ANRV345-BI77-28 ARI 1 May 2008 13:16

CFTR Functionand Prospects for TherapyJohn R. RiordanDepartment of Biochemistry and Biophysics, Cystic Fibrosis Treatment and ResearchCenter, School of Medicine, University of North Carolina at Chapel Hill,North Carolina 27599; email: jack riordan@med.unc.edu

Annu. Rev. Biochem. 2008. 77:701–26

First published online as a Review in Advance onFebruary 27, 2008

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev.biochem.75.103004.142532

Copyright c© 2008 by Annual Reviews.All rights reserved

0066-4154/08/0707-0701$20.00

Key Words

ABC protein, anion channel, cystic fibrosis, ER quality control,molecular therapy, protein misfolding

AbstractMutations in the gene coding for the cystic fibrosis transmembraneconductance regulator (CFTR) epithelial anion channel cause cys-tic fibrosis (CF). The multidomain integral membrane glycopro-tein, a member of the adenine nucleotide-binding cassette (ABC)transporter family, conserved in metazoan salt-transporting tissues,is required to control ion and fluid homeostasis on epithelial sur-faces. This review considers different therapeutic strategies that havearisen from knowledge of CFTR structure and function as well asits biosynthetic processing, intracellular trafficking, and turnover.

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FurtherANNUALREVIEWS

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CFTR: cysticfibrosistransmembraneconductanceregulator

�F508: deletion ofphenylalanine atposition 508 of theCFTR amino acidsequence

ABC: adeninenucleotide-bindingcassette

Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 702CYSTIC FIBROSIS . . . . . . . . . . . . . . . . . 702ABC TRANSPORTERS . . . . . . . . . . . . 702CFTR STRUCTURE . . . . . . . . . . . . . . 703CFTR FUNCTION . . . . . . . . . . . . . . . . 704

Primary Anion Channel Function . 705Mechanism of Channel

Regulation . . . . . . . . . . . . . . . . . . . . 706Secondary Functions . . . . . . . . . . . . . 707

BIOSYNTHETIC PROCESSINGAND INTRACELLULARTRAFFICKING . . . . . . . . . . . . . . . . . 708Synthesis, Conformational

Maturation, and EndoplasmicReticulum Export . . . . . . . . . . . . . 709

Role of Molecular Chaperones . . . . 711Traffic in Endocytic and Distal

Secretory Pathways . . . . . . . . . . . . 713PROSPECTS FOR THERAPY . . . . . 715

CFTR Gene Replacement . . . . . . . . 715Suppression of Premature

Stop Mutations . . . . . . . . . . . . . . . . 716Restoration of CFTR Folding

and Function . . . . . . . . . . . . . . . . . . 716Modulation of Other Epithelial

Ion Transport Pathways. . . . . . . . 718CONCLUSIONS AND

OUTLOOK . . . . . . . . . . . . . . . . . . . . . 718

INTRODUCTION

The purpose of this review is to summarizewhat has been learned over the past 20 yearsabout the cystic fibrosis transmembrane con-ductance regulator (CFTR) and to considerhow this knowledge is being applied to the de-velopment of new therapies for cystic fibrosis(CF). The basic ideas are to consider what theCFTR is, what it does, what is wrong with itin disease, and what might be done to fix it.

CYSTIC FIBROSIS

The clinical features and history of the dis-ease since its recognition as a discrete en-

tity in 1938 are extensively described else-where, including several recent, excellentconcise reviews (1–3). From our present per-spective, it is clear that virtually all indi-viduals diagnosed clinically with CF havemutations in both CFTR gene alleles. Al-though approximately 1500 different disease-associated mutations have been identified,a single codon deletion, �F508, is by farthe most common and is present on atleast one allele in approximately 90% of thepatients in some populations (http://www.genet.sickkids.on.ca/cftr). Thus, althoughthere is a wide range in severity and manifes-tations of this disease that are dependent onother genetic and environmental influences,mutations in the CFTR gene are the funda-mental cause of the disease. The CFTR gly-coprotein, essential in the apical membrane ofepithelial cells to maintain ion and fluid home-ostasis, is unique as it is the only member ofthe large adenine nucleotide-binding cassette(ABC) protein family known to function as anion channel. The absence of this precisely reg-ulated anion channel activity results in the fail-ure of ionic and water homeostasis on exocrineepithelial surfaces. This causes accumulationsof macromolecular secretions, which are de-hydrated and in an altered physical state. Thisoccurs in most exocrine tissues but with themost serious consequences in the pancreas,where failure of bicarbonate-rich fluid and en-zyme secretion impair intestinal digestion andabsorption, and in the airways of the lung.There, viscous mucus accumulations and col-onization by microorganisms cause damaginginflammatory responses and loss of function.The lack of a CFTR chloride channel in thesweat duct blocks salt reabsorption, making itselevated concentration in sweat diagnostic ofthe disease.

ABC TRANSPORTERS

Members of this very large and ubiquitousfamily of membrane transporters were firstidentified in entirely separate studies of bac-terial nutrient uptake (4) and of the resistance

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of mammalian tumor cells to antineoplasticdrugs (5, 6). Sequences of the DNA coding forsubunits of the bacterial nutrient importers(7) indicated that they were constituted bysoluble nucleotide-binding and membrane-integrated subunits (in addition to a periplas-mic nutrient-binding subunit). The sequenceof the multidrug resistance P-glycoprotein,overexpressed in cancer drug-resistant cells,revealed the presence of domains similarto the nucleotide-binding and membrane-spanning subunits of the bacterial nutrientimporters, the bacterial hemolysin toxin ex-porter (8), and Drosophila eye color genes se-quenced earlier. Other individual transportproteins sharing these sequence similaritieswere discovered, including those that con-tribute to drug resistance or are mutated inseveral human genetic diseases (9).

The family name ABC was applied toreflect the presence in all members oftwo homologous nucleotide-binding domains(NBDs) (9). Genome sequencing has revealedthe presence of thousands of these domainswith 48 complete ABC proteins in the humangenome (10). Although only a few have beencharacterized biochemically and in membranetransport assays, ABC transporters may beconsidered transport ATPases as they trans-port substances against a concentration gra-dient, and their ATPase activities are stim-ulated by the compound transported. Eachtransporter exhibits internal symmetry of pri-mary structure with one membrane-spanningdomain (MSD) and one NBD in the N-and C-terminal halves. In many ABC trans-porters, both of the ATP-binding sites arehydrolytic, whereas in others, including thehuman ABCC subfamily to which the CFTRbelongs, hydrolysis occurs at only one of thesites (11).

Understanding of the fundamental dualityin the structure of ABC transporters becamemuch clearer when it was found that both ofthe two ATP-binding sites are composite sitescontributed to by residues from each of theNBDs (12–15). Each complete nucleotide-binding site is formed at the interface between

NBD: nucleotide-binding domain

MSD: membrane-spanning domain

CL: cytoplasmicloop

the NBDs, with the binding altering their spa-tial relationship to each other as well as coin-ciding with molecular rearrangements withinthe domains (16–18). Hydrolysis and disso-ciation of products may then enable returnto the initial unbound state. The nucleotide-induced changes in the NBDs may be coupledto conformational alterations in the MSDs,contributing to transport. A possible struc-tural basis of such coupling became evidentfrom the atomic structures of several completebacterial ABC transporters (19–22). These re-veal associations between three-dimensional(3D) structural elements in NBDs that changeposition on ATP binding and others (so-called coupling helices) in cytoplasmic loops(CLs) separating membrane-spanning he-lices. The propagation of these impliedmolecular motions in NBDs, through thecoupling helices, to the membrane region maycontribute to the transmembrane transportevent.

CFTR STRUCTURE

High-resolution structures of eukaryotic ABCtransporters have not been determined yet,primarily because of limitations in generatinghomogeneous monodisperse preparations ofsufficient quality and quantity for large-scalecrystallization trials. The CFTR is especiallydemanding in this regard because of its lownatural abundance and low level of expressionin heterologous expression systems in whichit is fully active functionally. So far, sufficientamounts of the CFTR from mammalian cellexpression systems have been purified to gen-erate two-dimensional crystalline arrays (23)as well as single particles analyzed by electronmicroscopy to provide low-resolution 3Dstructural information (Figure 1). The low-resolution structure obtained shows strongsimilarity to that of P-glycoprotein generatedby similar methods (24). Two distinct crys-tal forms are observed, possibly reflecting dif-ferent nucleotide-bound states. The locationof the R domain relative to the membrane-associated and nucleotide-binding regions

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2 nm

Bipartatesubstructure

Interface

Density ( )

2.0

2.5

3.0

3.5

4.0

Figure 1CFTR structure by cryoelectron microscopy, dimeric CFTR particles as purified from detergent, and theCFTR crystallized as a monomer (23). Slices through the 3D structure taken perpendicular to the C2axis which is tilted slightly toward the viewer and to the left. The interface apparent between the twomonomers in the structure is indicated by the dashed line. The bipartite substructure of the upper regionis indicated by the double arrow in the second panel from the left. The gray surface of the structure isdrawn at a density equivalent to 2σ; above the mean. Color coding of the internal structure revealed bythe slices is as shown on the right. Figure provided by Robert C. Ford.

PKA: protein kinaseA

is indicated by the position of site-specificnano-gold labeling (25). Crystals are currentlybeing analyzed by cryoelectron microscopy toobtain higher-resolution structures. A high-resolution structure has been obtained forthe isolated nucleotide-binding domain 1(NBD1) of the CFTR synthesized in bacteria(26). It has the same basic fold as that of theNBDs of many bacterial ABC proteins deter-mined earlier. Most significantly the Phe508residue occupies a position on the surface ofthe wild-type domain, and its absence has onlyminor effects on the domain structure (27), asindicated in Figure 2.

The R domain, which separates NBD1 andMSD2 in the primary structure, is highly un-structured and distinguished primarily by aconserved set of phosphorylation sites, whichcontrol the activation state of the channel(28). Whereas this 200-residue region is theleast conserved part of the protein sequence,both the relative positions of the phosphory-lation sites (29) and the order-disorder pat-tern across the sequence are highly con-served and hence functionally significant(T. Hegedus, unpublished observations).Phosphorylation by protein kinase A (PKA)reduces the already low α-helical content ofthe domain (30, 31), and a highly informa-

tive NMR study has recently shown that theα-helical propensities of stretches of residuesadjacent to many of the individual phospho-rylation sites are reduced when these sites arephosphorylated (32). Importantly, there is acorresponding diminution in the interactionof several of these stretches with NBD1 onphosphorylation. However, only the R do-main and NBD1 were present in these exper-iments, and R domain phosphorylation mayhave structural impact involving other parts ofthe molecule as well. Although a 3D structureof the full-length CFTR, at sufficient resolu-tion to enable clear definition of the R domain,is required to further understand its mech-anism of action, computational studies of itsstructure can be informative (T. Hegedus, un-published observations).

CFTR FUNCTION

Despite its architectural similarity to othermembers of the large ABC transporter family,the CFTR is functionally distinct in perform-ing as an ion channel. Apparently conforma-tional movements within and between the twoNBDs are coupled to rearrangements amongmembrane-spanning sequences, which, ratherthan changing affinities and sidedness of

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allocrite-binding sites, shift the equilibriumbetween the actively gating (open) and quiet(closed) ion pore. Such a device would gateand conduct anions continually were it notfor the unique R domain, which, until phos-phorylated by PKA, restrains channel gating.Thus, the probability of channel opening iscontrolled by the extent of R domain phos-phorylation at multiple sites, reflecting thebalance between protein kinases and the phos-phatases acting on these sites. In the reabsorp-tive duct of the sweat gland, for example, PKAapparently normally dominates, resulting in acontinually active channel favoring maximalsalt recovery. In contrast, in secretory epithe-lia, the channel may be kept unphosphory-lated and inactive until stimuli elevating cyclicAMP make the action of PKA exceed that ofthe phosphatases.

Primary Anion Channel Function

When first identified at the primary structurelevel, it was stated that the CFTR might eitherbe the chloride channel defective in CF or aregulator of a separate channel protein (33).Although there are many reports of influencesof the CFTR on other channels and trans-porters (34, 35), strong evidence indicates thatthe CFTR polypeptide itself mediates a char-acteristic epithelial anion conductance. Prop-erties of its tightly regulated single-channelconductance have been thoroughly character-ized (36–38) and are not detailed here. It be-haves as an ohmic low-conductance channelcharacteristic of that observed in the apicalmembrane of epithelial cells of the tissues af-fected in the disease (39). This pathway for themovement of chloride ions is required in ei-ther the secretion or absorption of salt acrossthese epithelia (40). The CFTR also plays animportant role in HCO3

− secretion becauseit is permeant to the anion (41) and because itprobably stimulates Cl−/HCO3

− exchangers(42). The most obvious manifestation of theloss of this function is the impaired pancre-atic HCO3

− secretion in patients, but there is

F409F433

F429

R555R553

G550F508

Figure 2Comparison of nucleotide-binding domain 1 (NBD1) structure of thewild-type and �F508 CFTR. Structures for the wild-type (2BBO, red ) and�F508 (1XMJ, purple) CFTR were aligned, and the indicated mutationsaiding crystallization were reverted using PyMol (http://pymol.sourceforge.net/). The regulatory insertion (404–435), which is missingfrom the NBD1 crystal structure, is inserted from a loop database search(SYBL,Tripos Inc., CA). That loop (cyan) contains two α-helixes in goodagreement with hydrophobic patch analysis of this region (189). Thesolubilizing and rescue mutations are numbered and represented by sticks(light and dark green). Phe508 is colored yellow. The major differencesbetween the wild-type and mutant domains, exhibited by differentconformations of the two loops, are indicated with thick arrows. Figureprovided by Tamas Hegedus.

also reduced pH in the epithelial surface liq-uid of other tissues (43). The failure to alka-linize the fluid into which they are secretedmay be an important factor preventing thenormal processing of mucins and contribut-ing to their hyperviscosity (44). Although reg-ulation of ionic balance, hydration, and pHadjustment are believed to contribute, there

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remains an urgent need to further elucidatethe exact mechanisms whereby the wild-typeCFTR enables the achievement of the normalphysical state of mucins as they are secreted.

Mechanism of Channel Regulation

The CFTR is atypical both as an ion channeland as an ABC protein, having adopted the ba-sic ABC transporter structural architecture togenerate a ligand-gated channel whose levelof activity is quantitatively controlled by thephosphorylation state of its unique R domain.Phosphorylation by PKA has only a minor ef-fect on CFTR ATPase activity (45) and ap-parently does not act primarily by influencingbinding or hydrolysis of the ATP ligand (11)but does promote the association of the twoNBDs (46). However, this latter effect is notentirely responsible for channel activation byphosphorylation because that occurs with ac-tive C-terminally truncated constructs lackingNBD2 (47, 48). R domain phosphorylation al-ters its conformation as well as contacts withother parts of the protein (30–32, 49).

The relationship between binding and hy-drolysis of the ATP ligand and channel gat-ing also has not yet been entirely elucidated.From a thermodynamic perspective, interpre-tations have been somewhat confounded fromthe outset by assumptions about the role ofthe ATPase activity of the protein. An anal-ogy with ABC transporters that couple ATPhydrolysis to the transport of substances (all-ocrites) and the observation that hydrolyzablenucleoside triphosphates were preferred lig-ands (50) led to suggestions that CFTR chan-nel opening was energetically coupled to ATPhydrolysis, a view that was elaborated by oth-ers (51). Observations of apparent nonequilib-rium in transitions between two open statesof slightly different conductance were inter-preted to mean that the gating process was notin thermal equilibrium (52). This nonequi-librium in gating seemed consistent with theprevious suggestion that gating was driven byATP hydrolysis. However, the apparent sec-ond open conductance state could only be ob-

served in strongly filtered records owing toa fast flickering mode too rapid for detec-tion of its fully open state, and kinetic anal-ysis with extended bandwidth provides no ev-idence of nonequilibrium in gating (53, 54).Indeed, there is strong evidence that CFTRsingle-channel gating is a reversible, ther-mally driven process (55) and that hydroly-sis is not required for gating (56). Gating andhydrolysis do not appear to coincide either ki-netically or energetically. It is now generallyagreed that hydrolysis is not involved in chan-nel opening (57, 58).

However, studies focusing primarily onthe role of the tightening and looseningof the association between the two NBDsof the CFTR have retained the interpretationthat channel closing is dependent on the en-ergy liberated by the cleavage of the terminalphosphate from ATP (59). This view is basedprimarily on the assumption that gating of theCFTR is not a thermodynamic equilibriumprocess and the observation of a higher ac-tivation enthalpy for closing (∼70 kJ/mol) inXenopus oocytes (60) than that (∼20 kJ/mol)determined earlier in planar lipid bilayers(55). It was suggested that the latter lowervalue may have reflected the measurement andanalysis of brief flickering transitions withinchannel open bursts rather than burst closures(60), but this is not the case, because the clos-ing rates yielding the lower values agree verywell with the burst durations determined bypatch-clamp analyses in several other labo-ratories (61). Thus, the discrepancy betweenthe different Ea values for channel closing re-mains to be explained. In any case, the esti-mated free energy input of ∼70 kJ/mol forchannel closing does not correspond well withthe free energy of ATP hydrolysis, and thereis no suggestion of the source of the additional35 kJ/mol (60). Moreover, in addition to chan-nel closing occurring in the absence of ATPhydrolysis, there is also evidence that chan-nel closing is not obligatorily dependent on achange in the association of the two NBDs.That is, channel opening and closing eventsoccur in the complete absence of NBD2 (47).

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Thus, even though a model with directone-to-one coupling between the formationand disruption of tight ATP-mediated inter-NBD association and channel opening andclosing, respectively, fits well with general in-terpretations of ABC protein function (16,62), it does not account for all of the cur-rent data. Initiation of channel opening doesnot require tightening of the interface be-tween NBDs, although this may prolong theopen state. The closing of channels, which aremaintained in the open state in this way, isfacilitated by hydrolysis of the NBD2-boundATP and probably also by the disengagementof the NBD1-bound ATP from the degener-ate signature sequence of NBD2. Similarly,the gating of the full-length channels in re-sponse to nonhydrolyzable ATP analogues asligands (56) may also reflect dynamic eventsat the interface between the NBDs, but inneither case is the hydrolysis per se an inte-gral part of the gating mechanism. One reasonthat the precise relationship between the en-zymatic and channel activities of the CFTRare not yet more fully understood is that theenzymatic characterization of the protein isstill very limited (11, 45, 63, 64), and there isuncertainty as to whether activity assayed withthe purified, reconstituted protein accuratelyreflects that in its native environment.

One very interesting proposal is that chan-nel activity may correlate more closely withadenylate kinase than ATPase activity of theprotein (65). Apparent adenylate kinase ac-tivity has been assayed with isolated CFTRNBDs, but not with full-length protein(66, 67). Effects of nucleotide mixtures ofadenylate kinase substrates and productson CFTR activity in patch-clamp exper-iments have been described (65). It hasbeen suggested that channel gating would bethermodynamically more compatible with adependence on adenylate kinase than ATPaseactivity (65). However, there is no ther-modynamic incompatibility in a so-calledhydrolyzable-ligand-gated process in whichhydrolysis of the ATP ligand is a simple switchenabling release of structural strain in the pro-

tein rather than an energy source (61). Never-theless, lending credence to the possible im-portance of adenylate kinase activity of otherABC proteins, as well as the CFTR, is therecent description of the presence and role ofthis activity in the Mre11/Rad 50 DNA repaircomplex (68).

Secondary Functions

In addition to its well-established ion chan-nel function, the CFTR has been proposed tohave many other roles and either directly orindirectly impacts other cellular proteins andfunctions (34). Thus, the term pleiotropic hasbeen applied to the CFTR, but it is uncer-tain if this is appropriate. There are certainlydownstream effects in addition to altered an-ion permeation owing to CFTR function anddysfunction. However, it remains a challengeto identify at what level a given downstreamalteration is connected to the CFTR proteinitself or the anion conductance that it medi-ates. From the CF disease perspective, the in-fluence of the lack of the CFTR on conduc-tive epithelial Na+ permeability mediated byENaC channels has received a great deal ofemphasis (69, 70). This is because the con-tinued or enhanced Na+ absorption is pri-marily responsible for the dehydration of theairway surface, which impairs mucociliaryclearance (71). The importance of this effectis emphasized by the fact that transgenic over-expression of an ENaC subunit in mouse lungmimics the dehydration and mucus plugging,which occurs in the CF lung. (72).

In addition to serving as a chloride chan-nel itself, the CFTR may also regulate otherchloride channels, but these have not beenidentified at the protein level (73). There isstrong evidence that the CFTR has evolvedas an epithelial ion channel and that it is moststrongly expressed in the salt-transporting ep-ithelia of teleosts, elasmobranchs, amphibia,avia, and mammals. Nevertheless, in someassays of CFTR RNA or protein, its pres-ence has been detected in nonpolarized cells,including those of the immune system. For

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example, digestion of phagocytosed materialby alveolar macrophages was reported to bedefective in CF because of the absence ofthe CFTR from the membrane of the phago-some vacuole where it normally enables acid-ification by providing a counterion pathway(74). Detection of the CFTR in neutrophilsand other hemopoietic cells has also been de-scribed (75). The possibility of a direct con-tribution of the CFTR to the function ofimmune or inflammatory cells is an impor-tant issue that needs to be clearly resolved be-cause of the apparent hyperinflammatory sta-tus of patients’ tissues prior to infection (76).However, it is still unclear whether this is dueto inherent changes in the inflammatory cellsthemselves or a reaction to the altered stateof epithelial surfaces owing to ionic and fluidchanges and mucus accretion. The failure ofthe CF lung to clear infectious microorgan-isms is generally attributed to impaired mu-cociliary clearance, but in addition, the CFTRhas been proposed to serve as a receptor forPseudomonas aeruginosa (77). In this mecha-nism, binding of the microbe to the receptoris required for its internalization and stimula-

Wild type ΔF508

Figure 3Localization of the wild-type and �F508 CFTR in highly differentiatedprimary human epithelial cells. The wild-type CFTR is present and the�F508 CFTR is absent from the apical surface of ciliated airway cells.Fully differentiated primary cultures were adenovirally transduced toexpress a tagged CFTR protein, and the CFTR was detected in isolatedcells using a mouse antibody recognizing the tag, followed by goatantimouse Alexa Fluor 288 lgG conjugate. The immunofluorescence isshown in overlay with differential interference contrast and nuclearstaining by TO-PRO 3 iodide. Figure provided by Martina Gentzsch.

tion of the cytokine secretion response neces-sary for clearance (78).

In addition to chloride and bicarbonate,believed to be the main physiological anionspassing through the CFTR, prevention of thepassage of other permeant species in CF alsomay contribute to the disease phenotype. Animportant example from the infectious diseaseperspective is the apparent failure of trans-port of the thiocyanate anion onto the surfaceof CF airway epithelium (79). This can re-sult in the lack of oxidative generation of an-tibacterial hypothiocyanite, which may nor-mally help eliminate Staphylococcus aureus andP. aeruginosa.

BIOSYNTHETIC PROCESSINGAND INTRACELLULARTRAFFICKING

When the CFTR gene was initially identified,the absence of the codon for Phe508 was rec-ognized on the sequencing of cDNA synthe-sized from the sweat gland RNA of a patient(33). This deletion was then detected in ap-proximately two thirds of CF chromosomes(80). Heterologous expression of full-length�F508 CFTR cDNA indicated that, althoughthe glycoprotein was synthesized, it acquiredonly core and not complex oligosaccharidechains and failed to be transported to the cellsurface (81). These findings have been con-firmed and extended in many different mam-malian cell expression systems. The likelihoodthat the biosynthetic arrest of �F508 oc-curred in vivo in patients’ tissues and was notjust an artifact of heterologous overexpres-sion was supported by the observation thatthe protein did not reach the apical membraneof sweat duct cells in fresh skin biopsies (82).More recently this has been verified in intes-tine and airway from patients (83, 84). Thedifferential localization of the wild-type andmutant protein is illustrated in Figure 3.

Expression of �F508 in Xenopus oocytes(85) and insect cells (86), which are main-tained at lower temperatures than mammaliancells, resulted in at least partial maturation

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and some activity of the mutant proteinat the plasma membrane. This suggestionthat the mutation is temperature sensitivewas confirmed by the observation of similarbehavior when mammalian cells expressingthe mutant protein were shifted to lower tem-perature (87). This partial rescue by temper-ature manipulation presaged similar effectsof treatment of cells with osmolytes, such asglycerol and other so-called chemical chaper-ones (88). This, in turn, encouraged searchesfor small-molecule rescuing agents that mayultimately be effective in the treatment ofpatients (89). These efforts, which are on-going, provide hope for the development ofdrugs to overcome the misprocessing of mu-tant CFTRs.

Synthesis, ConformationalMaturation, and EndoplasmicReticulum Export

Extensive metabolic labeling and pulse-chaseexperiments on the CFTR heterologously ex-pressed in a variety of mammalian cells haverevealed that 5 to 10 minutes are required fora complete core-glycosylated CFTR polypep-tide to be formed (90). Although unglyco-sylated chains can be synthesized in vitro,none are formed in cells, indicating that N-glycosylation of the two sites in EL4 occurscotranslationally (81). Transformation to aform in which the high-mannose oligosaccha-ride chains have been trimmed and extendedto large complex chains takes longer, with re-sultant higher-molecular-weight species firstappearing only after nearly a half hour of chasetime (91). Complete transformation requiresat least two hours, and during this period,only approximately one third of the precursornascent chains are converted to mature prod-uct (92). The remainder is ubiquitylated anddegraded by the 26S proteasome (93, 94). In-deed, ubiquitylation begins before translationis complete (95), and it is now realized thatthe CFTR is scrutinized by complex qualitycontrol systems during its synthesis and as-sembly as well as throughout its lifetime in

the cell. These systems deal with most mem-brane and secretory proteins, but the CFTRseems to have particular difficulty in achievinga state that satisfies all criteria for export fromthe endoplasmic reticulum (ER). Wild-typeversions of other ABC transporters such as P-glycoprotein and MRP1, for example, matureand are exported with nearly 100% efficiencycompared to the ∼33% for the CFTR in thesame cells (96). Interestingly, however, dele-tion of the counterpart of Phe508 in each ofthese ABC proteins has the same effect as inthe CFTR, which is completely blocked mat-uration and ER export (97).

The most obvious feature distinguishingthe CFTR from other ABC proteins is theR domain, which is largely unstructured (32),and, therefore, possibly may contribute to thediminished efficiency of CFTR maturation.However, simple deletion of large portionsof the R domain does not improve this ef-ficiency and, in fact, reduces it further (98).Both intradomain folding and interdomainassembly, not unexpectedly, are required toachieve a state competent for ER export (47,99) as recognized by COP II constituents,which coat vesicles that bud from ER exit sites.The vesicle coat protein sec24 recognizes adiacidic exit code (DAD) of three residues inthe NBD1 of the CFTR, which when mu-tated prevents ER export (100). It is easy toimagine that �F508 or other processing mu-tants might conformationally mask this exitsignal or possibly others and thereby elicitthe same effect as inactivating it directly bymutagenesis. However, there are likely addi-tional dimensions to the recognition mecha-nisms during the dynamic interplay betweenprogression toward a native state and taggingfor a degradative fate. The role of molecularchaperones in this balancing act is outlined ina section below.

When the nascent CFTR was first rec-ognized as a substrate for ubiquitylation anddegradation by the proteosome (93), it wasdisappointing to observe that proteasome in-hibitors did not promote maturation and ERexport. A similar result was recently reported

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with the yeast Yor1p ABC protein into whichthe equivalent of the �F508 mutation was in-troduced (101). Thus, inhibition of endoplas-mic reticulum-associated protein degradation(ERAD) generally does not promote ER ex-port, although there is one report that thismay occur to a limited extent (102). Never-theless, to identify additional points of pos-sible intervention, it is necessary to elucidatethe different components of the quality con-trol systems and the sequence in which theyevaluate the state of the polypeptide and di-rect its elimination if it has not yet reacheda mature state. The Cyr laboratory (103) hascontributed substantially to the current un-derstanding of these events. Having earlierdemonstrated the involvement of a ubiqui-tylation complex that interacts with Hsc70in �F508 degradation (104, 105), this groupmore recently characterized a second complexcontaining the E3 ubiquitin ligase, RMA1,the E2 ubiquitin conjugating enzyme, Ubc6e,and the membrane-associated Derlin-1 (99).Their data and those of a parallel study (106)indicate that the latter complex, which alsoassociates with the AAA-ATPase P97 [theyeast Cdc48 counterpart (107)], recognizedthe nascent CFTR early in its synthesis. Thefirst CFTR MSD appears to be the target, per-haps initially of Derlin-1 if integration withthe second MSD has not yet occurred. Sig-nificantly, this targeting of the �F508 CFTRoccurs even when both MSDs have alreadybeen synthesized, implying that the mutationmay disrupt their assembly as is also indicatedby other studies (47).

Although this impact of �F508 is observedin truncation constructs in which NBD2 isnot present (47), the mutation is also knownto prevent compact folding of that domain inthe full-length protein (108). Indeed, Youngeret al. (99) postulate that this may be whatis detected by the first quality control com-plex, which they characterized involving theE2 UbcH5, E3 CHIP, the chaperone Hsc70,and its cochaperone, Hdj2. In this scenario,there is first scrutiny of MSD1 by the Derlin-1/RMA1 membrane-associated complex fol-

lowed by recognition of incompletely foldedNBD2 by the cytoplasmic Hsc70/CHIP com-plex. There may be additional contributorsto the efficient ERAD of incompletely oraberrantly assembled nascent CFTRs. For ex-ample, as mentioned in the chaperone sec-tion below, although the lectin chaperone-based conformation-sensing system of theER lumen is not a primary determinant ofCFTR quality control, overexpression of themannose-binding EDEM (ER degradationenhancing α-mannoside-like protein) doesaccelerate degradation (107). This pathway,which is known to direct the retrotransloca-tion, ubiquitylation, and proteolysis of othersecretory glycoproteins (109), may help en-sure that little unprocessed CFTR accumu-lates in the ER.

Because of its dominant contribution todisease, most attention has been focused onthe impact of �F508 on CFTR folding andassembly. Replacement of the Phe normallypresent at this position by each of all otheramino acids, rather than simple deletion,revealed that the presence of many differ-ent residues were compatible with substan-tial folding of the isolated NBD1 domain andthe full-length protein (108, 110). The pres-ence of the amino acid backbone regardlessof the side chain, with the exception of thatof tryptophan, enabled refolding of bacteri-ally expressed NBD1. The requirements formaturation of a full-length CFTR were some-what more stringent with charged residuesand large hydrophobics other than Phe, al-lowing very little formation of mature protein.These observations imply that the side chainmay contribute to an interdomain interaction.Although the variants that matured showedsome level of channel activity (108), the Phearomatic side chain apparently plays a specificrole in CFTR channel gating kinetics (111).In light of earlier evidence that the overallconformation of the �F508 CFTR is grosslyaltered as reflected by susceptibility to lim-ited proteolysis (112), the finding that the 3Dstructure of the �F508 NBD was little alteredwas somewhat surprising (27). However, this

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was also found to be the case with the isolateddomains containing either F508S or F508Rsubstitutions (110). In both of these variants,only a local surface perturbation around the508 position was evident, invoking the ideathat the main impact of the mutation may bein disrupting a crucial interaction between thissmall patch on the surface of NBD1 and an-other part of the molecule.

Consistent with this, Du et al. (108) foundthat �F508 caused little change in the pro-tease sensitivity of NBD1 but instead causedNBD2 to become much more sensitive to di-gestion. This could reflect impairment of a di-rect NBD1/NBD2 interaction or an indirecteffect of disruption of an NBD1 associationelsewhere in the molecule. �F508 has beenfound to increase the protease sensitivity ofMSD1, to which it is linearly adjacent, as wellas of NBD2 (47). However, possible sites ofspecific interaction of the Phe508 containinga surface patch of NBD1 have not yet beenidentified. Because �F508 has essentially thesame effect on a maturation-competent C-terminally truncated form of the CFTR fromwhich NBD2 is entirely absent as on the full-length protein (47), it seems that such sitesare likely within the membrane-spanning do-mains. Sequences in the CLs of both MSD1and MSD2 may interact with NBD1 (20). Ofthe several hundred disease-associated mis-sense mutants in the CFTR, many causingmisfolding and missassembly, several occur inCLs, with an especially high incidence in CL4(113). Some of these CL4 substitutions couldpreclude an important interaction betweenthis loop and NBD1 as suggested schemat-ically in Figure 4. Thus, many process-ing mutations may disrupt the crucial struc-tural interface between the cytoplasmic andmembrane-integrated domains of the CFTR.

Role of Molecular Chaperones

As the nascent CFTR polypeptide is synthe-sized, it encounters molecular chaperones onboth sides of the ER membrane. Initially, in-teractions of the immature form of both the

Wild type

MSD1

NBD2

NBD1 R

ΔF508

MSD1

MSD2

Figure 4Illustration of wild-type and �F508 CFTR domain assembly.Requirements for specific interactions between NBD1 and MSD2 andNBD2 and MSD1 are emphasized. The Phe508 deletion precludes the firstof these interactions, which in turn prevents the normal integration of theMSDs, and NBD2 is unable to associate with MSD1. Adapted and revisedfrom Reference 47. Figure provided by Lisa Brown.

wild-type and �F508 CFTR, with the majorcytoplasmic chaperone Hsp70 (114), and thelumen-facing chaperone of the ER membranecalnexin (115) were detected. Not unexpect-edly because most of the CFTR mass is consti-tuted by the large cytoplasmic domains, whichmust be folded and assembled correctly, mul-tiple cytoplasmic chaperones and cochaper-ones have since been implicated in these pro-cesses (116). An Hsp70 cochaperone of the

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Hsp40 class, Hdj-2, was shown to diminishaggregation and promote an early stage ofassembly, possibly by recruiting Hsc70 tothe cytoplasmic surface of the ER mem-brane (105). The nucleotide exchange factorHspBP1 aids Hsp70-supported CFTR fold-ing (117). In addition to the positive role ofHsc70 and its cochaperones in maturation ofCFTR cytoplasmic domains, they also com-plex with an E2 ubiquitin-conjugating en-zyme Ubc H5 and the E3 ubiquitin ligaseCHIP to direct ubiquitylation, which targetsthe nascent CFTR for proteasomal degra-dation (104). Although the nascent �F508CFTR has a somewhat greater tendency thanthe wild type to participate in these Hsc70-based complexes, neither simple up- or down-regulation of the chaperone significantly pro-motes maturation of the mutant (118). A drugwith some ability to counter Hsp70 inter-actions also had minimal if any effect on�F508 maturation (119). The fact that alarge proportion of wild-type nascent chainsundergo a similar set of interactions and adegradative fate probably means that veryfine-tuned manipulation of many of thesesteps would be required to shift the foldingyield of the mutant to a level nearer the wildtype.

Significantly, progress toward that end hasrecently been made by manipulations of theother major cytoplasmic chaperone complex,downstream from Hsc70, the Hsp90 sys-tem (116). Indeed, earlier work showed thatthe Hsp90 inhibitor geldanamycin completelyprevented the maturation of both the wild-type and �F508 CFTR, resulting in the rapidproteasomal degradation of immature nascentchains (96). A detailed proteomic analysis ofCFTR-binding partners revealed a predomi-nance of known members of the Hsp90 chap-erone complex, especially in association with�F508 (116). Several of these members wereup- and downregulated to test whether moresubtle modulation of this network might pro-mote CFTR maturation or stability, which iscompletely disrupted by inhibition of Hsp90

function with an ansamycin drug (96). RNAiknockdown of the p23 cochaperone, believedto be involved in Hsp90 client loading, fur-ther destabilized the mutant nascent chains,whereas overexpression had a modest stabi-lizing influence without any promotion ofmaturation. FKBP8, a member of the im-munophilin family, involved in folding ofother Hsp90 clients, further reduced theamount of the immature CFTR.

Shifting the level of the Ahal cochaperone,which stimulates Hsp90 ATPase activity, incontrast to p23, which inhibits activity, hada more pronounced impact on levels of bothimmature and apparently mature �F508 pro-teins. Whereas Ahal overexpression dimin-ished the amounts of both forms, reductionof the cochaperone level with RNAi increasedboth, resulting in some of the functionalCFTR at the cell surface. This provides proofof principle that modulation of the nascentCFTR chaperone environment can provide adegree of rescue of the �F508 CFTR. It is in-teresting that this has been possible with theHsp90 network, which is dedicated to alteringconformations of proteins that have alreadyreached a basal folded state. Thus, the CFTRis a member of the somewhat selective Hsp90clientele. Significantly, related ABC proteins,including P-glycoprotein and multidrug resis-tance protein 1, are not dependent on Hsp90for their maturation and stability (96). Thus,there is an apparent correlation between theinefficient maturation of the CFTR and itsdependence on Hsp90.

However, the nascent CFTR has beenshown to interact with other cytoplasmicchaperones in addition to the major Hsp70-and Hsp90-based systems, and it is con-ceivable that their manipulation might alsoaugment maturation of the mutant CFTR.Examples include the Hdj-2-related cysteine-string protein, which seems to interact withboth the ER and post-Golgi forms of theCFTR (106), and small Hsps, including αA-crystallin, which may contribute to ERAD ofthe misfolded CFTR (120).

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Multiple cytoplasmic chaperones andcochaperones act coordinately and sequen-tially in appraising and influencing the as-sembly of the large CFTR domains at thecytoplasmic surface of the ER; however, theinfluence of luminal ER chaperones is lessclear. Both calnexin and calreticulin bind thecore N-linked oligosaccharide chains on thefourth extracytoplasmic loop (115) as doesEDEM (107). Although these lectin chaper-ones together with key glycosidases and gly-cosyl transferases constitute a conformation-sensing mechanism that determines the fate ofmany glycoproteins during biogenesis (109),this is apparently not a rate-limiting pathwayfor the CFTR. This can be concluded be-cause the wild-type protein can be exportedfrom the ER entirely without glycosylation,whereas unglycosylated �F508 still cannot(121).

Nevertheless, there have been several at-tempts to promote “release” of the mutantfrom the ER by perturbing interactions withthe membrane-bound calnexin. Because itand its luminal counterpart, calreticulin, arecalcium-dependent chaperones, thapsigarintreatment to deplete ER calcium was reportedto cause some maturation (122) as was anotheragent, curcumin (123). Although the lattercompound may interact with the CFTR (48,124) as with many other proteins, an ability topromote �F508 maturation and stability hasnot been generally confirmed (125) nor has aninfluence on the interaction between calnexinand the nascent CFTR (126). Knockdown ofcalnexin by RNAi also did not elicit �F508maturation (127). Calreticulin, which is trans-ported from the ER to the cell surface, hasbeen reported to promote CFTR transport tothat location where it is rapidly endocytosedand degraded (128).

Even though the luminally exposed loopsbetween membrane-spanning sequences ofthe CFTR are quite short, additional ER lu-minal chaperones including GRP78, GRP75,reticulocalbin, and calumenin were iden-tified in immunoprecipitates from CFTR-

CCV: clathrin-coated vesicle

expressing cells (116). These interactions andtheir possible roles of nascent CFTR process-ing remain to be explored further.

Traffic in Endocytic andDistal Secretory Pathways

The population of wild-type CFTRmolecules that are exported from theER and reach the Golgi and post-Golgicompartments is quite stable with a half-lifeof approximately 16 h (90, 91). However,the cell surface CFTR pool was very rapidlyinternalized at a rate of approximately 10%per minute (129). Because the biosyntheticrate is very much less than that, this indicatedthat the internalized protein must be recycledto the surface. This was shown to be thecase in various cell types using differentmethodologies (130, 131). The “in” route hasbeen extensively characterized and appears tofollow, at least primarily, the clathrin-coatedvesicle (CCV) endocytic pathway (132). The“out” route is beginning to be better under-stood (see below). The CFTR was detected inisolated CCVs (133) and visualized in endo-somes by immunocytochemistry (134). Themajor functional implications of this entryand exit cycle are with respect to regulationof the amounts of cell surface CFTR activityand the fact that it is strongly perturbed bythe �F508 mutation. Lukacs and coworkers(132) observed a phosphorylation influenceon clathrin-dependent CFTR endocytosis,and some investigations have emphasizedthe role of a shift in the balance betweenthe plasma membrane and intracellularvesicular pools by PKA phosphorylation(135). However, the extent to which thismechanism contributes to epithelial PKA-stimulated chloride secretion compared tothe activation of individual CFTR channelsalready in the plasma membrane is not yetclear.

What is clear is that when �F508 andother processing variants of the CFTR havereached the plasma membrane, either in cells

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grown at reduced temperature or becauseof genetic or pharmacological manipulation,they are thermally unstable (111, 136) andagain very rapidly removed from the surface(137). This reduction in surface steady-stateamount has been attributed to acceleratedendocytosis in one study (138) and to fail-ure of recycling in another (131). The differ-ence may reflect the different cell types used,but it will be important to resolve this is-sue in the fully differentiated epithelial cellsof the tissues most affected by the disease.Binding of tyrosine- and dileucine-based sig-nals in the CFTR C-terminal extension tothe μ 2 subunit of the AP-2 clathrin adap-tor complex initiates clathrin-mediated endo-cytosis (139). Notwithstanding the fact that�F508 and other mutations impacting CFTRstructure cause enhanced clearance from thecell surface, introduction of endocytic motifselsewhere in the protein than the C termi-nus apparently can also augment endocytosis(140).

In addition to trafficking between theplasma membrane, early endosomes, and re-cycling endosomes, the CFTR also may passinto late endosomes, multivesicular bodies,and lysosomes for degradation, and indeedthe latter routing is the ultimate fate of theendocytosed �F508 CFTR (141). Details ofthe recognition mechanisms that are able todistinguish the wild-type from the mutantCFTR and thereby determine the directionstaken are not yet entirely understood, but theLukacs laboratory (131) has demonstrated theinvolvement of ubiquitylation and other fac-tors that participate in routing of the ubiqui-tylated substrate. This ubiquitylation and itsrecognition appear responsible for the mu-tant protein proceeding to lysosomal degrada-tion, using an Rme-1-dependent mechanism,rather than being recycled like the wild type(130).

The actin-based cytoskeleton plays a ma-jor role in the endocytic trafficking of theCFTR as it does of other internalized cargo.Myosin VI participates in endocytic uptake,perhaps by providing an actin-binding mo-

tor to drive directional movement of CCVs(142), whereas myosin Vb may act in ananalogous fashion in trafficking from recy-cling endosomes back to the plasma mem-brane (143). In both cases, filamentous actinassemblies are required. Disruption of theseassemblies by several different means hasrecently been shown to influence both inter-nalization and recycling of the CFTR (144).However, the net effect was a large reduc-tion in the size of the cell surface CFTRpool, apparently because of augmented inter-nalization. The later effect was interpretedas the result of freeing of a cytoskeletallyconstrained plasma membrane pool, whichcould be recruited more readily into clathrin-coated pits (144). It is not clear how to rec-oncile this increased internalization on dis-ruption of the actin cytoskeleton if it isrequired in the endocytosis-promoting actionof myosin VI (142). Populations of CFTRmolecules constrained in their mobility havebeen demonstrated in independent studies us-ing different methodologies (145, 146). Con-nections of the CFTR to the actin cytoskele-ton may be mediated by both PDZ-domainproteins binding at the C terminus (147) andfilamin at the N terminus (148). Internaliza-tion and transport to lysosomes for degrada-tion are promoted when filamin binding isprevented.

The entire significance of the extensivetrafficking and scrutiny of the CFTR in theendocytic and post-Golgi compartments maynot yet be fully appreciated. It clearly providesadditional mechanisms of quality control be-yond the apparently very stringent ones ap-plied at the ER during synthesis and assembly.There is evidence that the mutant CFTR canimpact the trafficking of other cellular con-stituents, including lipids, if it gains accessto the distal compartments (149). However,distal quality control may provide an elab-orate mechanism to control the turnover ofthe mature ion channel. Perhaps, changes inthe molecule, which may occur as it ages inan environment probably not perfectly pro-tected from damaging chemical species, are

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continuously monitored for disposal. In thisway, either genetic or environmental per-turbations would be similarly handled by ahighly evolved mechanism, probably sharingfeatures with systems that distinguish occu-pied and unoccupied cell surface receptors(150).

PROSPECTS FOR THERAPY

In addition to advancing the understandingof several aspects of human genetics and themolecular and cellular biology of epithelialion and fluid homeostasis, knowledge of theCFTR gene and protein has provided newopportunities to develop rational therapeu-tic approaches to the treatment of CF. Thosecurrently in progress include CFTR gene re-placement, suppression of nonsense muta-tions, restoration of folding and function ofmutant CFTR channels, activation of alter-native chloride channels, inhibition of sodiumabsorption, and other ionic and osmotic ma-nipulations to normalize fluid and bicarbon-ate levels on epithelial surfaces. These newerstrategies are intended to complement currenttreatments, which combat mucus viscosity andbacterial infection (151, 152).

CFTR Gene Replacement

Efforts to develop gene replacement ther-apy for CF have been ongoing since shortlyafter the gene was discovered. Both non-replicating viral vectors and DNA-lipid com-plexes have been employed but without defini-tive evidence of persistent functional efficacy.However, substantial progress has been madein understanding the limitations to the suc-cess of the early attempts. The target of theaerosolized vector is the apical surface of air-way and submucosal gland epithelial cells.The lack of receptors for adenovirus or adeno-associated virus serotypes, initially employed,precluded significant gene transfer into thesetarget cells. However, there are receptorsfor alternative adeno-associated virus (AAV)

serotypes, and these have been employed fordelivery of CFTR cDNA to cultured cellsand the airways of mice and macaques (153,154). The limited size of the AAV genome alsoconstrains the size of the expression cassettethat can be incorporated. Both the CFTRcDNA and enhancer/promoter elements havebeen minimized. In one approach, N-terminaltruncations of the CFTR cDNA, removingeither the first 117 or 264 amino acids ofthe protein, were employed (154). Althoughthese N-terminally truncated CFTRs appearcapable of generating chloride channel ac-tivity (155), their ability to mature and traf-fick to the surface of human cells is expectedto be compromised. In an alternative strat-egy, in addition to minimizing the length ofa CMV promoter, an intron and an interven-ing sequence element, a short region of the Rdomain not essential to maturation or func-tion, were deleted (153). Exposure to AAV5used for packaging of this construct impartedtransepithelial chloride transport on CF pri-mary epithelial cell cultures.

These and other improvements to AAV-based vectors provide hope that greater clini-cal benefit may occur in future clinical trials.Although host immune response to AAV vec-tors appears to be less of a problem than withother viral gene transfer vectors, Limberisand colleagues (156) have recently observeda T cell response to the CFTR protein it-self in mice to which CFTR cDNA was de-livered with a viral vector. Although there islikely to be tolerance to the CFTR in mostpatients who are candidates for gene ther-apy, this would presumably not be the casewith infrequent homozygous null mutations;hence, CFTR-specific T cell responses wouldneed to be monitored in such cases. Still ofmore immediate concern, however, are prac-tical issues of efficient delivery and persistentexpression in a setting in which pathologicalchanges, including inflammation and infec-tion, probably have already occurred. Theseissues still need to be effectively dealt with inparallel with the ongoing improvements andinnovation in vector development (157).

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Suppression of PrematureStop Mutations

Like gene replacement therapy, promotion ofread-through translation of transcripts withnonsense mutations is a conceptually simpleapproach to therapy for genetic diseases, suchas CF. Nearly 10% of patients possess an al-lele with a premature stop codon in the CFTRcoding sequence. However, in some popula-tions, such as of those of Ashkenazi Jewishorigin, this is the predominant type of mu-tation. The lack of CFTR protein generallyresults in a severe disease phenotype (158).Amino glycoside antibiotics can enable low-frequency insertion of an amino acid at the po-sition of the stop codon, resulting in synthesisof a small amount of full-length protein (159).This has been observed to occur to some ex-tent in cultured cells (160) and in transgenicmice expressing common CF-associated stopmutations (161).

Gentamycin application directly to thenasal mucosa of such patients also yielded en-couraging results, causing a significant de-crease in transepithelial potential differenceand an apparent increase in the full-lengthCFTR as detected by immunocytochemicalstaining (162). This study focused on a singlestop mutation common in the Israeli popula-tion (W1282X), and the nasal potential dif-ference measurements, all made at a singlecenter, were statistically quite uniform. In-terestingly, this truncation in the middle ofNBD2 is compatible with conformationalmaturation and a low level of chloride channelfunction (47, 163). A more recent multicen-ter study of patients with different nonsensemutations did not detect significant improve-ments of nasal potential difference or changesin the amount or localization of CFTR pro-tein in similarly treated nasal epithelial cells(164). There were several technical differ-ences in the two studies that could contributeto the apparently disparate results, but theoutcome is suggestive that truncations remov-ing more than just NBD2 from the proteinmay be less responsive to these aminoglyco-

sides. However, effectiveness even in patientswith the W1282X mutation alone would rep-resent a highly significant accomplishment iflong-term delivery of an appropriate com-pound, producing clinical benefit without ma-jor side effects, can be achieved. This ap-proach is illustrative of the likely evolution ofpatient- or population-specific therapies forCF where different treatments will be effec-tive for different genotypes.

Restoration of CFTR Foldingand Function

Because �F508- and other CFTR-processingmutant proteins are synthesized and ca-pable of functioning under certain condi-tions, there are intensive efforts to identifysmall molecules that can promote process-ing. High-throughput screens have employedassays that measure CFTR-mediated halideefflux from cells using a halide-sensitive flu-orescent protein (165) or CFTR-dependentchanges in membrane potential (166). A strat-egy employed by Verkman and collaborators(167) has proceeded through several stagesand proven very effective thus far. In thefirst stages, both inhibitors (168) and acti-vators (169) of the wild-type CFTR chan-nel activity were identified. In addition toverifying the utility of the assay for screen-ing, the two classes of inhibitors discoveredmay potentially block the chronically acti-vated CFTR in secretory diarrheas (168) andalso help confirm that membrane permeabil-ity changes, caused by positively acting mod-ulators of the mutant CFTRs, are actuallyoccurring through CFTRs. The two types ofinhibitors apparently have different modes ofaction: Thiazolidinones cause a greatly pro-longed channel closed state (168), and glycinehydrazides (170) strongly reduce the meanopen time.

Cells expressing the �F508 CFTR werefirst screened using the same assay for so-called potentiators, compounds that could in-crease the activity of the mutant channel, par-tially rescued by culture at low temperature

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(171, 172). Different classes of effective com-pounds able to activate several disease-causingmutants as well as �F508 were identified, in-cluding tetrahydrobenzothiophenes, sulfon-amides, and phenylglycines. In the highly sig-nificant next step, �F508 CFTR-expressingcells were exposed to test compounds forlonger periods (∼18 h) during which suf-ficient co- and posttranslational processing,transport to, and accumulation at the cellsurface could occur (89). Several classes ofcompounds, achieving this end at micro-molar concentrations or less, were found.Among these were bisaminomethylbithia-zoles, aminoarylthiazoles, and quinazoliny-laminopyrimidinones, which over a period ofseveral hours have a similar effect as growthof cells at 27◦C. Rescue was confirmed by theappearance of CFTR-mediated current andmature proteins at the cell surface. Some cor-rective effect was detected in differentiatedbronchial epithelial cells expressing �F508.Structure-activity relationship studies and op-timization of the lead compounds and scaf-folds identified should yield compounds ap-propriate for clinical trials.

A parallel screening and drug developmentprogram by another group has also yieldedboth potentiator and corrector compounds(166). A quinazolinone scaffold and deriva-tives from the later group have been fairly ex-tensively characterized in cell systems (173).One of these, VRT-325, causes partial matu-ration of �F508 and some other misprocess-ing mutants, but is nonspecific, also acting onthe related P-glycoprotein multidrug trans-porter (174) and unrelated hERG potassiumchannels (166). It is also highly hydropho-bic and toxic to cells. However, additionalcompounds more suitable for optimization asdrug candidates are in development. A pyra-zole compound, VRT-532, also identified inthis program, potentiated the activity of res-cued �F508. Thus, the progress of these twogroups has provided proof of principle andvalidated approaches for the discovery of smallmolecules to combat misprocessing and dys-

function of the mutant CFTRs by employ-ing high-throughput cell-based screening ofCFTR function. Recently, additional com-pounds, able to promote the appearance ofthe �F508 CFTR protein at the cell surface,have also been reported (175), as have a va-riety of compounds selected arbitrarily or insmall biased screens (176).

The modes of action of the effective po-tentiators and correctors identified so far havenot yet been established, and there is no di-rect evidence that they interact directly withthe CFTR. However, interesting observationsfrom the Clarke laboratory (177) have indi-cated that some pairs of the compounds haveadditive corrective effects, suggesting differ-ent sites of action. Furthermore, one mem-ber of this corrector pair was originally iden-tified as a potentiator, indicating that someagents may have both effects. Together withits specificity of action on the CFTR, but noton P-glycoprotein, this provides suggestiveevidence of direct binding to the CFTR.

Not surprisingly, many of the smallmolecules turned up in corrector screens arevery hydrophobic, having entered cells to gainaccess to the ER. As mentioned, the VRT-325corrector has this property, making it fairlytoxic to cells and nonspecific as well as in-fluencing other membrane proteins. Never-theless, its apparent action on the MSDs ofthe CFTR is quite informative (178), con-sistent with the facts that the �F508 muta-tion in NBD1 disrupts folding of the MSDs(47, 99) and that rescue can be achieved byovercoming this effect. By contrast, at leastsome potentiators have been reported to actat the level of the cytoplasmic NBDs (179).Such agents if able to repair the functionaldefect caused by an NBD mutation, such as�F508, and in so doing also prevent the sec-ondary MSD disruption, might be preferableto membrane active agents for several reasons.The main disadvantage of the latter group,in addition to their possible toxicity and lackof selectivity, could be that while circumvent-ing quality control they may leave functional

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regulation uncorrected. Moreover, becausethere are redundant quality control systemsdetecting the state of both membrane and cy-toplasmic domains (99), it will be necessaryto correct changes in both with individual orcombined agents.

Although there are suggestions that smallamounts of �F508 protein may normallyreach the cell surface in some patients (180),it is unlikely that this is sufficient to pro-vide normal function even if it were fully ac-tive. Therefore, most effort is appropriatelyfocused on overcoming ER retention andgetting misprocessed mutants to the plasmamembrane. However in addition to a kineticdefect in folding and assembly, the Phe508deletion results in a thermally unstable pro-tein (111, 136). Both the functional and bio-chemical half-lives of the rescued mutantprotein are greatly shortened. Thus, ideal cor-rective compounds need to stabilize the pro-tein as well as restore folding and function.It is known that the folding yield and sub-sequent stability of CFTR variants are notstrictly coupled (108) and that the VRT-325corrector caused only a partial extension of thelifetime of the �F508 protein that it rescued(166). Another corrector appeared unable tostabilize the surface mutant protein that it hadfreed from ER retention (89). Cellular manip-ulations, such as inhibition of endocytosis, arecapable of stabilizing the �F508 CFTR at thecell surface (181), but these types of unspecificmanipulations are impractical therapeutically.To be entirely effective, chemical agents indi-vidually, or in combination, will have to cor-rect folding, assembly, function, and stabilityin a selective and nontoxic manner. Althougha tall order, this now appears to be withinthe combined drug development capabilitiesof academic and industrial laboratories.

Modulation of Other EpithelialIon Transport Pathways

The CFTR provides a crucial regulatory stepin epithelial anion (Cl−, HCO−

3, and perhapsothers) permeability. Its complex regulatory

properties enable it to gauge the level of secre-tion or reabsorption required in different ep-ithelial tissues. However, there are additionalanion channels in at least some of these epithe-lia, which can and do contribute to secretion.For example in the crucial airways, calcium-activated chloride channels, which can be acti-vated by purinergic receptors, provide poten-tial alternative pathways to the CFTR (182,183). Nucleotide analogue agonists of thecoupled P2Y2 receptors are currently in clini-cal trials (184). Also contributing to the debil-itating dehydration of the airway surface is thefact that Na+ absorption continues unabatedin the face of inadequate Cl− secretion (72).Therefore, Na+ channel inhibitors have thepotential to ameliorate this situation, and theclassic ameloride inhibitor has been observedto do this in the short term (185). More po-tent and longer-lived ENaC inhibitors havebeen developed and are also in clinical trials(186). Thus, judicious dosing with alternativeanion channel activators and ENaC inhibitorsor other agents that intercede in the biochem-ical and cellular pathways that control themmay be able to partially mimic the role of theCFTR.

The feasibility of such non-CFTR-basedapproaches is illustrated by the effects ofadding osmotically active agents to the air-ways (187). The increased water that followsosmotically with the addition of hypertonicsaline causes increased mucociliary clearanceand improved lung function (188). Althoughrestoration of normal CFTR function by anymeans is clearly the ultimate objective, thesealternative methods of generating a more nor-mally balanced epithelial surface hydrationalso offer extremely important approaches tocombating CF lung disease.

CONCLUSIONS AND OUTLOOK

The CFTR has provided a focus for an everbroadening array of studies of CF and a spec-trum of less severe “CFTR-opathies.” Muchhas been learned, but there remain many cru-cial unresolved issues. A frequently expressed

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frustration in the field is that defects in theprimary function of the CFTR do not seemto link simply to the myriad pathophysio-logical features of the disease. This some-times leads to proposals that the protein musthave alternate or additional functions. De-termining whether this is the case, or if thelinkages with downstream effects have notyet been dissected out, can be quite chal-lenging. Cl− is quantitatively the predomi-nant permeant anion. Yet HCO3

− is of greatimportance, and its role in mucin process-ing is under intense investigation. Other an-ions, including SCN−, may be importantin the innate immunity of the airway. Themechanisms underlying the interplay betweenCFTR- and ENaC-mediated Na+ permeabil-ity in airway epithelia have yet to be re-solved and are necessary to fully understandhow ion and liquid homeostasis is maintainedthere.

The CFTR protein itself has revealed un-expected and fascinating properties, but itsmechanism of action remains incompletelyunderstood. This is partly because of diffi-culties in reproducibly obtaining large sta-ble preparations of purified and reconstitutedprotein for rigorous characterization of itsenzymology, channel activity, and physicalproperties. The 3D structure at atomic reso-lution must overcome several technical chal-lenges. However, the resolution obtained byelectron crystallography of both the wild-type

and important variants such as �F508 willbe improved and, in combination with bioin-formatic and computational methods, yielduseful structural models. The focus of thesemodels on alterations in intermediate foldingstates or perturbed interdomain associationsmay enable rational structure-based drug de-sign. Because both the dynamics and disorderinherent in the CFTR are among the imped-iments to X-ray crystallography, it is likelythat continued major advances will be madein NMR studies of the type that have recentlyprovided deeper insight into the action of theunique R domain.

As a firmly established therapeutic targetin CF (and a spectrum of less severe clini-cal conditions) mutant CFTR correction canbe pursued by all available experimental high-throughput strategies such as whole-genomesiRNA and others. The recent demonstrationthat quantitative downregulation of a specificHsp 90 cochaperone could provide partial res-cue of �F508 provides encouragement thatadditional subtle manipulations of the nascentCFTR chaperome may provide a means forgeneration of sufficient mature protein. Cur-rently, there are indications of a possible re-naissance in gene therapy efforts in CF, andsignificant advances may be anticipated. Theway forward is persistence in the pursuit ofthe defective mechanisms that must be over-come or supplanted to alleviate the symptomsof this disease.

SUMMARY POINTS

1. Although influenced by other environmental and genetic factors, CF may still beconsidered a single-gene defect disease. Therefore, understanding and restoration ofthe function of the CFTR are reasonable approaches to therapy.

2. As an ion channel, the CFTR’s distinguishing feature is its ability to hydrolyze itsbound ligand rather than respond to changes in bulk ligand concentrations as is doneby all other known ligand-gated channels.

3. Essential for utilization of the basic ABC transporter architecture as an ion channel isthe precisely graded control of gating by the phosphorylation state of multiple sitesin the unique disordered R domain.

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4. A large number of known mutations have been detected in CF patients, so therapeuticstrategies can be tailored to these mutation types.

5. As a complex multidomain membrane protein, the CFTR is assembled inefficientlyduring biosynthesis, and many mutations, including �F508, present in ∼90% ofCF patients, are unable to mature conformationally and succumb to quality controlmechanisms in the ER and endocytic compartments.

6. First generations of small molecules that can rescue these processing mutants andaugment channel function have been identified, and extensive efforts are being devotedto further the discovery and development of effective pharmaceuticals.

7. There is also ongoing progress in gene replacement methodologies, which will beeffective for all mutation types, and treatments to suppress nonsense mutations andmanipulate other ion transport pathways, which influence hydration of epithelial sur-faces to either mimic the CFTR or compensate for its absence.

FUTURE ISSUES

1. The molecular mechanism of action of CFTR is still far from completely resolved.The specific features of the protein that enable it to operate as an ion channel whileother proteins, of generally similar structure, perform active transport need to beclarified. How similar or different are the allosteric coupling events between NBDsand MSDs that drive active transport and those that determine the gating state ofthe CFTR channel? What is the molecular structure of the CFTR anion selectivepore? How does the unique R domain provide overriding control of the basic ABCtransporter mechanism? High-resolution 3D structures of the CFTR protein at dif-ferent stages of its catalytic and gating cycles as well as different phosphorylationstates will be necessary, but not entirely sufficient, to answer these questions. The dy-namic structural changes underlying functional transitions will have to be discernedfrom complementary approaches including kinetic analyses of CFTR’s enzymatic andsingle-channel activities as well as spectroscopic and computational methods.

2. Success in the development of molecular therapies for CF will require new break-throughs in gene replacement technology, further elucidation of the misfolding andmissassembly of �F508 CFTR, as well as the cellular mechanisms that recognize themutant protein and determine its fate. In addition, there is a need for further progressin understanding and manipulating major phenotypic modifications downstream frommutant CFTR, particularly alterations in the physical properties of mucins.

DISCLOSURE STATEMENT

J.R.R. has commercial interest in a company targeting mutant CFTRs.

ACKNOWLEDGMENT

The author’s laboratory is supported by grants from the NIH and the Cystic Fibrosis Foun-dation. I thank members of the Riordan laboratory for critical reading of the manuscript andAnne Edwards for its assembly.

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LITERATURE CITED

1. Davis PB. 2006. Am. J. Respir. Crit. Care Med. 173:475–822. Quinton PM. 2007. Physiology 22:212–253. Accurso FJ. 2007. Am. J. Respir. Crit. Care Med. 175:754–574. Higgins CF, Haag PD, Nikaido K, Ardeshir F, Garcia G, Ames GF. 1982. Nature

298:723–275. Gerlach JH, Endicott JA, Juranka PF, Henderson G, Sarangi F, et al. 1986. Nature

324:485–896. Gros P, Croop J, Housman D. 1986. Cell 47:371–807. Higgins CF, Hiles ID, Whalley K, Jamieson DJ. 1985. EMBO J. 4:1033–408. Felmlee T, Pellett S, Welch RA. 1985. J. Bacteriol. 163:94–1059. Holland B, Higgins CF, Kuchler K, Cole SPC, eds. 2003. ABC Proteins: From Bacteria to

Man. New York: Elsevier Sci.10. Dean M. 2005. Methods Enzymol. 400:409–2911. Aleksandrov L, Aleksandrov AA, Chang XB, Riordan JR. 2002. J. Biol. Chem. 277:15419–

2512. Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, et al. 2000. Cell 101:789–80013. Karpowich N, Martsinkevich O, Millen L, Yuan YR, Dai PL, et al. 2001. Structure 9:571–

8614. Moody JE, Millen L, Binns D, Hunt JF, Thomas PJ. 2002. J. Biol. Chem. 277:21111–1415. Smith PC, Karpowich N, Millen L, Moody JE, Rosen J, et al. 2002. Mol. Cell 10:139–4916. Chen J, Lu G, Lin J, Davidson AL, Quiocho FA. 2003. Mol. Cell 12:651–6117. Zaitseva J, Jenewein S, Wiedenmann A, Benabdelhak H, Holland IB, Schmitt L. 2005.

Biochemistry 44:9680–9018. Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, et al. 2006. EMBO J.

25:3432–4319. Locher KP, Lee AT, Rees DC. 2002. Science 296:1091–9820. Dawson RJ, Locher KP. 2006. Nature 443:180–8521. Pinkett HW, Lee AT, Lum P, Locher KP, Rees DC. 2007. Science 315:373–7722. Hollenstein K, Frei DC, Locher KP. 2007. Nature 446:213–1623. Rosenberg MF, Callaghan R, Modok S, Higgins CF, Ford RC. 2005. J. Biol. Chem.

280:2857–6224. Rosenberg MF, Callaghan R, Modok S, Higgins CF, Ford RC. 2005. J. Biol. Chem.

280:2857–6225. Awayn NH, Rosenberg MF, Kamis AB, Aleksandrov LA, Riordan JR, Ford RC. 2005.

Biochem. Soc. Trans. 33:996–9926. Lewis HA, Buchanan SG, Burley SK, Conners K, Dickey M, et al. 2004. EMBO J.

23:282–9327. Lewis HA, Zhao X, Wang C, Sauder JM, Rooney I, et al. 2005. J. Biol. Chem. 280:1346–5328. Hegedus T, Riordan JR. 2006. Cent. Euro. J. Biol. 1:29–4229. Dulhanty AM, Riordan JR. 1994. FEBS Lett. 343:109–1430. Dulhanty AM, Riordan JR. 1994. Biochemistry 33:4072–7931. Ostedgaard LS, Baldursson O, Vermeer DW, Welsh MJ, Robertson AD. 2000. Proc. Natl.

Acad. Sci. USA 97:5657–6232. Baker JM, Hudson RP, Kanelis V, Choy WY, Thibodeau PH, et al. 2007. Nat. Struct.

Mol. Biol. 14:738–4533. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, et al. 1989. Science 245:1066–

73

www.annualreviews.org • CFTR Function and Prospects for Therapy 721

Ann

u. R

ev. B

ioch

em. 2

008.

77:7

01-7

26. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by U

nive

rsity

of

Haw

aii a

t Man

oa L

ibra

ry o

n 01

/25/

11. F

or p

erso

nal u

se o

nly.

ANRV345-BI77-28 ARI 1 May 2008 13:16

34. Kunzelmann K. 2001. News Physiol. Sci. 16:167–7035. Lee MG, Wigley WC, Zeng W, Noel LE, Marino CR, et al. 1999. J. Biol. Chem.

274:3414–2136. Venglarik CJ, Schultz BD, Frizzell RA, Bridges RJ. 1994. J. Gen. Physiol. 104:123–4637. Zou X, Hwang TC. 2001. Biochemistry 40:5579–8638. Hanrahan JW, Wioland MA. 2004. Proc. Am. Thorac. Soc. 1:17–2139. Hanrahan JW, Kone Z, Mathews CJ, Luo J, Jia Y, Linsdell P. 1998. Methods Enzymol.

293:169–9440. Hanrahan JW, Tabcharani JA, Becq F, Mathews CJ, Augustinas O, et al. 1995. Soc. Gen.

Physiol. Ser. 50:125–3741. Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, Bridges RJ. 1999. J. Gen.

Physiol. 113:743–6042. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. 2001. Nature 410:94–

9743. Coakley RD, Grubb BR, Paradiso AM, Gatzy JT, Johnson LG, et al. 2003. Proc. Natl.

Acad. Sci. USA 100:16083–8844. Krouse ME, Talbott JF, Lee MM, Joo NS, Wine JJ. 2004. Am. J. Physiol. Lung Cell Mol.

Physiol. 287:L1274–8345. Li C, Ramjeesingh M, Wang W, Garami E, Hewryk M, et al. 1996. J. Biol. Chem.

271:28463–6846. Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC. 2006. EMBO J.

25:4728–3947. Cui L, Aleksandrov L, Chang XB, Hou YX, He L, et al. 2007. J. Mol. Biol. 365:981–9448. Wang W, Bernard K, Li G, Kirk KL. 2007. J. Biol. Chem. 282:4533–4449. Grimard V, Li C, Ramjeesingh M, Bear CE, Goormaghtigh E, Ruysschaert JM. 2004. J.

Biol. Chem. 279:5528–3650. Anderson MP, Berger HA, Rich DP, Gregory RJ, Smith AE, Welsh MJ. 1991. Cell

67:775–8451. Baukrowitz T, Hwang TC, Nairn AC, Gadsby DC. 1994. Neuron 12:473–8252. Gunderson KL, Kopito RR. 1995. Cell 82:231–3953. Ishihara H, Welsh MJ. 1997. Am. J. Physiol. 273:C1278–8954. Hennager DJ, Ikuma M, Hoshi T, Welsh MJ. 2001. Proc. Natl. Acad. Sci. USA 98:3594–9955. Aleksandrov AA, Riordan JR. 1998. FEBS Lett. 431:97–10156. Aleksandrov AA, Chang X, Aleksandrov L, Riordan JR. 2000. J. Physiol. 528(Part 2):259–

6557. Vergani P, Nairn AC, Gadsby DC. 2003. J. Gen. Physiol. 121:17–3658. Berger AL, Ikuma M, Welsh MJ. 2005. Proc. Natl. Acad. Sci. USA 102:455–6059. Vergani P, Lockless SW, Nairn AC, Gadsby DC. 2005. Nature 433:876–8060. Csanady L, Nairn AC, Gadsby DC. 2006. J. Gen. Physiol. 128:523–3361. Aleksandrov AA, Aleksandrov LA, Riordan JR. 2007. Pflugers Arch. 453:693–70262. Locher KP. 2004. Curr. Opin. Struct. Biol. 14:426–3163. Rosenberg MF, Kamis AB, Aleksandrov LA, Ford RC, Riordan JR. 2004. J. Biol. Chem.

279:39051–5764. Ketchum CJ, Rajendrakumar GV, Maloney PC. 2004. Biochemistry 43:1045–5365. Randak C, Welsh MJ. 2003. Cell 115:837–5066. Gross CH, Abdul-Manan N, Fulghum J, Lippke J, Liu X, et al. 2006. J. Biol. Chem.

281:4058–6867. Randak C, Neth P, Auerswald EA, Eckerskorn C, Assfalg-Machleidt I, Machleidt W.

1997. FEBS Lett. 410:180–86

722 Riordan

Ann

u. R

ev. B

ioch

em. 2

008.

77:7

01-7

26. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by U

nive

rsity

of

Haw

aii a

t Man

oa L

ibra

ry o

n 01

/25/

11. F

or p

erso

nal u

se o

nly.

ANRV345-BI77-28 ARI 1 May 2008 13:16

68. Bhaskara V, Dupre A, Lengsfeld B, Hopkins BB, Chan A, et al. 2007. Mol. Cell 25:647–6169. Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, et al. 1995. Science 269:847–5070. Boucher RC. 2007. J. Intern. Med. 261:5–1671. Boucher RC. 2007. Trends Mol. Med. 13:231–4072. Mall M, Grubb BR, Harkema JR, O’Neal WK, Boucher RC. 2004. Nat. Med. 10:487–9373. Gabriel SE, Clarke LL, Boucher RC, Stutts MJ. 1993. Nature 363:263–6874. Di A, Brown ME, Deriy LV, Li C, Szeto FL, et al. 2006. Nat. Cell Biol. 8:933–4475. Painter RG, Valentine VG, Lanson NA Jr, Leidal K, Zhang Q, et al. 2006. Biochemistry

45:10260–6976. Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DWH. 1995. Am. J.

Respir. Crit. Care Med. 151:1075–8277. Pier GB, Grout M, Zaidi TS, Olsen JC, Johnson LG, et al. 1996. Science 271:64–6778. Kowalski MP, Dubouix-Bourandy A, Bajmoczi M, Golan DE, Zaidi T, et al. 2007. Science

317:130–3279. Moskwa P, Lorentzen D, Excoffon KJ, Zabner J, McCray PB Jr, et al. 2007. Am. J. Respir.

Crit. Care Med. 175:174–8380. Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, et al. 1989. Science

245:1073–8081. Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, et al. 1990. Cell 63:827–3482. Kartner N, Augustinas O, Jensen TJ, Naismith AL, Riordan JR. 1992. Nat. Genet. 1:321–

2783. Mall M, Kreda SM, Mengos A, Jensen TJ, Hirtz S, et al. 2004. Gastroenterology 126:32–4184. Kreda SM, Mall M, Mengos A, Rochelle L, Yaskaskas J, et al. 2005. Mol. Biol. Cell 16:2154–

6785. Drumm ML, Wilkinson DJ, Smit LS, Worrell RT, Strong TV, et al. 1991. Science

254:1797–9986. Li C, Ramjeesingh M, Reyes E, Jensen T, Chang X-B, et al. 1993. Nat. Genet. 3:311–1687. Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ. 1992. Nature

358:761–6488. Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ. 1996. Cell Stress Chap-

erones 1:117–2589. Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, et al. 2005. J. Clin. Investig.

115:2564–7190. Ward CL, Kopito RR. 1994. J. Biol. Chem. 269:25710–1891. Lukacs GL, Mohamed A, Kartner N, Chang X-B, Riordan JR, Grinstein S. 1994. EMBO

J. 13:6076–8692. Gelman MS, Kopito RR. 2003. Methods Mol. Biol. 232:27–3793. Ward CL, Omura S, Kopito RR. 1995. Cell 83:121–2794. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR. 1995. Cell 83:129–

3595. Sato S, Ward CL, Kopito RR. 1998. J. Biol. Chem. 273:7189–9296. Loo MA, Jensen TJ, Cui L, Hou Y, Chang XB, Riordan JR. 1998. EMBO J. 17:6879–8797. Loo TW, Bartlett MC, Clarke DM. 2004. J. Biol. Chem. 279:38395–40198. Baldursson O, Ostedgaard LS, Rokhlina T, Cotten JF, Welsh MJ. 2001. J. Biol. Chem.

276:1904–1099. Younger JM, Chen L, Ren HY, Rosser MF, Turnbull EL, et al. 2006. Cell 126:571–82

100. Wang X, Matteson J, An Y, Moyer B, Yoo JS, et al. 2004. J. Cell Biol. 167:65–74101. Pagant S, Kung L, Dorrington M, Lee MC, Miller EA. 2007. Mol. Biol. Cell 18:3398–413

www.annualreviews.org • CFTR Function and Prospects for Therapy 723

Ann

u. R

ev. B

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ANRV345-BI77-28 ARI 1 May 2008 13:16

102. Vij N, Fang S, Zeitlin PL. 2006. J. Biol. Chem. 281:17369–78103. Cyr DM. 2005. Nat. Struct. Mol. Biol. 12:2–3104. Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM. 2001. Nat. Cell Biol. 3:100–

5105. Meacham GC, Lu Z, King S, Sorscher E, Tousson A, Cyr DM. 1999. EMBO J. 18:1492–

505106. Zhang H, Schmidt BZ, Sun F, Condliffe SB, Butterworth MB, et al. 2006. J. Biol. Chem.

281:11312–21107. Gnann A, Riordan JR, Wolf DH. 2004. Mol. Biol. Cell 15:4125–35108. Du K, Sharma M, Lukacs GL. 2005. Nat. Struct. Mol. Biol. 12:17–25109. Parodi AJ. 2000. Annu. Rev. Biochem. 69:69–93110. Thibodeau PH, Brautigam CA, Machius M, Thomas PJ. 2005. Nat. Struct. Mol. Biol.

12:10–16111. Cui L, Aleksandrov L, Hou YX, Gentzsch M, Chen JH, et al. 2006. J. Physiol. 572:347–58112. Zhang F, Kartner N, Lukacs GL. 1998. Nat. Struct. Biol. 5:180–83113. Seibert FS, Loo TW, Clarke DM, Riordan JR. 1997. J. Bioenerg. Biomembr. 29:429–42114. Yang Y, Janich S, Cohn JA, Wilson JM. 1993. Proc. Natl. Acad. Sci. USA 90:9480–84115. Pind S, Riordan JR, Williams DB. 1994. J. Biol. Chem. 269:12784–88116. Wang X, Venable J, LaPointe P, Hutt DM, Koulov AV, et al. 2006. Cell 127:803–15117. Alberti S, Bohse K, Arndt V, Schmitz A, Hohfeld J. 2004. Mol. Biol. Cell 15:4003–10118. Choo-Kang LR, Zeitlin PL. 2001. Am. J. Physiol. Lung Cell Mol. Physiol. 281:L58–68119. Jiang C, Fang SL, Xiao YF, O’Connor SP, Nadler SG, et al. 1998. Am. J. Physiol.

275:C171–78120. Ahner A, Nakatsukasa K, Zhang H, Frizzell RA, Brodsky JL. 2007. Mol. Biol. Cell 18:806–

14121. Marshall J, Fang S, Ostedgaard LS, O’Riordan CR, Ferrara D, et al. 1994. J. Biol. Chem.

269:2987–95122. Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, et al. 2002. Nat. Med.

8:485–92123. Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, et al. 2004. Science 304:600–2124. Berger AL, Randak CO, Ostedgaard LS, Karp PH, Vermeer DW, Welsh MJ. 2005.

J. Biol. Chem. 280:5221–26125. Loo TW, Bartlett MC, Clarke DM. 2004. Biochem. Biophys. Res. Commun. 325:580–85126. Grubb BR, Gabriel SE, Mengos A, Gentzsch M, Randell SH, et al. 2006. Am. J. Respir.

Cell Mol. Biol. 34:355–63127. Farinha CM, Amaral MD. 2005. Mol. Cell. Biol. 25:5242–52128. Harada K, Okiyoneda T, Hashimoto Y, Ueno K, Nakamura K, et al. 2006. J. Biol. Chem.

281:12841–48129. Prince LS, Workman RB Jr, Marchase RB. 1994. Proc. Natl. Acad. Sci. USA 91:5192–96130. Picciano JA, Ameen N, Grant B, Bradbury NA. 2003. Am. J. Physiol. Cell Physiol.

285:C1009–18131. Sharma M, Pampinella F, Nemes C, Benharouga M, So J, et al. 2004. J. Cell Biol. 164:923–

33132. Lukacs GL, Segal G, Kartner N, Grinstein S, Zhang F. 1997. Biochem. J. 328:353–61133. Bradbury NA, Cohn JA, Venglarik CJ, Bridges RJ. 1994. J. Biol. Chem. 269:8296–302134. Webster P, Vanacore L, Nairn AC, Marino CR. 1994. Am. J. Physiol. 267:C340–48135. Howard M, Jiang X, Stolz DB, Hill WG, Johnson JA, et al. 2000. Am. J. Physiol. Cell

Physiol. 279:C375–82

724 Riordan

Ann

u. R

ev. B

ioch

em. 2

008.

77:7

01-7

26. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by U

nive

rsity

of

Haw

aii a

t Man

oa L

ibra

ry o

n 01

/25/

11. F

or p

erso

nal u

se o

nly.

ANRV345-BI77-28 ARI 1 May 2008 13:16

136. Sharma M, Benharouga M, Hu W, Lukacs GL. 2001. J. Biol. Chem. 276:8942–50137. Lukacs GL, Chang X-B, Bear C, Kartner N, Mohamed A, et al. 1993. J. Biol. Chem.

268:21592–98138. Swiatecka-Urban A, Brown A, Moreau-Marquis S, Renuka J, Coutermarsh B, et al. 2005.

J. Biol. Chem. 280:36762–72139. Weixel KM, Bradbury NA. 2001. J. Biol. Chem. 276:46251–59140. Silvis MR, Picciano JA, Bertrand C, Weixel K, Bridges RJ, Bradbury NA. 2003. J. Biol.

Chem. 278:11554–60141. Gentzsch M, Chang XB, Cui L, Wu Y, Ozols VV, et al. 2004. Mol. Biol. Cell 15:2684–96142. Swiatecka-Urban A, Boyd C, Coutermarsh B, Karlson KH, Barnaby R, et al. 2004. J.

Biol. Chem. 279:38025–31143. Swiatecka-Urban A, Talebian L, Kanno E, Moreau-Marquis S, Coutermarsh B, et al.

2007. J. Biol. Chem. 282:23725–36144. Ganeshan R, Nowotarski K, Di A, Nelson DJ, Kirk KL. 2007. Biochim. Biophys. Acta

1773:192–200145. Bates IR, Hebert B, Luo Y, Liao J, Bachir AI, et al. 2006. Biophys. J. 91:1046–58146. Jin S, Haggie PM, Verkman AS. 2007. Biophys. J. 93:1079–88147. Guggino WB. 2004. Proc. Am. Thorac. Soc. 1:28–32148. Thelin WR, Chen Y, Gentzsch M, Kreda SM, Sallee JL, et al. 2007. J. Clin. Investig.

117:364–74149. Gentzsch M, Choudhury A, Chang XB, Pagano RE, Riordan JR. 2007. J. Cell Sci.

120:447–55150. Huang F, Kirkpatrick D, Jiang X, Gygi S, Sorkin A. 2006. Mol. Cell 21:737–48151. Fuchs HJ, Borowitz DS, Christiansen DH, Morris EM, Nash ML, et al. 1994. N. Engl.

J. Med. 331:637–42152. Bonsignore CL. 1998. Pediatr. Nurs. 24:258–59153. Ostedgaard LS, Rokhlina T, Karp PH, Lashmit P, Afione S, et al. 2005. Proc. Natl. Acad.

Sci. USA 102:2952–57154. Fischer AC, Smith CI, Cebotaru L, Zhang X, Askin FB, et al. 2007. Mol. Ther. 15:756–63155. Carroll TP, Morales MM, Fulmer SB, Allen SS, Flotte TR, et al. 1995. J. Biol. Chem.

270:11941–46156. Limberis MP, Figueredo J, Calcedo R, Wilson JM. 2007. Mol. Ther. 9:1694–97157. Copreni E, Penzo M, Carrabino S, Conese M. 2004. Gene Ther. 11(Suppl. 1):S67–75158. Kerem E, Kerem B. 1996. Pediatr. Pulmonol. 22:387–95159. Howard M, Frizzell RA, Bedwell DM. 1996. Nat. Med. 2:467–69160. Bedwell DM, Kaenjak A, Benos DJ, Bebok Z, Bubien JK, et al. 1997. Nat. Med. 3:1280–84161. Du M, Jones JR, Lanier J, Keeling KM, Lindsey JR, et al. 2002. J. Mol. Med. 80:595–604162. Wilschanski M, Yahav Y, Yaacov Y, Blau H, Bentur L, et al. 2003. N. Engl. J. Med.

349:1433–41163. Rowe SM, Rab A, Zsuzsa B, Byram K, Li Y, et al. 2006. Pediatr. Pulmonol. Suppl. 29:227164. Clancy JP, Rowe SM, Bebok Z, Aitken ML, Gibson R, et al. 2007. Am. J. Respir. Cell Mol.

Biol. 37:57–66165. Galietta LV, Jayaraman S, Verkman AS. 2001. Am. J. Physiol. Cell Physiol. 281:C1734–42166. Van Goor F, Straley KS, Cao D, Gonzalez J, Hadida S, et al. 2006. Am. J. Physiol. Lung

Cell Mol. Physiol. 290:L1117–30167. Verkman AS, Lukacs GL, Galietta LJ. 2006. Curr. Pharm. Des. 12:2235–47168. Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, et al. 2002. J. Clin. Investig.

110:1651–58

www.annualreviews.org • CFTR Function and Prospects for Therapy 725

Ann

u. R

ev. B

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nive

rsity

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aii a

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ibra

ry o

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/25/

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or p

erso

nal u

se o

nly.

ANRV345-BI77-28 ARI 1 May 2008 13:16

169. Ma T, Vetrivel L, Yang H, Pedemonte N, Zegarra-Moran O, et al. 2002. J. Biol. Chem.277:37235–41

170. Muanprasat C, Sonawane ND, Salinas D, Taddei A, Galietta LJ, Verkman AS. 2004.J. Gen. Physiol. 124:125–37

171. Yang H, Shelat AA, Guy RK, Gopinath VS, Ma T, et al. 2003. J. Biol. Chem. 278:35079–85172. Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, et al. 2005. Mol.

Pharmacol. 67:1797–807173. Loo TW, Bartlett MC, Wang Y, Clarke DM. 2006. Biochem. J. 395:537–42174. Wang Y, Bartlett MC, Loo TW, Clarke DM. 2006. Mol. Pharmacol. 70:297–302175. Carlile GW, Robert R, Zhang D, Teske KA, Luo Y, et al. 2007. ChemBioChem 8:1012–20176. Noel S, Faveau C, Norez C, Rogier C, Mettey Y, Becq F. 2006. J. Pharmacol. Exp. Ther.

319:349–59177. Wang Y, Loo TW, Bartlett MC, Clarke DM. 2007. Biochem. J. 406:257–63178. Wang Y, Loo TW, Bartlett MC, Clarke DM. 2007. Mol. Pharmacol. 71:751–58179. Moran O, Galietta LJ, Zegarra-Moran O. 2005. Cell. Mol. Life Sci. 62:446–60180. Kalin N, Claass A, Sommer M, Puchelle E, Tummler B. 1999. J. Clin. Investig. 103:1379–

89181. Okiyoneda T, Lukacs GL. 2007. Biochim. Biophys. Acta 1773:476–79182. Stutts MJ, Chinet TC, Mason SJ, Fullton JM, Clarke LL, Boucher RC. 1992. Proc. Natl.

Acad. Sci. USA 89:1621–25183. Knowles MR, Clarke LL, Boucher RC. 1992. Chest 101(3 Suppl.):S60–63184. Deterding R, Retsch-Bogart G, Milgram L, Gibson R, Daines C, et al. 2005. Pediatr.

Pulmonol. 39:339–48185. Hofmann T, Stutts MJ, Ziersch A, Ruckes C, Weber WM, et al. 1998. Am. J. Respir. Crit.

Care Med. 157:1844–49186. Hirsh AJ, Sabater JR, Zamurs A, Smith RT, Paradiso AM, et al. 2004. J. Pharmacol. Exp.

Ther. 311:929–38187. Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, et al. 2006. N. Engl. J. Med.

354:229–40188. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. 2006.

N. Engl. J. Med. 354:241–50189. Callebaut I, Eudes R, Mornon JP, Lehn P. 2004. Cell. Mol. Life Sci. 61:230–42

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Annual Review ofBiochemistry

Volume 77, 2008Contents

Prefatory Chapters

Discovery of G Protein SignalingZvi Selinger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

Moments of DiscoveryPaul Berg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 14

Single-Molecule Theme

In singulo Biochemistry: When Less Is MoreCarlos Bustamante � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 45

Advances in Single-Molecule Fluorescence Methodsfor Molecular BiologyChirlmin Joo, Hamza Balci, Yuji Ishitsuka, Chittanon Buranachai,and Taekjip Ha � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 51

How RNA Unfolds and RefoldsPan T.X. Li, Jeffrey Vieregg, and Ignacio Tinoco, Jr. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 77

Single-Molecule Studies of Protein FoldingAlessandro Borgia, Philip M. Williams, and Jane Clarke � � � � � � � � � � � � � � � � � � � � � � � � � � � � �101

Structure and Mechanics of Membrane ProteinsAndreas Engel and Hermann E. Gaub � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �127

Single-Molecule Studies of RNA Polymerase: Motoring AlongKristina M. Herbert, William J. Greenleaf, and Steven M. Block � � � � � � � � � � � � � � � � � � � �149

Translation at the Single-Molecule LevelR. Andrew Marshall, Colin Echeverría Aitken, Magdalena Dorywalska,and Joseph D. Puglisi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �177

Recent Advances in Optical TweezersJeffrey R. Moffitt, Yann R. Chemla, Steven B. Smith, and Carlos Bustamante � � � � � �205

Recent Advances in Biochemistry

Mechanism of Eukaryotic Homologous RecombinationJoseph San Filippo, Patrick Sung, and Hannah Klein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �229

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Structural and Functional Relationships of the XPF/MUS81Family of ProteinsAlberto Ciccia, Neil McDonald, and Stephen C. West � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �259

Fat and Beyond: The Diverse Biology of PPARγ

Peter Tontonoz and Bruce M. Spiegelman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �289

Eukaryotic DNA Ligases: Structural and Functional InsightsTom Ellenberger and Alan E. Tomkinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

Structure and Energetics of the Hydrogen-Bonded Backbonein Protein FoldingD. Wayne Bolen and George D. Rose � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �339

Macromolecular Modeling with RosettaRhiju Das and David Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �363

Activity-Based Protein Profiling: From Enzyme Chemistryto Proteomic ChemistryBenjamin F. Cravatt, Aaron T. Wright, and John W. Kozarich � � � � � � � � � � � � � � � � � � � � � �383

Analyzing Protein Interaction Networks Using Structural InformationChristina Kiel, Pedro Beltrao, and Luis Serrano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �415

Integrating Diverse Data for Structure Determinationof Macromolecular AssembliesFrank Alber, Friedrich Förster, Dmitry Korkin, Maya Topf, and Andrej Sali � � � � � � � �443

From the Determination of Complex Reaction Mechanismsto Systems BiologyJohn Ross � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �479

Biochemistry and Physiology of Mammalian SecretedPhospholipases A2

Gerard Lambeau and Michael H. Gelb � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �495

Glycosyltransferases: Structures, Functions, and MechanismsL.L. Lairson, B. Henrissat, G.J. Davies, and S.G. Withers � � � � � � � � � � � � � � � � � � � � � � � � � � �521

Structural Biology of the Tumor Suppressor p53Andreas C. Joerger and Alan R. Fersht � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �557

Toward a Biomechanical Understanding of Whole Bacterial CellsDylan M. Morris and Grant J. Jensen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �583

How Does Synaptotagmin Trigger Neurotransmitter Release?Edwin R. Chapman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �615

Protein Translocation Across the Bacterial Cytoplasmic MembraneArnold J.M. Driessen and Nico Nouwen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �643

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Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms,Connected Processes, and DiseasesRoland Lill and Ulrich Mühlenhoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �669

CFTR Function and Prospects for TherapyJohn R. Riordan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �701

Aging and Survival: The Genetics of Life Span Extensionby Dietary RestrictionWilliam Mair and Andrew Dillin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �727

Cellular Defenses against Superoxide and Hydrogen PeroxideJames A. Imlay � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �755

Toward a Control Theory Analysis of AgingMichael P. Murphy and Linda Partridge � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �777

Indexes

Cumulative Index of Contributing Authors, Volumes 73–77 � � � � � � � � � � � � � � � � � � � � � � � �799

Cumulative Index of Chapter Titles, Volumes 73–77 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �803

Errata

An online log of corrections to Annual Review of Biochemistry articles may be foundat http://biochem.annualreviews.org/errata.shtml

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