CFTR Function

ANRV345-BI77-28 ARI 1 May 2008 13:16
ANNUAL
REVIEWS Further CFTR Function
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Annu. Rev. Biochem. 2008.77:701-726. Downloaded from www.annualreviews.org
Department of Biochemistry and Biophysics, Cystic Fibrosis Treatment and Research

by University of Hawaii at Manoa Library on 01/25/11. For personal use only.
Center, 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 Key Words

First published online as a Review in Advance on ABC protein, anion channel, cystic fibrosis, ER quality control,
February 27, 2008
molecular therapy, protein misfolding
The Annual Review of Biochemistry is online at
biochem.annualreviews.org Abstract
This article’s doi: Mutations in the gene coding for the cystic fibrosis transmembrane
10.1146/annurev.biochem.75.103004.142532
conductance regulator (CFTR) epithelial anion channel cause cys-
Copyright c 2008 by Annual Reviews. tic fibrosis (CF). The multidomain integral membrane glycopro-
All rights reserved
tein, a member of the adenine nucleotide-binding cassette (ABC)
0066-4154/08/0707-0701$20.00 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 have
arisen from knowledge of CFTR structure and function as well as
its biosynthetic processing, intracellular trafficking, and turnover.
701
ANRV345-BI77-28 ARI 1 May 2008 13:16
tity in 1938 are extensively described else-

Contents where, including several recent, excellent
concise reviews (1–3). From our present per-
INTRODUCTION . . . . . . . . . . . . . . . . . 702
spective, it is clear that virtually all indi-
CYSTIC FIBROSIS . . . . . . . . . . . . . . . . . 702
viduals diagnosed clinically with CF have
ABC TRANSPORTERS . . . . . . . . . . . . 702
mutations in both CFTR gene alleles. Al-
CFTR STRUCTURE . . . . . . . . . . . . . . 703
though approximately 1500 different disease-
CFTR FUNCTION . . . . . . . . . . . . . . . . 704
associated mutations have been identified,
Primary Anion Channel Function . 705
a single codon deletion, F508, is by far
Mechanism of Channel
the most common and is present on at
Regulation . . . . . . . . . . . . . . . . . . . . 706
least one allele in approximately 90% of the
Secondary Functions . . . . . . . . . . . . . 707
patients in some populations (http://www.
BIOSYNTHETIC PROCESSING
genet.sickkids.on.ca/cftr). Thus, although
AND INTRACELLULAR
there is a wide range in severity and manifes-

TRAFFICKING . . . . . . . . . . . . . . . . . 708
tations of this disease that are dependent on

Synthesis, Conformational
other genetic and environmental influences,
Maturation, and Endoplasmic
mutations in the CFTR gene are the funda-
Reticulum Export . . . . . . . . . . . . . 709
mental cause of the disease. The CFTR gly-
Role of Molecular Chaperones . . . . 711
coprotein, essential in the apical membrane of
Traffic in Endocytic and Distal
epithelial cells to maintain ion and fluid home-
Secretory Pathways . . . . . . . . . . . . 713
ostasis, is unique as it is the only member of
PROSPECTS FOR THERAPY . . . . . 715
the large adenine nucleotide-binding cassette
CFTR Gene Replacement . . . . . . . . 715
(ABC) protein family known to function as an
Suppression of Premature
ion channel. The absence of this precisely reg-
Stop Mutations . . . . . . . . . . . . . . . . 716
ulated anion channel activity results in the fail-
Restoration of CFTR Folding
ure of ionic and water homeostasis on exocrine
and Function . . . . . . . . . . . . . . . . . . 716
epithelial surfaces. This causes accumulations
Modulation of Other Epithelial
of macromolecular secretions, which are de-
Ion Transport Pathways. . . . . . . . 718
hydrated and in an altered physical state. This
CONCLUSIONS AND
occurs in most exocrine tissues but with the
OUTLOOK . . . . . . . . . . . . . . . . . . . . . 718
most serious consequences in the pancreas,
where failure of bicarbonate-rich fluid and en-
zyme secretion impair intestinal digestion and
INTRODUCTION absorption, and in the airways of the lung.
The purpose of this review is to summarize There, viscous mucus accumulations and col-
what has been learned over the past 20 years onization by microorganisms cause damaging
CFTR: cystic about the cystic fibrosis transmembrane con- inflammatory responses and loss of function.
fibrosis
transmembrane
ductance regulator (CFTR) and to consider The lack of a CFTR chloride channel in the
conductance how this knowledge is being applied to the de- sweat duct blocks salt reabsorption, making its
regulator velopment of new therapies for cystic fibrosis elevated concentration in sweat diagnostic of
F508: deletion of (CF). The basic ideas are to consider what the the disease.
phenylalanine at CFTR is, what it does, what is wrong with it
position 508 of the in disease, and what might be done to fix it.
CFTR amino acid ABC TRANSPORTERS
sequence
Members of this very large and ubiquitous
ABC: adenine CYSTIC FIBROSIS family of membrane transporters were first
nucleotide-binding
cassette
The clinical features and history of the dis- identified in entirely separate studies of bac-
ease since its recognition as a discrete en- terial nutrient uptake (4) and of the resistance
702 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
of mammalian tumor cells to antineoplastic the NBDs, with the binding altering their spa-
drugs (5, 6). Sequences of the DNA coding for tial relationship to each other as well as coin-
subunits of the bacterial nutrient importers ciding with molecular rearrangements within
NBD: nucleotide-
(7) indicated that they were constituted by the domains (16–18). Hydrolysis and disso- binding domain
soluble nucleotide-binding and membrane- ciation of products may then enable return
MSD: membrane-
integrated subunits (in addition to a periplas- to the initial unbound state. The nucleotide- spanning domain
mic nutrient-binding subunit). The sequence induced changes in the NBDs may be coupled
CL: cytoplasmic
of the multidrug resistance P-glycoprotein, to conformational alterations in the MSDs, loop
overexpressed in cancer drug-resistant cells, contributing to transport. A possible struc-
revealed the presence of domains similar tural basis of such coupling became evident
to the nucleotide-binding and membrane- from the atomic structures of several complete
spanning subunits of the bacterial nutrient bacterial ABC transporters (19–22). These re-
importers, the bacterial hemolysin toxin ex- veal associations between three-dimensional
porter (8), and Drosophila eye color genes se- (3D) structural elements in NBDs that change
quenced earlier. Other individual transport position on ATP binding and others (so-
proteins sharing these sequence similarities called coupling helices) in cytoplasmic loops
were discovered, including those that con- (CLs) separating membrane-spanning he-
tribute to drug resistance or are mutated in lices. The propagation of these implied
several human genetic diseases (9). molecular motions in NBDs, through the
The family name ABC was applied to coupling helices, to the membrane region may
reflect the presence in all members of contribute to the transmembrane transport
two homologous nucleotide-binding domains event.
(NBDs) (9). Genome sequencing has revealed
the presence of thousands of these domains
with 48 complete ABC proteins in the human CFTR STRUCTURE
genome (10). Although only a few have been High-resolution structures of eukaryotic ABC
characterized biochemically and in membrane transporters have not been determined yet,
transport assays, ABC transporters may be primarily because of limitations in generating
considered transport ATPases as they trans- homogeneous monodisperse preparations of
port substances against a concentration gra- sufficient quality and quantity for large-scale
dient, and their ATPase activities are stim- crystallization trials. The CFTR is especially
ulated by the compound transported. Each demanding in this regard because of its low
transporter exhibits internal symmetry of pri- natural abundance and low level of expression
mary structure with one membrane-spanning in heterologous expression systems in which
domain (MSD) and one NBD in the N- it is fully active functionally. So far, sufficient
and C-terminal halves. In many ABC trans- amounts of the CFTR from mammalian cell
porters, both of the ATP-binding sites are expression systems have been purified to gen-
hydrolytic, whereas in others, including the erate two-dimensional crystalline arrays (23)
human ABCC subfamily to which the CFTR as well as single particles analyzed by electron
belongs, hydrolysis occurs at only one of the microscopy to provide low-resolution 3D
sites (11). structural information (Figure 1). The low-
Understanding of the fundamental duality resolution structure obtained shows strong
in the structure of ABC transporters became similarity to that of P-glycoprotein generated
much clearer when it was found that both of by similar methods (24). Two distinct crys-
the two ATP-binding sites are composite sites tal forms are observed, possibly reflecting dif-
contributed to by residues from each of the ferent nucleotide-bound states. The location
NBDs (12–15). Each complete nucleotide- of the R domain relative to the membrane-
binding site is formed at the interface between associated and nucleotide-binding regions
www.annualreviews.org • CFTR Function and Prospects for Therapy 703

ANRV345-BI77-28 ARI 1 May 2008 13:16
Bipartate
e substructure
rfac
Inte
2 nm
Density ( )
4.0
3.5
3.0
2.5
2.0
Figure 1
CFTR structure by cryoelectron microscopy, dimeric CFTR particles as purified from detergent, and the
CFTR crystallized as a monomer (23). Slices through the 3D structure taken perpendicular to the C2
axis which is tilted slightly toward the viewer and to the left. The interface apparent between the two
monomers in the structure is indicated by the dashed line. The bipartite substructure of the upper region
is indicated by the double arrow in the second panel from the left. The gray surface of the structure is
drawn at a density equivalent to 2σ; above the mean. Color coding of the internal structure revealed by
the slices is as shown on the right. Figure provided by Robert C. Ford.
is indicated by the position of site-specific tive NMR study has recently shown that the
nano-gold labeling (25). Crystals are currently α-helical propensities of stretches of residues
being analyzed by cryoelectron microscopy to adjacent to many of the individual phospho-
obtain higher-resolution structures. A high- rylation sites are reduced when these sites are
resolution structure has been obtained for phosphorylated (32). Importantly, there is a
the isolated nucleotide-binding domain 1 corresponding diminution in the interaction
(NBD1) of the CFTR synthesized in bacteria of several of these stretches with NBD1 on
(26). It has the same basic fold as that of the phosphorylation. However, only the R do-
NBDs of many bacterial ABC proteins deter- main and NBD1 were present in these exper-
mined earlier. Most significantly the Phe508 iments, and R domain phosphorylation may
residue occupies a position on the surface of have structural impact involving other parts of
the wild-type domain, and its absence has only the molecule as well. Although a 3D structure
minor effects on the domain structure (27), as of the full-length CFTR, at sufficient resolu-
indicated in Figure 2. tion to enable clear definition of the R domain,
The R domain, which separates NBD1 and is required to further understand its mech-
MSD2 in the primary structure, is highly un- anism of action, computational studies of its
structured and distinguished primarily by a structure can be informative (T. Hegedus, un-
conserved set of phosphorylation sites, which published observations).
control the activation state of the channel
(28). Whereas this 200-residue region is the
least conserved part of the protein sequence, CFTR FUNCTION
both the relative positions of the phosphory- Despite its architectural similarity to other
lation sites (29) and the order-disorder pat- members of the large ABC transporter family,
tern across the sequence are highly con- the CFTR is functionally distinct in perform-
served and hence functionally significant ing as an ion channel. Apparently conforma-
(T. Hegedus, unpublished observations). tional movements within and between the two
Phosphorylation by protein kinase A (PKA) NBDs are coupled to rearrangements among
PKA: protein kinase
A
reduces the already low α-helical content of membrane-spanning sequences, which, rather
the domain (30, 31), and a highly informa- than changing affinities and sidedness of
704 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
allocrite-binding sites, shift the equilibrium

between the actively gating (open) and quiet
F429
(closed) ion pore. Such a device would gate
F409
and conduct anions continually were it not F433
for the unique R domain, which, until phos-
phorylated by PKA, restrains channel gating.
Thus, the probability of channel opening is
controlled by the extent of R domain phos-
phorylation at multiple sites, reflecting the
balance between protein kinases and the phos-
phatases acting on these sites. In the reabsorp-
tive duct of the sweat gland, for example, PKA
apparently normally dominates, resulting in a
continually active channel favoring maximal

salt recovery. In contrast, in secretory epithe-

lia, the channel may be kept unphosphory-
lated and inactive until stimuli elevating cyclic
G550
AMP make the action of PKA exceed that of F508
R553
the phosphatases.
R555
Primary Anion Channel Function

When first identified at the primary structure
level, it was stated that the CFTR might either
be the chloride channel defective in CF or a
regulator of a separate channel protein (33). Figure 2
Although there are many reports of influences Comparison of nucleotide-binding domain 1 (NBD1) structure of the
wild-type and F508 CFTR. Structures for the wild-type (2BBO, red ) and
of the CFTR on other channels and trans-
F508 (1XMJ, purple) CFTR were aligned, and the indicated mutations
porters (34, 35), strong evidence indicates that aiding crystallization were reverted using PyMol (http://pymol.
the CFTR polypeptide itself mediates a char- sourceforge.net/). The regulatory insertion (404–435), which is missing
acteristic epithelial anion conductance. Prop- from the NBD1 crystal structure, is inserted from a loop database search
erties of its tightly regulated single-channel (SYBL,Tripos Inc., CA). That loop (cyan) contains two α-helixes in good
agreement with hydrophobic patch analysis of this region (189). The
conductance have been thoroughly character-
solubilizing and rescue mutations are numbered and represented by sticks
ized (36–38) and are not detailed here. It be- (light and dark green). Phe508 is colored yellow. The major differences
haves as an ohmic low-conductance channel between the wild-type and mutant domains, exhibited by different
characteristic of that observed in the apical conformations of the two loops, are indicated with thick arrows. Figure
membrane of epithelial cells of the tissues af- provided by Tamas Hegedus.
fected in the disease (39). This pathway for the
movement of chloride ions is required in ei-
ther the secretion or absorption of salt across also reduced pH in the epithelial surface liq-
these epithelia (40). The CFTR also plays an uid of other tissues (43). The failure to alka-
important role in HCO3 − secretion because linize the fluid into which they are secreted
it is permeant to the anion (41) and because it may be an important factor preventing the
probably stimulates Cl− /HCO3 − exchangers normal processing of mucins and contribut-
(42). The most obvious manifestation of the ing to their hyperviscosity (44). Although reg-
loss of this function is the impaired pancre- ulation of ionic balance, hydration, and pH
atic HCO3 − secretion in patients, but there is adjustment are believed to contribute, there

ANRV345-BI77-28 ARI 1 May 2008 13:16
remains an urgent need to further elucidate served in strongly filtered records owing to
the exact mechanisms whereby the wild-type a fast flickering mode too rapid for detec-
CFTR enables the achievement of the normal tion of its fully open state, and kinetic anal-
physical state of mucins as they are secreted. ysis with extended bandwidth provides no ev-
idence of nonequilibrium in gating (53, 54).
Indeed, there is strong evidence that CFTR
Mechanism of Channel Regulation single-channel gating is a reversible, ther-
The CFTR is atypical both as an ion channel mally driven process (55) and that hydroly-
and as an ABC protein, having adopted the basis is not required for gating (56). Gating and
sic ABC transporter structural architecture to hydrolysis do not appear to coincide either ki-
generate a ligand-gated channel whose level netically or energetically. It is now generally
of activity is quantitatively controlled by the agreed that hydrolysis is not involved in chan-
phosphorylation state of its unique R domain. nel opening (57, 58).
Phosphorylation by PKA has only a minor ef- However, studies focusing primarily on
fect on CFTR ATPase activity (45) and ap- the role of the tightening and loosening
parently does not act primarily by influencing of the association between the two NBDs
binding or hydrolysis of the ATP ligand (11) of the CFTR have retained the interpretation
but does promote the association of the two that channel closing is dependent on the en-
NBDs (46). However, this latter effect is not ergy liberated by the cleavage of the terminal
entirely responsible for channel activation by phosphate from ATP (59). This view is based
phosphorylation because that occurs with ac- primarily on the assumption that gating of the
tive C-terminally truncated constructs lacking CFTR is not a thermodynamic equilibrium
NBD2 (47, 48). R domain phosphorylation al- process and the observation of a higher ac-
ters its conformation as well as contacts with tivation enthalpy for closing (∼70 kJ/mol) in
other parts of the protein (30–32, 49). Xenopus oocytes (60) than that (∼20 kJ/mol)
The relationship between binding and hy- determined earlier in planar lipid bilayers
drolysis of the ATP ligand and channel gat- (55). It was suggested that the latter lower
ing also has not yet been entirely elucidated. value may have reflected the measurement and
From a thermodynamic perspective, interpre- analysis of brief flickering transitions within
tations have been somewhat confounded from channel open bursts rather than burst closures
the outset by assumptions about the role of (60), but this is not the case, because the clos-
the ATPase activity of the protein. An anal- ing rates yielding the lower values agree very
ogy with ABC transporters that couple ATP well with the burst durations determined by
hydrolysis to the transport of substances (all- patch-clamp analyses in several other labo-
ocrites) and the observation that hydrolyzable ratories (61). Thus, the discrepancy between
nucleoside triphosphates were preferred lig- the different Ea values for channel closing re-
ands (50) led to suggestions that CFTR chan- mains to be explained. In any case, the esti-
nel opening was energetically coupled to ATP mated free energy input of ∼70 kJ/mol for
hydrolysis, a view that was elaborated by oth- channel closing does not correspond well with
ers (51). Observations of apparent nonequilib- the free energy of ATP hydrolysis, and there
rium in transitions between two open states is no suggestion of the source of the additional
of slightly different conductance were inter- 35 kJ/mol (60). Moreover, in addition to chan-
preted to mean that the gating process was not nel closing occurring in the absence of ATP
in thermal equilibrium (52). This nonequi- hydrolysis, there is also evidence that chan-
librium in gating seemed consistent with the nel closing is not obligatorily dependent on a
previous suggestion that gating was driven by change in the association of the two NBDs.
ATP hydrolysis. However, the apparent sec- That is, channel opening and closing events
ond open conductance state could only be ob- occur in the complete absence of NBD2 (47).
706 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
Thus, even though a model with direct tein rather than an energy source (61). Never-
one-to-one coupling between the formation theless, lending credence to the possible im-
and disruption of tight ATP-mediated inter- portance of adenylate kinase activity of other
NBD association and channel opening and ABC proteins, as well as the CFTR, is the
closing, respectively, fits well with general in- recent description of the presence and role of
terpretations of ABC protein function (16, this activity in the Mre11/Rad 50 DNA repair
62), it does not account for all of the cur- complex (68).
rent data. Initiation of channel opening does
not require tightening of the interface be-
tween NBDs, although this may prolong the Secondary Functions
open state. The closing of channels, which are In addition to its well-established ion chan-
maintained in the open state in this way, is nel function, the CFTR has been proposed to
facilitated by hydrolysis of the NBD2-bound have many other roles and either directly or
ATP and probably also by the disengagement indirectly impacts other cellular proteins and
of the NBD1-bound ATP from the degener- functions (34). Thus, the term pleiotropic has
ate signature sequence of NBD2. Similarly, been applied to the CFTR, but it is uncer-
the gating of the full-length channels in re- tain if this is appropriate. There are certainly
sponse to nonhydrolyzable ATP analogues as downstream effects in addition to altered an-
ligands (56) may also reflect dynamic events ion permeation owing to CFTR function and
at the interface between the NBDs, but in dysfunction. However, it remains a challenge
neither case is the hydrolysis per se an inte- to identify at what level a given downstream
gral part of the gating mechanism. One reason alteration is connected to the CFTR protein
that the precise relationship between the en- itself or the anion conductance that it medi-
zymatic and channel activities of the CFTR ates. From the CF disease perspective, the in-
are not yet more fully understood is that the fluence of the lack of the CFTR on conduc-
enzymatic characterization of the protein is tive epithelial Na+ permeability mediated by
still very limited (11, 45, 63, 64), and there is ENaC channels has received a great deal of
uncertainty as to whether activity assayed with emphasis (69, 70). This is because the con-
the purified, reconstituted protein accurately tinued or enhanced Na+ absorption is pri-
reflects that in its native environment. marily responsible for the dehydration of the
One very interesting proposal is that chan- airway surface, which impairs mucociliary
nel activity may correlate more closely with clearance (71). The importance of this effect
adenylate kinase than ATPase activity of the is emphasized by the fact that transgenic over-
protein (65). Apparent adenylate kinase ac- expression of an ENaC subunit in mouse lung
tivity has been assayed with isolated CFTR mimics the dehydration and mucus plugging,
NBDs, but not with full-length protein which occurs in the CF lung. (72).
(66, 67). Effects of nucleotide mixtures of In addition to serving as a chloride chan-
adenylate kinase substrates and products nel itself, the CFTR may also regulate other
on CFTR activity in patch-clamp exper- chloride channels, but these have not been
iments have been described (65). It has identified at the protein level (73). There is
been suggested that channel gating would be strong evidence that the CFTR has evolved
thermodynamically more compatible with a as an epithelial ion channel and that it is most
dependence on adenylate kinase than ATPase strongly expressed in the salt-transporting ep-
activity (65). However, there is no ther- ithelia of teleosts, elasmobranchs, amphibia,
modynamic incompatibility in a so-called avia, and mammals. Nevertheless, in some
hydrolyzable-ligand-gated process in which assays of CFTR RNA or protein, its pres-
hydrolysis of the ATP ligand is a simple switch ence has been detected in nonpolarized cells,
enabling release of structural strain in the pro- including those of the immune system. For

ANRV345-BI77-28 ARI 1 May 2008 13:16
example, digestion of phagocytosed material tion of the cytokine secretion response neces-
by alveolar macrophages was reported to be sary for clearance (78).
defective in CF because of the absence of In addition to chloride and bicarbonate,
the CFTR from the membrane of the phago- believed to be the main physiological anions
some vacuole where it normally enables acid- passing through the CFTR, prevention of the
ification by providing a counterion pathway passage of other permeant species in CF also
(74). Detection of the CFTR in neutrophils may contribute to the disease phenotype. An
and other hemopoietic cells has also been de- important example from the infectious disease
scribed (75). The possibility of a direct con- perspective is the apparent failure of trans-
tribution of the CFTR to the function of port of the thiocyanate anion onto the surface
immune or inflammatory cells is an impor- of CF airway epithelium (79). This can re-
tant issue that needs to be clearly resolved be- sult in the lack of oxidative generation of an-
cause of the apparent hyperinflammatory sta- tibacterial hypothiocyanite, which may nor-
tus of patients’ tissues prior to infection (76). mally help eliminate Staphylococcus aureus and
However, it is still unclear whether this is due P. aeruginosa.

to inherent changes in the inflammatory cells
themselves or a reaction to the altered state
of epithelial surfaces owing to ionic and fluid BIOSYNTHETIC PROCESSING
changes and mucus accretion. The failure of AND INTRACELLULAR
the CF lung to clear infectious microorgan- TRAFFICKING
isms is generally attributed to impaired mu- When the CFTR gene was initially identified,
cociliary clearance, but in addition, the CFTR the absence of the codon for Phe508 was rec-
has been proposed to serve as a receptor for ognized on the sequencing of cDNA synthe-
Pseudomonas aeruginosa (77). In this mecha- sized from the sweat gland RNA of a patient
nism, binding of the microbe to the receptor (33). This deletion was then detected in ap-
is required for its internalization and stimula- proximately two thirds of CF chromosomes
(80). Heterologous expression of full-length
F508 CFTR cDNA indicated that, although
ΔF508 the glycoprotein was synthesized, it acquired
Wild type
only core and not complex oligosaccharide
chains and failed to be transported to the cell
surface (81). These findings have been con-
firmed and extended in many different mam-
malian cell expression systems. The likelihood
that the biosynthetic arrest of F508 oc-
curred in vivo in patients’ tissues and was not
just an artifact of heterologous overexpres-
sion was supported by the observation that
the protein did not reach the apical membrane
Figure 3 of sweat duct cells in fresh skin biopsies (82).
Localization of the wild-type and F508 CFTR in highly differentiated More recently this has been verified in intes-
primary human epithelial cells. The wild-type CFTR is present and the tine and airway from patients (83, 84). The
F508 CFTR is absent from the apical surface of ciliated airway cells. differential localization of the wild-type and
Fully differentiated primary cultures were adenovirally transduced to mutant protein is illustrated in Figure 3.
express a tagged CFTR protein, and the CFTR was detected in isolated
Expression of F508 in Xenopus oocytes
cells using a mouse antibody recognizing the tag, followed by goat
antimouse Alexa Fluor 288 lgG conjugate. The immunofluorescence is (85) and insect cells (86), which are main-
shown in overlay with differential interference contrast and nuclear tained at lower temperatures than mammalian
staining by TO-PRO 3 iodide. Figure provided by Martina Gentzsch. cells, resulted in at least partial maturation
708 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
and some activity of the mutant protein the cell. These systems deal with most mem-
at the plasma membrane. This suggestion brane and secretory proteins, but the CFTR
that the mutation is temperature sensitive seems to have particular difficulty in achieving
was confirmed by the observation of similar a state that satisfies all criteria for export from
behavior when mammalian cells expressing the endoplasmic reticulum (ER). Wild-type
the mutant protein were shifted to lower tem- versions of other ABC transporters such as P-
perature (87). This partial rescue by temper- glycoprotein and MRP1, for example, mature
ature manipulation presaged similar effects and are exported with nearly 100% efficiency
of treatment of cells with osmolytes, such as compared to the ∼33% for the CFTR in the
glycerol and other so-called chemical chaper- same cells (96). Interestingly, however, dele-
ones (88). This, in turn, encouraged searches tion of the counterpart of Phe508 in each of
for small-molecule rescuing agents that may these ABC proteins has the same effect as in
ultimately be effective in the treatment of the CFTR, which is completely blocked mat-
patients (89). These efforts, which are on- uration and ER export (97).
going, provide hope for the development of The most obvious feature distinguishing
drugs to overcome the misprocessing of mu- the CFTR from other ABC proteins is the
tant CFTRs. R domain, which is largely unstructured (32),
and, therefore, possibly may contribute to the
diminished efficiency of CFTR maturation.
Synthesis, Conformational However, simple deletion of large portions
Maturation, and Endoplasmic of the R domain does not improve this ef-
Reticulum Export ficiency and, in fact, reduces it further (98).
Extensive metabolic labeling and pulse-chase Both intradomain folding and interdomain
experiments on the CFTR heterologously ex- assembly, not unexpectedly, are required to
pressed in a variety of mammalian cells have achieve a state competent for ER export (47,
revealed that 5 to 10 minutes are required for 99) as recognized by COP II constituents,
a complete core-glycosylated CFTR polypep- which coat vesicles that bud from ER exit sites.
tide to be formed (90). Although unglyco- The vesicle coat protein sec24 recognizes a
sylated chains can be synthesized in vitro, diacidic exit code (DAD) of three residues in
none are formed in cells, indicating that N- the NBD1 of the CFTR, which when mu-
glycosylation of the two sites in EL4 occurs tated prevents ER export (100). It is easy to
cotranslationally (81). Transformation to a imagine that F508 or other processing mu-
form in which the high-mannose oligosaccha- tants might conformationally mask this exit
ride chains have been trimmed and extended signal or possibly others and thereby elicit
to large complex chains takes longer, with re- the same effect as inactivating it directly by
sultant higher-molecular-weight species first mutagenesis. However, there are likely addi-
appearing only after nearly a half hour of chase tional dimensions to the recognition mecha-
time (91). Complete transformation requires nisms during the dynamic interplay between
at least two hours, and during this period, progression toward a native state and tagging
only approximately one third of the precursor for a degradative fate. The role of molecular
nascent chains are converted to mature prod- chaperones in this balancing act is outlined in
uct (92). The remainder is ubiquitylated and a section below.
degraded by the 26S proteasome (93, 94). In- When the nascent CFTR was first rec-
deed, ubiquitylation begins before translation ognized as a substrate for ubiquitylation and
is complete (95), and it is now realized that degradation by the proteosome (93), it was
the CFTR is scrutinized by complex quality disappointing to observe that proteasome in-
control systems during its synthesis and as- hibitors did not promote maturation and ER
sembly as well as throughout its lifetime in export. A similar result was recently reported

ANRV345-BI77-28 ARI 1 May 2008 13:16
with the yeast Yor1p ABC protein into which lowed by recognition of incompletely folded
the equivalent of the F508 mutation was in- NBD2 by the cytoplasmic Hsc70/CHIP com-
troduced (101). Thus, inhibition of endoplas- plex. There may be additional contributors
mic reticulum-associated protein degradation to the efficient ERAD of incompletely or
(ERAD) generally does not promote ER ex- aberrantly assembled nascent CFTRs. For ex-
port, although there is one report that this ample, as mentioned in the chaperone sec-
may occur to a limited extent (102). Never- tion below, although the lectin chaperone-
theless, to identify additional points of pos- based conformation-sensing system of the
sible intervention, it is necessary to elucidate ER lumen is not a primary determinant of
the different components of the quality con- CFTR quality control, overexpression of the
trol systems and the sequence in which they mannose-binding EDEM (ER degradation
evaluate the state of the polypeptide and di- enhancing α-mannoside-like protein) does
rect its elimination if it has not yet reached accelerate degradation (107). This pathway,
a mature state. The Cyr laboratory (103) has which is known to direct the retrotransloca-
contributed substantially to the current un- tion, ubiquitylation, and proteolysis of other
derstanding of these events. Having earlier secretory glycoproteins (109), may help en-
demonstrated the involvement of a ubiqui- sure that little unprocessed CFTR accumu-
tylation complex that interacts with Hsc70 lates in the ER.
in F508 degradation (104, 105), this group Because of its dominant contribution to
more recently characterized a second complex disease, most attention has been focused on
containing the E3 ubiquitin ligase, RMA1, the impact of F508 on CFTR folding and
the E2 ubiquitin conjugating enzyme, Ubc6e, assembly. Replacement of the Phe normally
and the membrane-associated Derlin-1 (99). present at this position by each of all other
Their data and those of a parallel study (106) amino acids, rather than simple deletion,
indicate that the latter complex, which also revealed that the presence of many differ-
associates with the AAA-ATPase P97 [the ent residues were compatible with substan-
yeast Cdc48 counterpart (107)], recognized tial folding of the isolated NBD1 domain and
the nascent CFTR early in its synthesis. The the full-length protein (108, 110). The pres-
first CFTR MSD appears to be the target, per- ence of the amino acid backbone regardless
haps initially of Derlin-1 if integration with of the side chain, with the exception of that
the second MSD has not yet occurred. Sig- of tryptophan, enabled refolding of bacteri-
nificantly, this targeting of the F508 CFTR ally expressed NBD1. The requirements for
occurs even when both MSDs have already maturation of a full-length CFTR were some-
been synthesized, implying that the mutation what more stringent with charged residues
may disrupt their assembly as is also indicated and large hydrophobics other than Phe, al-
by other studies (47). lowing very little formation of mature protein.
Although this impact of F508 is observed These observations imply that the side chain
in truncation constructs in which NBD2 is may contribute to an interdomain interaction.
not present (47), the mutation is also known Although the variants that matured showed
to prevent compact folding of that domain in some level of channel activity (108), the Phe
the full-length protein (108). Indeed, Younger aromatic side chain apparently plays a specific
et al. (99) postulate that this may be what role in CFTR channel gating kinetics (111).
is detected by the first quality control com- In light of earlier evidence that the overall
plex, which they characterized involving the conformation of the F508 CFTR is grossly
E2 UbcH5, E3 CHIP, the chaperone Hsc70, altered as reflected by susceptibility to lim-
and its cochaperone, Hdj2. In this scenario, ited proteolysis (112), the finding that the 3D
there is first scrutiny of MSD1 by the Derlin- structure of the F508 NBD was little altered
1/RMA1 membrane-associated complex fol- was somewhat surprising (27). However, this
710 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
was also found to be the case with the isolated Wild type ΔF508
domains containing either F508S or F508R
substitutions (110). In both of these variants, MSD1
only a local surface perturbation around the
508 position was evident, invoking the idea NBD1 R
that the main impact of the mutation may be
in disrupting a crucial interaction between this
small patch on the surface of NBD1 and an-
other part of the molecule.
Consistent with this, Du et al. (108) found
that F508 caused little change in the pro-
2
tease sensitivity of NBD1 but instead caused MSD
NBD2 to become much more sensitive to di-
gestion. This could reflect impairment of a di-

rect NBD1/NBD2 interaction or an indirect

effect of disruption of an NBD1 association
elsewhere in the molecule. F508 has been
found to increase the protease sensitivity of
MSD1, to which it is linearly adjacent, as well
as of NBD2 (47). However, possible sites of
specific interaction of the Phe508 containing
a surface patch of NBD1 have not yet been
identified. Because F508 has essentially the
NBD2
same effect on a maturation-competent C-
terminally truncated form of the CFTR from
which NBD2 is entirely absent as on the full-
length protein (47), it seems that such sites
are likely within the membrane-spanning do-
mains. Sequences in the CLs of both MSD1
and MSD2 may interact with NBD1 (20). Of
the several hundred disease-associated mis-
sense mutants in the CFTR, many causing Figure 4
misfolding and missassembly, several occur in Illustration of wild-type and F508 CFTR domain assembly.
CLs, with an especially high incidence in CL4 Requirements for specific interactions between NBD1 and MSD2 and
NBD2 and MSD1 are emphasized. The Phe508 deletion precludes the first
(113). Some of these CL4 substitutions could
of these interactions, which in turn prevents the normal integration of the
preclude an important interaction between MSDs, and NBD2 is unable to associate with MSD1. Adapted and revised
this loop and NBD1 as suggested schemat- from Reference 47. Figure provided by Lisa Brown.
ically in Figure 4. Thus, many process-
ing mutations may disrupt the crucial struc- wild-type and F508 CFTR, with the major
tural interface between the cytoplasmic and cytoplasmic chaperone Hsp70 (114), and the
membrane-integrated domains of the CFTR. lumen-facing chaperone of the ER membrane
calnexin (115) were detected. Not unexpect-
edly because most of the CFTR mass is consti-
Role of Molecular Chaperones tuted by the large cytoplasmic domains, which
As the nascent CFTR polypeptide is synthe- must be folded and assembled correctly, mul-
sized, it encounters molecular chaperones on tiple cytoplasmic chaperones and cochaper-
both sides of the ER membrane. Initially, in- ones have since been implicated in these pro-
teractions of the immature form of both the cesses (116). An Hsp70 cochaperone of the

ANRV345-BI77-28 ARI 1 May 2008 13:16
Hsp40 class, Hdj-2, was shown to diminish function with an ansamycin drug (96). RNAi
aggregation and promote an early stage of knockdown of the p23 cochaperone, believed
assembly, possibly by recruiting Hsc70 to to be involved in Hsp90 client loading, fur-
the cytoplasmic surface of the ER mem- ther destabilized the mutant nascent chains,
brane (105). The nucleotide exchange factor whereas overexpression had a modest stabi-
HspBP1 aids Hsp70-supported CFTR fold- lizing influence without any promotion of
ing (117). In addition to the positive role of maturation. FKBP8, a member of the im-
Hsc70 and its cochaperones in maturation of munophilin family, involved in folding of
CFTR cytoplasmic domains, they also com- other Hsp90 clients, further reduced the
plex with an E2 ubiquitin-conjugating en- amount of the immature CFTR.
zyme Ubc H5 and the E3 ubiquitin ligase Shifting the level of the Ahal cochaperone,
CHIP to direct ubiquitylation, which targets which stimulates Hsp90 ATPase activity, in
the nascent CFTR for proteasomal degra- contrast to p23, which inhibits activity, had
dation (104). Although the nascent F508 a more pronounced impact on levels of both
CFTR has a somewhat greater tendency than immature and apparently mature F508 pro-
the wild type to participate in these Hsc70- teins. Whereas Ahal overexpression dimin-
based complexes, neither simple up- or down- ished the amounts of both forms, reduction
regulation of the chaperone significantly proof the cochaperone level with RNAi increased
motes maturation of the mutant (118). A drug both, resulting in some of the functional
with some ability to counter Hsp70 inter- CFTR at the cell surface. This provides proof
actions also had minimal if any effect on of principle that modulation of the nascent
F508 maturation (119). The fact that a CFTR chaperone environment can provide a
large proportion of wild-type nascent chains degree of rescue of the F508 CFTR. It is in-
undergo a similar set of interactions and a teresting that this has been possible with the
degradative fate probably means that very Hsp90 network, which is dedicated to altering
fine-tuned manipulation of many of these conformations of proteins that have already
steps would be required to shift the folding reached a basal folded state. Thus, the CFTR
yield of the mutant to a level nearer the wild is a member of the somewhat selective Hsp90
type. clientele. Significantly, related ABC proteins,
Significantly, progress toward that end has including P-glycoprotein and multidrug resis-
recently been made by manipulations of the tance protein 1, are not dependent on Hsp90
other major cytoplasmic chaperone complex, for their maturation and stability (96). Thus,
downstream from Hsc70, the Hsp90 sys- there is an apparent correlation between the
tem (116). Indeed, earlier work showed that inefficient maturation of the CFTR and its
the Hsp90 inhibitor geldanamycin completely dependence on Hsp90.
prevented the maturation of both the wild- However, the nascent CFTR has been
type and F508 CFTR, resulting in the rapid shown to interact with other cytoplasmic
proteasomal degradation of immature nascent chaperones in addition to the major Hsp70-
chains (96). A detailed proteomic analysis of and Hsp90-based systems, and it is con-
CFTR-binding partners revealed a predomi- ceivable that their manipulation might also
nance of known members of the Hsp90 chap- augment maturation of the mutant CFTR.
erone complex, especially in association with Examples include the Hdj-2-related cysteine-
F508 (116). Several of these members were string protein, which seems to interact with
up- and downregulated to test whether more both the ER and post-Golgi forms of the
subtle modulation of this network might pro- CFTR (106), and small Hsps, including αA-
mote CFTR maturation or stability, which is crystallin, which may contribute to ERAD of
completely disrupted by inhibition of Hsp90 the misfolded CFTR (120).
712 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
Multiple cytoplasmic chaperones and expressing cells (116). These interactions and
cochaperones act coordinately and sequen- their possible roles of nascent CFTR process-
tially in appraising and influencing the as- ing remain to be explored further.
CCV: clathrin-
sembly of the large CFTR domains at the coated vesicle
cytoplasmic surface of the ER; however, the
influence of luminal ER chaperones is less Traffic in Endocytic and
clear. Both calnexin and calreticulin bind the Distal Secretory Pathways
core N-linked oligosaccharide chains on the The population of wild-type CFTR
fourth extracytoplasmic loop (115) as does molecules that are exported from the
EDEM (107). Although these lectin chaper- ER and reach the Golgi and post-Golgi
ones together with key glycosidases and gly- compartments is quite stable with a half-life
cosyl transferases constitute a conformation- of approximately 16 h (90, 91). However,
sensing mechanism that determines the fate of the cell surface CFTR pool was very rapidly
many glycoproteins during biogenesis (109), internalized at a rate of approximately 10%

this is apparently not a rate-limiting pathway per minute (129). Because the biosynthetic
for the CFTR. This can be concluded be- rate is very much less than that, this indicated
cause the wild-type protein can be exported that the internalized protein must be recycled
from the ER entirely without glycosylation, to the surface. This was shown to be the
whereas unglycosylated F508 still cannot case in various cell types using different
(121). methodologies (130, 131). The “in” route has
Nevertheless, there have been several at- been extensively characterized and appears to
tempts to promote “release” of the mutant follow, at least primarily, the clathrin-coated
from the ER by perturbing interactions with vesicle (CCV) endocytic pathway (132). The
the membrane-bound calnexin. Because it “out” route is beginning to be better under-
and its luminal counterpart, calreticulin, are stood (see below). The CFTR was detected in
calcium-dependent chaperones, thapsigarin isolated CCVs (133) and visualized in endo-
treatment to deplete ER calcium was reported somes by immunocytochemistry (134). The
to cause some maturation (122) as was another major functional implications of this entry
agent, curcumin (123). Although the latter and exit cycle are with respect to regulation
compound may interact with the CFTR (48, of the amounts of cell surface CFTR activity
124) as with many other proteins, an ability to and the fact that it is strongly perturbed by
promote F508 maturation and stability has the F508 mutation. Lukacs and coworkers
not been generally confirmed (125) nor has an (132) observed a phosphorylation influence
influence on the interaction between calnexin on clathrin-dependent CFTR endocytosis,
and the nascent CFTR (126). Knockdown of and some investigations have emphasized
calnexin by RNAi also did not elicit F508 the role of a shift in the balance between
maturation (127). Calreticulin, which is trans- the plasma membrane and intracellular
ported from the ER to the cell surface, has vesicular pools by PKA phosphorylation
been reported to promote CFTR transport to (135). However, the extent to which this
that location where it is rapidly endocytosed mechanism contributes to epithelial PKA-
and degraded (128). stimulated chloride secretion compared to
Even though the luminally exposed loops the activation of individual CFTR channels
between membrane-spanning sequences of already in the plasma membrane is not yet
the CFTR are quite short, additional ER lu- clear.
minal chaperones including GRP78, GRP75, What is clear is that when F508 and
reticulocalbin, and calumenin were iden- other processing variants of the CFTR have
tified in immunoprecipitates from CFTR- reached the plasma membrane, either in cells

ANRV345-BI77-28 ARI 1 May 2008 13:16
grown at reduced temperature or because tor to drive directional movement of CCVs

of genetic or pharmacological manipulation, (142), whereas myosin Vb may act in an
they are thermally unstable (111, 136) and analogous fashion in trafficking from recy-
again very rapidly removed from the surface cling endosomes back to the plasma mem-
(137). This reduction in surface steady-state brane (143). In both cases, filamentous actin
amount has been attributed to accelerated assemblies are required. Disruption of these
endocytosis in one study (138) and to fail- assemblies by several different means has
ure of recycling in another (131). The differ- recently been shown to influence both inter-
ence may reflect the different cell types used, nalization and recycling of the CFTR (144).
but it will be important to resolve this is- However, the net effect was a large reduc-
sue in the fully differentiated epithelial cells tion in the size of the cell surface CFTR
of the tissues most affected by the disease. pool, apparently because of augmented inter-
Binding of tyrosine- and dileucine-based sig- nalization. The later effect was interpreted
nals in the CFTR C-terminal extension to as the result of freeing of a cytoskeletally

the μ 2 subunit of the AP-2 clathrin adap- constrained plasma membrane pool, which
tor complex initiates clathrin-mediated endo- could be recruited more readily into clathrin-
cytosis (139). Notwithstanding the fact that coated pits (144). It is not clear how to rec-
F508 and other mutations impacting CFTR oncile this increased internalization on dis-
structure cause enhanced clearance from the ruption of the actin cytoskeleton if it is
cell surface, introduction of endocytic motifs required in the endocytosis-promoting action
elsewhere in the protein than the C termi- of myosin VI (142). Populations of CFTR
nus apparently can also augment endocytosis molecules constrained in their mobility have
(140). been demonstrated in independent studies us-
In addition to trafficking between the ing different methodologies (145, 146). Con-
plasma membrane, early endosomes, and re- nections of the CFTR to the actin cytoskele-
cycling endosomes, the CFTR also may pass ton may be mediated by both PDZ-domain
into late endosomes, multivesicular bodies, proteins binding at the C terminus (147) and
and lysosomes for degradation, and indeed filamin at the N terminus (148). Internaliza-
the latter routing is the ultimate fate of the tion and transport to lysosomes for degrada-
endocytosed F508 CFTR (141). Details of tion are promoted when filamin binding is
the recognition mechanisms that are able to prevented.
distinguish the wild-type from the mutant The entire significance of the extensive
CFTR and thereby determine the directions trafficking and scrutiny of the CFTR in the
taken are not yet entirely understood, but the endocytic and post-Golgi compartments may
Lukacs laboratory (131) has demonstrated the not yet be fully appreciated. It clearly provides
involvement of ubiquitylation and other fac- additional mechanisms of quality control be-
tors that participate in routing of the ubiqui- yond the apparently very stringent ones ap-
tylated substrate. This ubiquitylation and its plied at the ER during synthesis and assembly.
recognition appear responsible for the mu- There is evidence that the mutant CFTR can
tant protein proceeding to lysosomal degrada- impact the trafficking of other cellular con-
tion, using an Rme-1-dependent mechanism, stituents, including lipids, if it gains access
rather than being recycled like the wild type to the distal compartments (149). However,
(130). distal quality control may provide an elab-
The actin-based cytoskeleton plays a ma- orate mechanism to control the turnover of
jor role in the endocytic trafficking of the the mature ion channel. Perhaps, changes in
CFTR as it does of other internalized cargo. the molecule, which may occur as it ages in
Myosin VI participates in endocytic uptake, an environment probably not perfectly pro-
perhaps by providing an actin-binding mo- tected from damaging chemical species, are
714 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
continuously monitored for disposal. In this serotypes, and these have been employed for
way, either genetic or environmental per- delivery of CFTR cDNA to cultured cells
turbations would be similarly handled by a and the airways of mice and macaques (153,
highly evolved mechanism, probably sharing 154). The limited size of the AAV genome also
features with systems that distinguish occu- constrains the size of the expression cassette
pied and unoccupied cell surface receptors that can be incorporated. Both the CFTR
(150). cDNA and enhancer/promoter elements have
been minimized. In one approach, N-terminal
truncations of the CFTR cDNA, removing
PROSPECTS FOR THERAPY either the first 117 or 264 amino acids of
the protein, were employed (154). Although
In addition to advancing the understanding
these N-terminally truncated CFTRs appear
of several aspects of human genetics and the
capable of generating chloride channel ac-
molecular and cellular biology of epithelial
tivity (155), their ability to mature and traf-

ion and fluid homeostasis, knowledge of the
fick to the surface of human cells is expected

CFTR gene and protein has provided new
to be compromised. In an alternative strat-
opportunities to develop rational therapeu-
egy, in addition to minimizing the length of
tic approaches to the treatment of CF. Those
a CMV promoter, an intron and an interven-
currently in progress include CFTR gene re-
ing sequence element, a short region of the R
placement, suppression of nonsense muta-
domain not essential to maturation or func-
tions, restoration of folding and function of
tion, were deleted (153). Exposure to AAV5
mutant CFTR channels, activation of alter-
used for packaging of this construct imparted
native chloride channels, inhibition of sodium
transepithelial chloride transport on CF pri-
absorption, and other ionic and osmotic ma-
mary epithelial cell cultures.
nipulations to normalize fluid and bicarbon-
These and other improvements to AAV-
ate levels on epithelial surfaces. These newer
based vectors provide hope that greater clini-
strategies are intended to complement current
cal benefit may occur in future clinical trials.
treatments, which combat mucus viscosity and
Although host immune response to AAV vec-
bacterial infection (151, 152).
tors appears to be less of a problem than with
other viral gene transfer vectors, Limberis
and colleagues (156) have recently observed
CFTR Gene Replacement a T cell response to the CFTR protein it-
Efforts to develop gene replacement ther- self in mice to which CFTR cDNA was de-
apy for CF have been ongoing since shortly livered with a viral vector. Although there is
after the gene was discovered. Both non- likely to be tolerance to the CFTR in most
replicating viral vectors and DNA-lipid com- patients who are candidates for gene ther-
plexes have been employed but without defini- apy, this would presumably not be the case
tive evidence of persistent functional efficacy. with infrequent homozygous null mutations;
However, substantial progress has been made hence, CFTR-specific T cell responses would
in understanding the limitations to the suc- need to be monitored in such cases. Still of
cess of the early attempts. The target of the more immediate concern, however, are prac-
aerosolized vector is the apical surface of air- tical issues of efficient delivery and persistent
way and submucosal gland epithelial cells. expression in a setting in which pathological
The lack of receptors for adenovirus or adeno- changes, including inflammation and infec-
associated virus serotypes, initially employed, tion, probably have already occurred. These
precluded significant gene transfer into these issues still need to be effectively dealt with in
target cells. However, there are receptors parallel with the ongoing improvements and
for alternative adeno-associated virus (AAV) innovation in vector development (157).

ANRV345-BI77-28 ARI 1 May 2008 13:16
Suppression of Premature sides. However, effectiveness even in patients

Stop Mutations with the W1282X mutation alone would rep-
resent a highly significant accomplishment if
Like gene replacement therapy, promotion of
long-term delivery of an appropriate com-
read-through translation of transcripts with
pound, producing clinical benefit without ma-
nonsense mutations is a conceptually simple
jor side effects, can be achieved. This ap-
approach to therapy for genetic diseases, such
proach is illustrative of the likely evolution of
as CF. Nearly 10% of patients possess an al-
patient- or population-specific therapies for
lele with a premature stop codon in the CFTR
CF where different treatments will be effec-
coding sequence. However, in some popula-
tive for different genotypes.
tions, such as of those of Ashkenazi Jewish
origin, this is the predominant type of mu-
tation. The lack of CFTR protein generally Restoration of CFTR Folding
results in a severe disease phenotype (158). and Function
Amino glycoside antibiotics can enable low- Because F508- and other CFTR-processing
frequency insertion of an amino acid at the po- mutant proteins are synthesized and ca-
sition of the stop codon, resulting in synthesis pable of functioning under certain condi-
of a small amount of full-length protein (159). tions, there are intensive efforts to identify
This has been observed to occur to some ex- small molecules that can promote process-
tent in cultured cells (160) and in transgenic ing. High-throughput screens have employed
mice expressing common CF-associated stop assays that measure CFTR-mediated halide
mutations (161). efflux from cells using a halide-sensitive flu-
Gentamycin application directly to the orescent protein (165) or CFTR-dependent
nasal mucosa of such patients also yielded en- changes in membrane potential (166). A strat-
couraging results, causing a significant de- egy employed by Verkman and collaborators
crease in transepithelial potential difference (167) has proceeded through several stages
and an apparent increase in the full-length and proven very effective thus far. In the
CFTR as detected by immunocytochemical first stages, both inhibitors (168) and acti-
staining (162). This study focused on a single vators (169) of the wild-type CFTR chan-
stop mutation common in the Israeli popula- nel activity were identified. In addition to
tion (W1282X), and the nasal potential dif- verifying the utility of the assay for screen-
ference measurements, all made at a single ing, the two classes of inhibitors discovered
center, were statistically quite uniform. In- may potentially block the chronically acti-
terestingly, this truncation in the middle of vated CFTR in secretory diarrheas (168) and
NBD2 is compatible with conformational also help confirm that membrane permeabil-
maturation and a low level of chloride channel ity changes, caused by positively acting mod-
function (47, 163). A more recent multicen- ulators of the mutant CFTRs, are actually
ter study of patients with different nonsense occurring through CFTRs. The two types of
mutations did not detect significant improve- inhibitors apparently have different modes of
ments of nasal potential difference or changes action: Thiazolidinones cause a greatly pro-
in the amount or localization of CFTR pro- longed channel closed state (168), and glycine
tein in similarly treated nasal epithelial cells hydrazides (170) strongly reduce the mean
(164). There were several technical differ- open time.
ences in the two studies that could contribute Cells expressing the F508 CFTR were
to the apparently disparate results, but the first screened using the same assay for so-
outcome is suggestive that truncations remov- called potentiators, compounds that could in-
ing more than just NBD2 from the protein crease the activity of the mutant channel, par-
may be less responsive to these aminoglyco- tially rescued by culture at low temperature
716 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
(171, 172). Different classes of effective com- function of the mutant CFTRs by employ-
pounds able to activate several disease-causing ing high-throughput cell-based screening of
mutants as well as F508 were identified, in- CFTR function. Recently, additional com-
cluding tetrahydrobenzothiophenes, sulfon- pounds, able to promote the appearance of
amides, and phenylglycines. In the highly sig- the F508 CFTR protein at the cell surface,
nificant next step, F508 CFTR-expressing have also been reported (175), as have a va-
cells were exposed to test compounds for riety of compounds selected arbitrarily or in
longer periods (∼18 h) during which suf- small biased screens (176).
ficient co- and posttranslational processing, The modes of action of the effective po-
transport to, and accumulation at the cell tentiators and correctors identified so far have
surface could occur (89). Several classes of not yet been established, and there is no di-
compounds, achieving this end at micro- rect evidence that they interact directly with
molar concentrations or less, were found. the CFTR. However, interesting observations
Among these were bisaminomethylbithia- from the Clarke laboratory (177) have indi-
zoles, aminoarylthiazoles, and quinazoliny- cated that some pairs of the compounds have
laminopyrimidinones, which over a period of additive corrective effects, suggesting differ-
several hours have a similar effect as growth ent sites of action. Furthermore, one mem-
of cells at 27◦ C. Rescue was confirmed by the ber of this corrector pair was originally iden-
appearance of CFTR-mediated current and tified as a potentiator, indicating that some
mature proteins at the cell surface. Some cor- agents may have both effects. Together with
rective effect was detected in differentiated its specificity of action on the CFTR, but not
bronchial epithelial cells expressing F508. on P-glycoprotein, this provides suggestive
Structure-activity relationship studies and op- evidence of direct binding to the CFTR.
timization of the lead compounds and scaf- Not surprisingly, many of the small
folds identified should yield compounds ap- molecules turned up in corrector screens are
propriate for clinical trials. very hydrophobic, having entered cells to gain
A parallel screening and drug development access to the ER. As mentioned, the VRT-325
program by another group has also yielded corrector has this property, making it fairly
both potentiator and corrector compounds toxic to cells and nonspecific as well as in-
(166). A quinazolinone scaffold and deriva- fluencing other membrane proteins. Never-
tives from the later group have been fairly ex- theless, its apparent action on the MSDs of
tensively characterized in cell systems (173). the CFTR is quite informative (178), con-
One of these, VRT-325, causes partial matu- sistent with the facts that the F508 muta-
ration of F508 and some other misprocess- tion in NBD1 disrupts folding of the MSDs
ing mutants, but is nonspecific, also acting on (47, 99) and that rescue can be achieved by
the related P-glycoprotein multidrug trans- overcoming this effect. By contrast, at least
porter (174) and unrelated hERG potassium some potentiators have been reported to act
channels (166). It is also highly hydropho- at the level of the cytoplasmic NBDs (179).
bic and toxic to cells. However, additional Such agents if able to repair the functional
compounds more suitable for optimization as defect caused by an NBD mutation, such as
drug candidates are in development. A pyra- F508, and in so doing also prevent the sec-
zole compound, VRT-532, also identified in ondary MSD disruption, might be preferable
this program, potentiated the activity of res- to membrane active agents for several reasons.
cued F508. Thus, the progress of these two The main disadvantage of the latter group,
groups has provided proof of principle and in addition to their possible toxicity and lack
validated approaches for the discovery of small of selectivity, could be that while circumvent-
molecules to combat misprocessing and dys- ing quality control they may leave functional

ANRV345-BI77-28 ARI 1 May 2008 13:16
regulation uncorrected. Moreover, because properties enable it to gauge the level of secre-
there are redundant quality control systems tion or reabsorption required in different ep-
detecting the state of both membrane and cy- ithelial tissues. However, there are additional
toplasmic domains (99), it will be necessary anion channels in at least some of these epithe-
to correct changes in both with individual or lia, which can and do contribute to secretion.
combined agents. For example in the crucial airways, calcium-
Although there are suggestions that small activated chloride channels, which can be acti-
amounts of F508 protein may normally vated by purinergic receptors, provide poten-
reach the cell surface in some patients (180), tial alternative pathways to the CFTR (182,
it is unlikely that this is sufficient to pro- 183). Nucleotide analogue agonists of the
vide normal function even if it were fully ac- coupled P2Y2 receptors are currently in clini-
tive. Therefore, most effort is appropriately cal trials (184). Also contributing to the debil-
focused on overcoming ER retention and itating dehydration of the airway surface is the
fact that Na+ absorption continues unabated
getting misprocessed mutants to the plasma

membrane. However in addition to a kinetic in the face of inadequate Cl− secretion (72).
defect in folding and assembly, the Phe508 Therefore, Na+ channel inhibitors have the
deletion results in a thermally unstable pro- potential to ameliorate this situation, and the
tein (111, 136). Both the functional and bio- classic ameloride inhibitor has been observed
chemical half-lives of the rescued mutant to do this in the short term (185). More po-
protein are greatly shortened. Thus, ideal cor- tent and longer-lived ENaC inhibitors have
rective compounds need to stabilize the pro- been developed and are also in clinical trials
tein as well as restore folding and function. (186). Thus, judicious dosing with alternative
It is known that the folding yield and sub- anion channel activators and ENaC inhibitors
sequent stability of CFTR variants are not or other agents that intercede in the biochem-
strictly coupled (108) and that the VRT-325 ical and cellular pathways that control them
corrector caused only a partial extension of the may be able to partially mimic the role of the
lifetime of the F508 protein that it rescued CFTR.
(166). Another corrector appeared unable to The feasibility of such non-CFTR-based
stabilize the surface mutant protein that it had approaches is illustrated by the effects of
freed from ER retention (89). Cellular manip- adding osmotically active agents to the air-
ulations, such as inhibition of endocytosis, are ways (187). The increased water that follows
capable of stabilizing the F508 CFTR at the osmotically with the addition of hypertonic
cell surface (181), but these types of unspecific saline causes increased mucociliary clearance
manipulations are impractical therapeutically. and improved lung function (188). Although
To be entirely effective, chemical agents indi- restoration of normal CFTR function by any
vidually, or in combination, will have to cor- means is clearly the ultimate objective, these
rect folding, assembly, function, and stability alternative methods of generating a more nor-
in a selective and nontoxic manner. Although mally balanced epithelial surface hydration
a tall order, this now appears to be within also offer extremely important approaches to
the combined drug development capabilities combating CF lung disease.
of academic and industrial laboratories.
CONCLUSIONS AND OUTLOOK

Modulation of Other Epithelial The CFTR has provided a focus for an ever
Ion Transport Pathways broadening array of studies of CF and a spec-
The CFTR provides a crucial regulatory step trum of less severe “CFTR-opathies.” Much
in epithelial anion (Cl− , HCO− 3 , and perhaps has been learned, but there remain many cru-
others) permeability. Its complex regulatory cial unresolved issues. A frequently expressed
718 Riordan
ANRV345-BI77-28 ARI 1 May 2008 13:16
frustration in the field is that defects in the and important variants such as F508 will
primary function of the CFTR do not seem be improved and, in combination with bioin-
to link simply to the myriad pathophysio- formatic and computational methods, yield
logical features of the disease. This some- useful structural models. The focus of these
times leads to proposals that the protein must models on alterations in intermediate folding
have alternate or additional functions. De- states or perturbed interdomain associations
termining whether this is the case, or if the may enable rational structure-based drug de-
linkages with downstream effects have not sign. Because both the dynamics and disorder
yet been dissected out, can be quite chal- inherent in the CFTR are among the imped-
lenging. Cl− is quantitatively the predomi- iments to X-ray crystallography, it is likely
nant permeant anion. Yet HCO3 − is of great that continued major advances will be made
importance, and its role in mucin process- in NMR studies of the type that have recently
ing is under intense investigation. Other an- provided deeper insight into the action of the
ions, including SCN− , may be important
unique R domain.
in the innate immunity of the airway. The As a firmly established therapeutic target
mechanisms underlying the interplay between in CF (and a spectrum of less severe clini-
CFTR- and ENaC-mediated Na+ permeabil- cal conditions) mutant CFTR correction can
ity in airway epithelia have yet to be re- be pursued by all available experimental high-
solved and are necessary to fully understand throughput strategies such as whole-genome
how ion and liquid homeostasis is maintained siRNA and others. The recent demonstration
there. that quantitative downregulation of a specific
The CFTR protein itself has revealed un- Hsp 90 cochaperone could provide partial res-
expected and fascinating properties, but its cue of F508 provides encouragement that
mechanism of action remains incompletely additional subtle manipulations of the nascent
understood. This is partly because of diffi- CFTR chaperome may provide a means for
culties in reproducibly obtaining large sta- generation of sufficient mature protein. Cur-
ble preparations of purified and reconstituted rently, there are indications of a possible re-
protein for rigorous characterization of its naissance in gene therapy efforts in CF, and
enzymology, channel activity, and physical significant advances may be anticipated. The
properties. The 3D structure at atomic reso- way forward is persistence in the pursuit of
lution must overcome several technical chal- the defective mechanisms that must be over-
lenges. However, the resolution obtained by come or supplanted to alleviate the symptoms
electron crystallography of both the wild-type of this disease.
SUMMARY POINTS
1. Although influenced by other environmental and genetic factors, CF may still be
considered a single-gene defect disease. Therefore, understanding and restoration of
the function of the CFTR are reasonable approaches to therapy.
2. As an ion channel, the CFTR’s distinguishing feature is its ability to hydrolyze its
bound ligand rather than respond to changes in bulk ligand concentrations as is done
by all other known ligand-gated channels.
3. Essential for utilization of the basic ABC transporter architecture as an ion channel is
the precisely graded control of gating by the phosphorylation state of multiple sites
in the unique disordered R domain.

ANRV345-BI77-28 ARI 1 May 2008 13:16
4. A large number of known mutations have been detected in CF patients, so therapeutic

strategies can be tailored to these mutation types.
5. As a complex multidomain membrane protein, the CFTR is assembled inefficiently
during biosynthesis, and many mutations, including F508, present in ∼90% of
CF patients, are unable to mature conformationally and succumb to quality control
mechanisms in the ER and endocytic compartments.
6. First generations of small molecules that can rescue these processing mutants and
augment channel function have been identified, and extensive efforts are being devoted
to further the discovery and development of effective pharmaceuticals.
7. There is also ongoing progress in gene replacement methodologies, which will be
effective for all mutation types, and treatments to suppress nonsense mutations and
manipulate 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 while
other proteins, of generally similar structure, perform active transport need to be
clarified. How similar or different are the allosteric coupling events between NBDs
and MSDs that drive active transport and those that determine the gating state of
the CFTR channel? What is the molecular structure of the CFTR anion selective
pore? How does the unique R domain provide overriding control of the basic ABC
transporter mechanism? High-resolution 3D structures of the CFTR protein at dif-
ferent stages of its catalytic and gating cycles as well as different phosphorylation
states will be necessary, but not entirely sufficient, to answer these questions. The dy-
namic structural changes underlying functional transitions will have to be discerned
from complementary approaches including kinetic analyses of CFTR’s enzymatic and
single-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 and
missassembly of F508 CFTR, as well as the cellular mechanisms that recognize the
mutant protein and determine its fate. In addition, there is a need for further progress
in understanding and manipulating major phenotypic modifications downstream from
mutant 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 and
Anne Edwards for its assembly.
720 Riordan
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726 Riordan
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Annual Review of
Biochemistry
Contents Volume 77, 2008
Prefatory Chapters
Discovery of G Protein Signaling
Zvi Selinger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1
Moments of Discovery
Paul Berg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 14
Single-Molecule Theme
In singulo Biochemistry: When Less Is More
Carlos Bustamante p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 45
Advances in Single-Molecule Fluorescence Methods
for Molecular Biology
Chirlmin Joo, Hamza Balci, Yuji Ishitsuka, Chittanon Buranachai,
and Taekjip Ha p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 51
How RNA Unfolds and Refolds
Pan T.X. Li, Jeffrey Vieregg, and Ignacio Tinoco, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 77
Single-Molecule Studies of Protein Folding
Alessandro Borgia, Philip M. Williams, and Jane Clarke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p101
Structure and Mechanics of Membrane Proteins
Andreas Engel and Hermann E. Gaub p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p127
Single-Molecule Studies of RNA Polymerase: Motoring Along
Kristina M. Herbert, William J. Greenleaf, and Steven M. Block p p p p p p p p p p p p p p p p p p p p149
Translation at the Single-Molecule Level
R. Andrew Marshall, Colin Echeverría Aitken, Magdalena Dorywalska,
and Joseph D. Puglisi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177
Recent Advances in Optical Tweezers
Jeffrey R. Moffitt, Yann R. Chemla, Steven B. Smith, and Carlos Bustamante p p p p p p205
Recent Advances in Biochemistry

Mechanism of Eukaryotic Homologous Recombination
Joseph San Filippo, Patrick Sung, and Hannah Klein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229
v
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Structural and Functional Relationships of the XPF/MUS81

Family of Proteins
Alberto Ciccia, Neil McDonald, and Stephen C. West p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p259
Fat and Beyond: The Diverse Biology of PPARγ
Peter Tontonoz and Bruce M. Spiegelman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p289
Eukaryotic DNA Ligases: Structural and Functional Insights
Tom Ellenberger and Alan E. Tomkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313
Structure and Energetics of the Hydrogen-Bonded Backbone
in Protein Folding
D. Wayne Bolen and George D. Rose p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339
Macromolecular Modeling with Rosetta
Rhiju Das and David Baker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363

Activity-Based Protein Profiling: From Enzyme Chemistry
to Proteomic Chemistry
Benjamin F. Cravatt, Aaron T. Wright, and John W. Kozarich p p p p p p p p p p p p p p p p p p p p p p383
Analyzing Protein Interaction Networks Using Structural Information
Christina Kiel, Pedro Beltrao, and Luis Serrano p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415
Integrating Diverse Data for Structure Determination
of Macromolecular Assemblies
Frank Alber, Friedrich Förster, Dmitry Korkin, Maya Topf, and Andrej Sali p p p p p p p p443
From the Determination of Complex Reaction Mechanisms
to Systems Biology
John Ross p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p479
Biochemistry and Physiology of Mammalian Secreted
Phospholipases A2
Gérard Lambeau and Michael H. Gelb p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p495
Glycosyltransferases: Structures, Functions, and Mechanisms
L.L. Lairson, B. Henrissat, G.J. Davies, and S.G. Withers p p p p p p p p p p p p p p p p p p p p p p p p p p p521
Structural Biology of the Tumor Suppressor p53
Andreas C. Joerger and Alan R. Fersht p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p557
Toward a Biomechanical Understanding of Whole Bacterial Cells
Dylan M. Morris and Grant J. Jensen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p583
How Does Synaptotagmin Trigger Neurotransmitter Release?
Edwin R. Chapman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p615
Protein Translocation Across the Bacterial Cytoplasmic Membrane
Arnold J.M. Driessen and Nico Nouwen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p643
vi Contents
AR345-FM ARI 7 May 2008 14:43
Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms,

Connected Processes, and Diseases
Roland Lill and Ulrich Mühlenhoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p669
CFTR Function and Prospects for Therapy
John R. Riordan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p701
Aging and Survival: The Genetics of Life Span Extension
by Dietary Restriction
William Mair and Andrew Dillin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p727
Cellular Defenses against Superoxide and Hydrogen Peroxide
James A. Imlay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p755
Toward a Control Theory Analysis of Aging
Michael P. Murphy and Linda Partridge p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p777
Indexes
Cumulative Index of Contributing Authors, Volumes 73–77 p p p p p p p p p p p p p p p p p p p p p p p p799

Cumulative Index of Chapter Titles, Volumes 73–77 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p803
Errata
An online log of corrections to Annual Review of Biochemistry articles may be found

at http://biochem.annualreviews.org/errata.shtml
Contents vii

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Department of Biochemistry and Biophysics, Cystic Fibrosis Treatment and Research

Center, School of Medicine, University of North Carolina at Chapel Hill,

Annu. Rev. Biochem. 2008. 77:701–26 Key Words

tity in 1938 are extensively described else-

there is a wide range in severity and manifes-

tations of this disease that are dependent on

www.annualreviews.org • CFTR Function and Prospects for Therapy 703

allocrite-binding sites, shift the equilibrium

continually active channel favoring maximal

salt recovery. In contrast, in secretory epithe-

Primary Anion Channel Function

www.annualreviews.org • CFTR Function and Prospects for Therapy 705

www.annualreviews.org • CFTR Function and Prospects for Therapy 707

However, it is still unclear whether this is due P. aeruginosa.

www.annualreviews.org • CFTR Function and Prospects for Therapy 709

gestion. This could reﬂect impairment of a di-

rect NBD1/NBD2 interaction or an indirect

www.annualreviews.org • CFTR Function and Prospects for Therapy 711

many glycoproteins during biogenesis (109), internalized at a rate of approximately 10%

www.annualreviews.org • CFTR Function and Prospects for Therapy 713

grown at reduced temperature or because tor to drive directional movement of CCVs

nals in the CFTR C-terminal extension to as the result of freeing of a cytoskeletally

tivity (155), their ability to mature and traf-

ﬁck to the surface of human cells is expected

www.annualreviews.org • CFTR Function and Prospects for Therapy 715

Suppression of Premature sides. However, effectiveness even in patients

www.annualreviews.org • CFTR Function and Prospects for Therapy 717

getting misprocessed mutants to the plasma

CONCLUSIONS AND OUTLOOK

www.annualreviews.org • CFTR Function and Prospects for Therapy 719

4. A large number of known mutations have been detected in CF patients, so therapeutic

faces to either mimic the CFTR or compensate for its absence.

10. Dean M. 2005. Methods Enzymol. 400:409–29

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

34. Kunzelmann K. 2001. News Physiol. Sci. 16:167–70

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

102. Vij N, Fang S, Zeitlin PL. 2006. J. Biol. Chem. 281:17369–78

136. Sharma M, Benharouga M, Hu W, Lukacs GL. 2001. J. Biol. Chem. 276:8942–50

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

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

Contents Volume 77, 2008

Recent Advances in Biochemistry

Structural and Functional Relationships of the XPF/MUS81

Rhiju Das and David Baker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363

Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms,

Michael P. Murphy and Linda Partridge p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p777

Cumulative Index of Contributing Authors, Volumes 73–77 p p p p p p p p p p p p p p p p p p p p p p p p799

An online log of corrections to Annual Review of Biochemistry articles may be found

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