lipidmetabolism is different in CF cells, but the exact classes oflipid supplementation that might restore the balance oflipids remain to be determined for human airway cells.These requirements might be different for different or-gans, thus adding to the difficulty of using this therapeuticstrategy.

LIPID PRODUCTS AND INFLAMMATION:TOO LITTLE RESOLUTION?

The response of the CF lung to infection is neutrophil-dominated, but complex interactions occur.35 It is unclearwhy a chronic monocyte/macrophage response does notdevelop over time, as is the usual consequence of an on-going proinflammatory stimulus. Recently, an excitingobservation was made with the finding that the resolutionof inflammation might be reduced in CF cells, thus


prolonging the proinflammatory, neutrophil-dominatedphase of inflammation.36 It is commonly held that arachi-donic acid is ‘‘bad,’’ i.e., proinflammatory, but recentunderstanding shows that this is too simplistic and that thefunctional outcome depends on the pathways used tooxidize this fatty acid, such that it no longer contains fourdouble bonds. These double bonds cause AA to fold backon itself into a C-shaped structure that brings the two armsof the C into close proximity. The addition of oxygenstabilizes this shape by cross-linking the arms (see below).Although the mechanisms are beyond the scope of thisreview, three therapeutic concepts need to be understood.Firstly, if AA is oxidized by the inducible enzyme cyclo-oxygenase 2 (COX-2), and provided a nonsteroidal drugsuch as aspirin is also present that is capable of acetylatingCOX-2, then the acetylated COX-2 generates novel co-valently modified AA products such as 15R-HETE thatfeed into the synthesis of multiple anti-inflammatoryclasses of lipids called lipoxins.36,37 This is relevantbecause acetylation alters the location of proteins withinmembranes, cosegregating proteins with the above minorlipid classes linked to the endocytotic process. For un-known reasons, these lipoxins are deficient in CF epithe-lium, and recent work suggests that these molecules assistin the resolution of neutrophil-dominated lung inflamma-tion in CF.38 It follows that in CF, lipoxin deficiencymaintains neutrophil influx. Secondly, compared to thegently curved C-shaped AA, the lipoxin molecule foldsback on itself yet further to form an A-shaped structure,with the two arms of the A held together by an oxygenbridge. This new shape interacts with specific receptorsthat are different from those binding AA-derived pros-taglandins via cyclooxygenase, for example. Thirdly,lipoxins are intercellular signaling molecules generatedby the sequential activities of enzymes that occur indifferent tissues. Thus they are generated by cross-talkbetween cells (e.g., neutrophils and epithelial cells).36–38

It might be relevant that many years ago, Kuitert et al.found that the mRNA for one of the rate-limiting stepsfrom AA to lipoxins A4/B4 was missing in CF neutrophilsand epithelial cells, but was unaffected in asthmatics andnormal controls.39 This is important information for in-terventions aimed at modulating CF by dietary means toincrease intake of o3 lipids, e.g., using fish diets, whereeven the type of fish might be critical.40–42 Similarconsiderations apply to the administration of anti-inflam-matory therapy with nonsteroidal drugs,43 which isclaimed to be of some benefit at high dosage. Clinicaltrialswith new stable lipoxin analogues thatwere shown tobe beneficial in mouse models38 are awaited. The mecha-nisms bywhich such a vast array of lipid anomalies can begenerated in CF epithelial cells still remain in the realm ofspeculation. There are some clues from the considerationof the diverse functions2 and protein-protein interactionsof CFTR.17

COLLABORATIVE FUNCTIONS OF CFTR

This discussion is to some extent speculative, and focuseson only one of the many proteins which bind to CFTR. Thisprotein, which acts as an amplifier ofmultiple cell functions,was chosen because it has direct interaction with CFTR toclose the channel following activation by a rise in cellularAMP concentration,17 and is a key factor in cellular fuelhomeostasis involving glucose and fat.CFTR has been ‘‘understood’’ since 1989, although this

series is intended to highlight the huge gains in knowledgesince then, and the gaps which remain. One CFTR-interacting protein was discovered around the same timeas CFTR, but only recently has any functional under-standing been attained. Near the C terminus (but away fromtheAP2 site), CFTR binds a protein kinase, AMP-activatedprotein kinase (AMPK).44,45 AMP concentrations riserapidly during cell stresses such as infection or osmoticload: put simply, AMPK acts as a gatekeeper formetabolism, e.g., by turning on the ATP supply by stimu-lating mitochondrial b-oxidation and reducing wastage ofATP by turning off the committed steps of fatty acidsynthesis that might be required, e.g., to generate newsurface membrane area during cell division or endocytosis.The balance between mitochondrial fatty-acid oxidationand de novo fatty-acid synthesis depends on the synthesis ofa slightly longer version of acetyl CoA (malonyl CoA) inthe cytosol.44 In mitochondrial fatty-acid oxidation, mal-onyl CoA has a critical role as gatekeeper for the rate ofmitochondrial entry for long-chain fatty acids, so that theycan participate in b-oxidation to generate energywithin themitochondrial matrix. Any process reducing malonyl CoAsynthesis enhances fatty-acid oxidation during cellularstress by permitting more fatty acid to enter eachmitochondrion to replenishATP levels by generating acetylCoA (AcCoA) fuel for the TCA cycle. As discussed earlier,DHAprecursors utilize the oxidative pathway to synthesizeDHA. Malonyl CoA can perform this gate-keeping rolebecause the CFTR-associated AMPK described abovephosphorylates and hence inactivates a filamentous cyto-solic enzyme (acetyl CoA carboxylase) that normallygeneratesmalonylCoA. If acetyl CoAcarboxylase is active(AMPK not present or not working), its synthetic product,malonyl CoA, stops fatty acids entering the mitochondrionon the carnitine shuttle. It is interesting to note the spatialcolocalization of AMPK toCFTR at the apical pole of cellswhere mitochondria are also concentrated, a zone throughwhich endosomes also cycle. In CF, a key unansweredquestion is the degree to which failure of CFTR traffic/endocytosis results in attenuation of the cytosolic effects ofthe AMPK-CFTR interaction due to the loss of CFTRprotein at the apicalmembrane.Does this have an impact onmitochondrial chain elongation for theDHAclass of lipids?The complexity of this problem should not be under-estimated, becauseAMPKisnot a simple enzyme.Not only

296 Mehta

does it come in two different isotypes,44,45 but as befits agatekeeper, it is subject tomany checks and balances beforeit is permitted to act.46 These include sensing the need for‘‘action’’ during metabolic stress as AMP concentrationsrise; for full activity, needing to be phosphorylated via anupstream protein kinase involved in cell polarity, small-bowel cancer, and the cell cycle;46 and being capableof activation using alternative routes by drugs of themetformin class (mechanism unknown). Controversially,CFTR was proposed as an AMP generator,47 but therelationship to AMPK is unknown. These actions, coupledto proposed roles for AMPK in the control of glucosemetabolism and the inhibition of the committed step offatty-acid synthesis in the cell cytosol (reduction ofmalonylCoAproduction from acetyl CoA carboxylase), promise anexciting future for studies on the role of this enzyme in CFinflammation. The metabolic disturbances in CF, and theirconsequences for inflammation, however, remain poorlyunderstood.48

CONCLUSIONS

CFTR is amultifunctional protein, and the accident thatits chloride channel activity was among the first of itsmany activities to be discovered should not obscure thou-ght about other capabilities.4,49 CFTR does not always actalone, but may provide the platform containing dockingsites which allow the localization of metabolic processesto the vicinity of the apical membrane. This could linkCFTR dysfunction to inflammation via the disruption offatty-acid metabolism. This idea is part of an emergingconcept of microanatomical phenotypes generated bygroups of proteins acting in concert.50,51 The molecularshuttles arriving and leaving from the CFTR platformwilltell us how this protein52 acts on processes that might beunrelated to the transport functions of CFTR.53 There isprecedent for the ideas promoted here: there is compellingevidence that enzymes that constitute recognized bio-chemical pathways such as glycolysis in red cells54,55 arenot free entities. Their known tight regulation that permitsfeeding of substrates between pathway enzymes by theirtethering to membrane ion channels is a concept that maybe extremely relevant to cystic fibrosis,54,55 because one ofthe very substances thought to be transported by CFTR,glutathione,11 can regulate protein/enzyme function. 55

Although speculative, it is not unlikely that the non-channel and control functions of CFTR may be as impor-tant in the pathogenesis of CF as the better-understoodtransporter activities.

ACKNOWLEDGMENTS

I thank the Wellcome Trust, CF Trust, and AnonymousTrust for generously supporting my laboratory for manyyears. Due to space limitations, I have had to be selective

in referencing material, and I apologize to those authorsnot quoted herein. I am grateful for critical discussionswith Professor Andrew Bush, Dr. C. Ghioni, and Dr. R.Muimo on the topic of lipid metabolism, and withProfessor C.J. Ellory on aspects of red-cell physiology.I am grateful to Vera Murray for excellent secretarialassistance.

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