Download 12 Insights into the mechanisms underlying CFTR channel activity

© The Authors Journal compilation © 2011 Biochemical Society
Essays Biochem. (2011) 50, 233–248; doi:10.1042/BSE0500233
12
Insights into the mechanisms
underlying CFTR channel
activity, the molecular
basis for cystic fibrosis and
strategies for therapy
Patrick Kim Chiaw1, Paul D.W. Eckford1 and
Christine E. Bear2
Programme in Molecular Structure and Function, Hospital for Sick
Children, 555 University Avenue, Toronto, ON, Canada, M5G 1X8
Abstract
Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator)
cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway
obstruction with recurrent airway inflammation and infection. Pulmonary
obstruction in CF has been linked to the loss of CFTR function as a regulated
Cl− channel on the lumen‑facing membrane of the epithelium lining the
airways. We have learned much about the molecular basis for nucleotideand phosphorylation‑dependent regulation of channel activity of the normal
(wild‑type) version of the CFTR protein through electrophysiological studies.
The major CF‑causing mutation, F508del‑CFTR, causes the protein to misfold
and be retained in the ER (endoplasmic reticulum). Importantly, recent
studies in cell culture have shown that retention in the ER can be ‘corrected’
1These
2To
authors are joint primary authors.
whom correspondence should be addressed (email [email protected]).
233
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Essays in Biochemistry volume 50 2011
through the application of certain small‑molecule modulators and, once at the
surface, the altered channel function of the major mutant can be ‘potentiated’,
pharmacologically. Importantly, two such small molecules, a ‘corrector’
(VX‑809) and a ‘potentiator’ (VX‑770) compound are undergoing clinical
trial for the treatment of CF. In this chapter, we describe recent discoveries
regarding the wild‑type CFTR and F508del‑CFTR protein, in the context
of molecular models based on X‑ray structures of prokaryotic ABC (ATPbinding cassette) proteins. Finally, we discuss the promise of small‑molecule
modulators to probe the relationship between structure and function in the
wild‑type protein, the molecular defects caused by the most common mutation
and the structural changes required to correct these defects.
Introduction
CF (cystic fibrosis), one of the most common genetic diseases in Caucasian
populations, is an autosomal recessive disease caused by mutations in both
alleles of the CFTR (cystic fibrosis transmembrane conductance regulator)
gene. CFTR is a multi‑spanning membrane protein of the ABC (ATP‑binding
cassette) superfamily C subclass (ABCC7), primarily expressed in apical
(lumen‑facing) membranes of epithelial tissues such as the airways, intestine,
pancreas and sweat ducts, where it controls transepithelial salt and fluid
movement [1]. Function and trafficking defects in mutant CFTR lead to
reduced secretion of Cl− and fluid in the airway, resulting in viscous airway
surface liquid. This increased viscosity prevents the cilia on the luminal
membrane from functioning properly, leading to defective mucociliary
clearance with bacterial colonization. Whereas CF patients frequently suffer
from pancreatic insufficiency (where the pancreas fails to deliver sufficient
digestive enzymes into the duodenum to break down food), intestinal
obstruction, male sterility and other effects, mortality in CF is primarily
caused by chronic lung infection [2].
In this chapter, we discuss the importance of structural information on
ABC proteins and homology models of the CFTR protein in enhancing our
understanding of ion permeation and gating by the CFTR channel. We also
discuss the potential value of small‑molecule ligands as new tools for identify‑
ing the molecular defects caused by disease‑causing mutations.
CFTR: a unique ABC protein which functions as a regulated
Cl− channel
As described in detail throughout this volume, ABC proteins are typically
active transporters, dependent on the binding/hydrolysis of ATP at their
NBDs (nucleotide‑binding domains) to drive conformational changes in their
MSDs (membrane‑spanning domains), also known as TMDs (transmembrane
domains), and the transport of substrate up a concentration gradient across
the membrane. ABC transporters are thought to bind substrates at a discrete
© The Authors Journal compilation © 2011 Biochemical Society
P.K. Chiaw, P.D.W. Eckford and C.E. Bear
235
binding site on one side of the membrane, and then bind/hydrolyse nucleotide
to induce a shift to an alternative conformation in which access to one side
of the membrane is occluded while access to the opposite site is opened [3].
This would occur along with a concomitant change in the binding site affinity
for the substrate, and the molecule would be released on the other side of the
membrane with a net hydrolysis of an ill‑defined number of ATP molecules.
Uniquely, CFTR (Figure 1) functions as a Cl− channel, not as a transporter.
In fact, electrophysiological measurements on purified protein nearly 20
years ago [4] showed that CFTR mediates Cl− electrodiffusion along its
electrochemical gradient.
The unique channel function of CFTR in a family of transporters con‑
tinues to garner considerable attention by membrane protein biophysicists
concerned with the structural determinants for channel compared with trans‑
porter function. The ClC family of membrane proteins resembles the ABC
family as it too comprises Cl− channels and transporters (recently reviewed
in [5]). Within the family of ClC proteins, very subtle structural differences
(substitution of a single residue in the case of the Escherichia coli ClC) are suf‑
ficient to convert a ClC transporter into a channel. It remains to be determined
whether this is the case for ABC proteins.
Although the high‑resolution structure of the full‑length CFTR pro‑
tein has not been solved, structures have been determined for NBD1 of
Figure 1. Schematic diagram of CFTR showing its domain organization
As a member of the ABC superfamily, CFTR (N‑terminal half is shown schematically in teal and
the C‑terminal half in pink) possesses two MSDs (MSD1 and MSD2, darker teal and pink respec‑
tively), two NBDs (NBD1 and NBD2, lighter teal and pink respectively) and the unique regula‑
tory R domain (orange) that is phosphorylated (green Ps) in the active protein. The MSDs are
linked to the NBDs via ICLs. Coupling helices in the ICLs are thought to interact with the NBDs
and are shown as cylinders that contact the NBDs. Phe508 (F508) is shown in yellow. Domain
structures are coloured to match the colouring of the models in Figure 2.
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Figure 2. Homology models of CFTR open and closed structures
Structural homology models of CFTR in the closed (left) and open (right) states, with the N‑ and
C‑terminal halves of the protein shown in teal and pink respectively. The R domain is shown in
orange and Phe508, which is located at the interface between NBD1 (light teal) and the TMDs, is
shown as a yellow space‑filling structure. ADP bound to the structures (one for the closed state
and two for the open state) is shown as a blue space‑filling structure. Opening of the channel
occurs with binding of ATP to the canonical nucleotide‑binding site as suggested diagrammati‑
cally (forward direction), allowing movement of Cl− ions through the channel, while hydrolysis
and release of ADP and Pi from the site (reverse direction) closes the channel to the flow of Cl−.
Models were rendered from the co‑ordinates of Mornon et al. [11].
CFTR [6,7], revealing properties of the nucleotide‑binding site of NBD1 at
the atomic level. Low‑resolution structures have been generated of purified
full‑length CFTR using cryo‑electron microscopy by Ford and colleagues
[8,9]. Overall, these models, generated in the presence and absence of ATP
analogues, suggest that long‑range conformational changes in the MSDs are
induced by ATP binding to the NBDs and phosphorylation of the R domain
(regulatory domain) [10].
Homology models of full‑length CFTR have been developed recently on
the basis of the structures of intact bacterial ABC transporters, together with
high‑resolution structural models of NBD1 [6,7]. Callebaut and co‑workers
constructed a model of the closed state of the CFTR channel [11] based on
the closed‑apo (inward‑facing) structure of Vibrio cholerae MsbA [12] (Figure
2, left). The weakly homologous N‑ (teal) and C‑ (pink) terminal halves of
CFTR are joined by a linker region that contains an R domain (orange) that is
thought to interact with the NBDs and regulate their interaction. ATP (dark
blue space‑filling model) has been modelled into the degenerate site (lacking
conservation of the Walker B motif and Walker C or ‘signature’ motif), and
© The Authors Journal compilation © 2011 Biochemical Society
P.K. Chiaw, P.D.W. Eckford and C.E. Bear
237
the clinically relevant Phe508 residue is shown in yellow space‑filling struc‑
ture to highlight its proximity to the NBD–MSD interface. Upon binding
of ATP to the canonical (possessing conserved Walker motifs) ATP‑binding
site, a rigid‑body movement occurs in NBD2 to bring the NBDs together
and probably opens the pore across the membrane. The open‑channel struc‑
ture of CFTR (Figure 2, right) was modelled by Callebaut and colleagues [11]
and by Riordan and colleagues [13] on the outward‑facing structure of the
Staphylococcus aureus Sav1866 exporter [14] and shows the interaction of
both nucleotide‑binding sites with ATP and the concomitant reorganization
of the MSDs in the protein upon binding of ATP to the canonical site. Upon
hydrolysis of ATP in the canonical site, the protein is expected to revert to
the closed state (Figure 2, left). The first full‑length mammalian ABC protein
structure, that of Pgp (P‑glycoprotein) (ABCB1), was recently solved [15],
and is in good agreement with the structures of bacterial ABC proteins such
as MsbA. Whereas the physiological relevance of the wide‑open inward‑facing
Pgp structure remains in question, the overall domain architecture is otherwise
very much as anticipated for both prokaryotic and eukaryotic ABC proteins
and indirectly supports the suitability of constructing CFTR models based on
prokaryotic protein structures.
The role of the NBD in CFTR channel gating
Typically, the two NBDs of ABC proteins interact in a head‑to‑tail
arrangement to form two nucleotide‑binding sites at their interface [16].
Although no structural data for a CFTR NBD1–NBD2 dimer exists, data
from NBD1–NBD1 homodimeric crystal structures do suggest that the CFTR
NBDs associate in the head‑to‑tail manner anticipated for all ABC proteins
[17]. For most ABC members (including Pgp), ATP hydrolysis occurs at
two symmetrical catalytic sites formed at the NBD dimer interface in an
alternating fashion. On the other hand, for CFTR and other members of the C
subfamily of ABC proteins [including MRP1 (multidrug‑resistance protein 1)],
hydrolysis occurs only at one of the two ATP‑binding sites, the ‘canonical’
catalytic site [18,19]. The other, the ‘degenerate’ ATP‑binding site, is defective
in ATP hydrolysis and is thought to be stably occupied by nucleotide.
This difference probably accounts for the much lower ATPase activity by
CFTR (~70 nmol/mg of protein per min) [20] relative to the activity of Pgp
(5000 nmol/mg of protein per min) [21]. Furthermore, using purified and
reconstituted CFTR protein, we provide direct evidence that the ATPase
activity of phosphorylated CFTR is diminished by mutations in either the
conserved Walker A or B motifs comprising the canonical catalytic site [20].
ATP binding and hydrolysis at this canonical catalytic site is associated
with gating of the CFTR channel [18,19]. Addition of MgATP to membrane
patches harbouring PKA (protein kinase A)‑phosphorylated CFTR leads to
burst‑like channel opening [22]. ATP hydrolysis at this catalytic site has been
linked to closure after the open channel burst in electrophysiological studies
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comparing gating kinetics in the normal protein and mutants lacking the key
residues in the canonical catalytic site [22]. There are several excellent reviews
of the putative mechanisms underlying ATP‑dependent gating and, recently, a
kinetic scheme supporting a strict coupling of ATP binding and hydrolysis to
gating has been proposed [19,23–25].
The role of the coupling helices in gating
As modelled by Riordan and co‑workers [13] and by Callebaut and
co‑workers [11] (Figure 2), the cytosolic connections between the
transmembrane segments in the membrane may be structured as lengthy
helical segments extending from the membrane domain, perpendicular to the
plane of the lipid bilayer. Shorter helical segments at the foot of these long
extensions and lying parallel to the surface of the NBD heterodimer have been
called the ‘coupling helices’. These coupling helices are thought to dock on to
hydrophobic patches on the NBDs. In the transition from ‘closed’ to ‘open’
conformations in the models of Callebaut and co‑workers, there is a sliding
of NBDs relative to one another upon ATP binding and/or hydrolysis at the
catalytic site [11]. These authors suggest that the coupling helices provide a
pivot point above which twisting along the long axis of the helical extension
and the transmembrane helices occurs. An ‘iris‑like’ opening mechanism has
been described in computational studies for other channels including the CorA
Mg2+ channel [26]. Recent studies by Kirk and colleagues lend support to the
idea that changes in the relative orientation of the long helical segments are
instrumental for channel gating and may occur subsequent to ATP binding
and/or hydrolysis by the NBDs [27].
The molecular basis for permeation through the CFTR
channel
Once the gate (or gates) are opened, anions (predominantly Cl− or HCO3−)
are conducted via intramembrane helices in the MSDs of CFTR [28]. To date,
although the identity of all of the helical segments which comprise the pore is
unknown, electrophysiological (patch‑clamp) and mutagenesis studies have
confirmed a critical role for the sixth transmembrane segment in contributing
to the conduction path [29–31]. Patch‑clamp studies of anion conduction also
support the idea that the pore in the membrane is asymmetric with a relatively
large cytosolic vestibule. Net positive charge on the surface of the inner
[comprising the cytosolic face of TMHs (transmembrane helices) 1, 5, 6 and
12] and the outer (comprising the external face of TMH6 and the extracellular
loops between TMH1–TMH2 and TMH11–TMH12) vestibules appears to
ensure efficient anion entry into the pore [32].
Using cysteine‑scanning mutagenesis and thiol chemistry together with
electrophysiological studies and molecular dynamics simulations, Dawson and
colleagues confirmed the apparent fidelity of the Sav1866 model in predicting
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P.K. Chiaw, P.D.W. Eckford and C.E. Bear
239
which residues in TMH6 face the aqueous pore of CFTR [33]. These findings
support the presence of a structural constriction of the pore close to the extra‑
cellular surface of the protein in the vicinity of Thr338 and Thr339 as described
previously by Linsdell [28]. Interestingly, accessibility of substituted cysteine
residues in TMH6 to thiol‑group‑specific reagents depends on ATP binding
to the NBDs and phosphorylation of the R domain [34], consistent with the
notion that the structure of the pore may be dynamic and regulated.
The role of the R domain in channel gating
In addition to the structural features shared with other ABC proteins, CFTR
also contains a unique 241‑residue R domain in the linker region that joins the
two halves of the protein [35] and is essential for its activity [36]. The highly
charged R domain possesses multiple phosphorylation sites for PKA and
other kinases including PKC (protein kinase C) and AMPK (AMP‑activated
protein kinase) [37–39]. As a result of elegant biochemical, structural
and computational studies, we now understand that the R domain is a flexible
disordered region of the protein which undergoes dynamic interactions with
other CFTR domains [40,41]. Although this domain is disordered, it possesses
regions with the propensity to form α‑helices in the non‑phosphorylated
state and PKA phosphorylation reduces this propensity. These findings are
consistent with other biophysical and computational studies showing that the
isolated R domain may become less compact with phosphorylation [42].
In the context of the full‑length CFTR protein, phosphorylation of the
R domain probably alters multiple interactions with other domains, with
certain interactions becoming weaker [41] and others acquiring higher affin‑
ity [39]. For example, phosphorylation may weaken interactions between the
R domain and patches on NBD1 (possibly including the regulatory insertion
and the regulatory extension) which, in the resting state, prevents heterodimeri‑
zation of NBD1 and NBD2, an important trigger for ATP‑dependent channel
opening [7,19,41,43]. On the other hand, phosphorylation may promote the
direct interactions between specific regions in the R domain and structure(s)
in the membrane domains, the N‑ and/or the C‑terminus. There appears to be
an interaction between regions of the R domain and acidic residues near the
N‑terminal domain that affect PKA‑dependent CFTR channel activity [44].
According to recent electron microscopy studies, the whole CFTR protein
becomes more compact after phosphorylation, supporting the idea that this
modification may enhance certain intramolecular interactions mediated by the
R domain [9].
Phosphorylation of the R domain modulates the activity of the protein in a
complex manner. It is well known that phosphorylation by PKA is absolutely
required for channel function [38,45,46] and occurs on at least nine sites in
the R domain and one in NBD1 [36,47]. Our biochemical studies show that the
apparent ATP affinity for the intrinsic ATPase activity of CFTR is also modu‑
lated by PKA‑dependent phosphorylation [48]. These biochemical findings are
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consistent with the role of phosphorylation in permitting heterodimerization
of NBD1 and NBD2, ATP binding to the catalytic site and ATP‑dependent
channel opening [43]. Ostedgaard et al. [40] were the first to present a model
of the R domain wherein its ‘disordered’ structure was critical in conferring
its biological activity. In this model, multiple phosphoserine residues in the
disordered R domain could function interchangeably to mediate the intramo‑
lecular interactions necessary for channel opening. Furthermore, these authors
suggested that, although no one phosphoserine residue is required, different
phosphoserine residues could have quantitatively different effects. In general
terms, this model is consistent with experimental data obtained by patch‑clamp
studies of the CFTR channel activity [36,43,46,49], as well as structural stud‑
ies of the isolated domain [41]. However, the relative role of each individual
PKA phosphosite remains unclear, with certain sites thought to be stimulatory,
whereas others are thought to be inhibitory to channel opening [49]. Therefore,
although there has been significant progress to date, the molecular basis for
phosphorylation‑dependent activation remains unclear.
Studies of disease‑causing mutations provide insight into
the role for domain–domain interactions underlying protein
folding
To date, over 1800 mutations have been detected in CF patients, with the
most prevalent mutation being the deletion of Phe 508 (F508del‑CFTR)
(http://www.genet.sickkids.on.ca/cftr/app). This mutation occurs on at
least one allele in ~70% of CF patients [50] and results in misfolding and
retention of the mutant protein in the ER (endoplasmic reticulum) [51]
(Figure 3). In the ER, the mutant protein is targeted for degradation via
the ERAD (ER‑associated degradation) pathway [52]. The molecular basis
for the misfolding of F508del‑CFTR remains poorly understood. X‑ray
crystallography studies by Lewis et al. [6] on the isolated mutant form of the
human NBD1 (hNBD1‑F508del) suggests that deletion of Phe 508 does not
impede the fold of the independent domain, but rather may alter the surface
topology of NBD1 in the immediate vicinity of the mutation site and hence
may alter domain–domain assembly. However, as many solubility‑enhancing
mutations were introduced in order to obtain the crystal structures of human
NBD1, the impact of deleting Phe 508 on this individual domain requires
further study. In full‑length CFTR, these solubility‑enhancing mutations
improve the processing and channel gating of F508del‑CFTR [53].
Homology models of CFTR based on the bacterial ABC transporter
Sav1866 illustrate a putative consequence of the Phe508 deletion in the context
of the full‑length protein (see Figures 1 and 2). According to both of these
models, Phe508 contributes to a hydrophobic groove on the surface of NBD1
with which the ‘coupling helix’ of ICL4 (fourth intracellular loop) normally
engages [11,13]. These models prompted the hypothesis that this particular
interaction may be perturbed in the case of the major mutant F508del‑CFTR.
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P.K. Chiaw, P.D.W. Eckford and C.E. Bear
241
Figure 3. Aberrant exposure of trafficking motifs leads to retention of F508del‑CFTR
in the ER
Left: cartoon showing the positive role of an ER forward trafficking motif (intense green sphere
and green arrow) in mediating ER exit of wild‑type CFTR. Retrograde trafficking of wild‑type
CFTR back to the ER from the Golgi may be minimized due to masking of an ER‑retention
motif (red outlined arrow and pale red sphere). Right: cartoon of F508del‑CFTR mistrafficking.
The majority of the mutant protein is misfolded and thus remains in the ER. Misfolding leads
to dysfunction of an ER forward trafficking motif (pale green sphere and green outlined arrow)
and enhanced retrograde trafficking via an exposed ER retention motif (intense red arrow and
intense red sphere).
Using a cysteine cross‑linking approach (on the background of the full‑length
protein lacking its endogenous cysteine residues), the research groups of
Riordan [13] and Clarke [54] showed that this interface was disrupted in
the F508del‑CFTR protein. Furthermore, it appears likely that additional
domain–domain interactions are modified in this mutant, perhaps second‑
ary to the loss of the specific interface described above. Limited proteolysis
studies performed by Du et al. [55] have also revealed that the F508del‑CFTR
mutation in NBD1 alters the assembly of the second half of the CFTR protein
and enhances protease‑susceptibility of both NBD2 and MSD2 in the context
of the full‑length mutant protein. Despite this progress, our understanding
of CFTR folding remains incomplete. For example, it still remains unclear
whether each domain folds independently of the others and then assembles
or requires association with disparate distant regions for a stable fold of each
domain to be achieved. Importantly, we have only looked at regions that
can be readily monitored. Hence, the role of the N‑ and C‑termini, the R
domain and the intracellular loops in folding, or misfolding in the case of
F508del‑CFTR, have not been examined in a systematic way.
As a consequence of protein misfolding and/or misassembly caused by
the Phe508 deletion, the presentation of both di‑acidic ER exit motifs and
di‑arginine ER retention/retrieval signals are altered, thus leading to ER reten‑
tion of the major mutant (Figure 3). Studies by Balch and co‑workers suggested
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that proper presentation of the ER exit motif, the di‑acidic motif D565AD, is
lacking, or masked, in F508del‑CFTR [56]. Hypothetically, masking of the
D565AD motif would, in turn, prevent association of the mutant protein with
the Sec24 protein and exit from the ER via COPII (coatamer protein II)‑coated
vesicles [56]. To further compound this defect in trafficking out of the ER, any
F508del‑CFTR protein which does manage to reach the Golgi is thought to be
sent back to the ER via retrograde trafficking by virtue of the aberrant expo‑
sure of di‑arginine (RXR) retention/retrieval motifs [57]. One such di‑arginine
motif (R553AR) appears to play a predominant role, as secondary site suppres‑
sor mutations (in yeast expression studies) have been identified which dis‑
rupt this motif and lead to partial rescue of the primary trafficking defect in
F508del‑CFTR [58]. Furthermore, in our own work, we showed that delivery
of a synthetic peptide bearing this motif (R553AR), via Tat (transactivator of
transcription)‑mediated transduction, partially rescued the F508del‑CFTR bio‑
synthetic defect, probably by competing with the retrograde trafficking machin‑
ery (Figure 3) [59]. Together, these studies support the idea that deletion of
Phe508 causes abnormal assembly of NBD1 with the rest of the protein leading
to a relative burial of D565AD and exposure of R553AR. As both of these motifs
reside in the helical subdomain of NBD1, we suggest that this subdomain may
exhibit an altered structure in the context of the whole molecule. In turn, this
putative defect may contribute to misassembly of NBD1 with other domains of
F508del‑CFTR or aberrant interactions with different proteins. Our studies of
the ATPase activity of reconstituted F508del‑CFTR revealed that it retains par‑
tial catalytic activity, arguing that any structural defect in the helical subdomain
(comprising part of the catalytic site) may be subtle.
Small‑molecule ligands identified through high‑throughput
chemical screens provide powerful new tools in studies of the
molecular defects caused by disease‑associated mutations
The first evidence that the trafficking defect of the Phe508 deletion could
be modified to allow partial escape from the ER and functional expression
on the cell surface was published by Welsh and colleagues almost 20
years ago [60]. These researchers showed that low‑temperature incubation
(e.g. 27°C) of cells expressing the major mutant was effective in causing
its expression on the cell surface [60]. Likewise, several small‑molecule
modulators, identified in high‑throughput screens of chemical libraries,
have been found to ‘rescue’ the trafficking defect [61–63]. High‑throughput
screens also led to the identification of ‘potentiator’ compounds which
enhance the channel activity of F508del‑CFTR once rescued to reach the
cell surface. Clinical trials of one corrector (VX‑809) and one potentiator
(VX‑770) compound are currently underway, and these trials are generating
considerable excitement in the field. To date, however, the molecular
target(s) for these compounds remains unknown, limiting future discovery of
novel compounds.
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243
It has been hypothesized that certain small‑molecule correctors act by
binding directly to the F508del‑CFTR protein to induce an ER-exit‑competent
fold [64]. Using the cysteine cross‑linking approach described above, Clarke
and colleagues have shown that the addition of the small‑molecule correctors
VRT‑325 and Corrector 4a restores normal interactions within the transmem‑
brane domains [64]. Such studies support the notion that these compounds may
promote ER exit by inducing a more compact fold in F508del‑CFTR. Recently,
we showed that addition of the corrector compound VRT‑325 (at concentrations
known to mediate biosynthetic rescue) to proteoliposomes bearing partially
purified F508del‑CFTR led to significant inhibition of its apparent affinity for
ATP and intrinsic ATPase activity [65]. At the same concentration, acute treat‑
ment of ‘temperature‑corrected’ cells with VRT‑325 exerted a modest inhibi‑
tory effect on channel activation. Using current models of CFTR structure and
function, we speculate that direct binding of VRT‑325 leads to a series of allos‑
teric transitions which significantly alter the dynamic conformational changes
required for continuous cycles of ATP binding and hydrolysis and, consequent‑
ly, inhibit ATP‑dependent channel gating. We speculate that this alteration of
conformational dynamics contributes to its activity as a ‘corrector’.
As mentioned above, small molecules with potentiator activity are capable
of increasing the channel activity of biosynthetically rescued F508del‑CFTR
[62,66,67]. Direct binding of the potentiator VRT‑532 to F508del‑CFTR was
supported in our activity assays of partially purified and functionally reconsti‑
tuted F508del‑CFTR [62,66,67]. We observed that VRT‑532 induced a more
modest effect than the corrector compound on the ATPase activity of partially
purified F508del‑CFTR. VRT‑532 binding did not significantly affect the
apparent ATP affinity, rather it lowered the rate of ATP turnover, an effect
predicted to stabilize the channel open state and consistent with the potentia‑
tor activity of VRT‑532. Clearly, future studies revealing the binding sites for
these small molecules on CFTR or F508del‑CFTR are essential for our under‑
standing of their mechanism of action and will provide insight into the mol‑
ecular basis for disease caused by the most common mutation.
Future directions and challenges
The goal of solving the molecular mechanisms of action of CFTR and the
consequences of disease‑causing mutations continues to be a daunting task.
Important novel research tools are being developed, such as high‑resolution
structures of other ABC family members (in different ligand‑bound states) that
lead to the generation of models which predict their molecular mechanism of
action. Such models have been informative with respect to our understanding of
the assembly of the multiple domains of CFTR. However, in our opinion, the
potential of these models in revealing the molecular mechanism of CFTR as a Cl−
channel will only be fully realized when novel biophysical methods for studying
conformational dynamics by the full‑length CFTR protein directly are developed
to complement single‑channel gating studies. Future progress in this field will
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Essays in Biochemistry volume 50 2011
be enhanced due to the identification of chemical ligands capable of promoting
specific channel gating transitions. Molecular mechanisms underlying normal
CFTR gating and the perturbations arising from disease‑causing mutations may
be revealed once the docking sites for small molecules are defined.
Summary
•
•
•
•
•
•
•
CFTR is an ABC‑type protein that is a unique channel‑forming
member of this transporter family.
A variety of CFTR mutations result in defective membrane traffick‑
ing and/or channel activity and lead to CF, a chronic and fatal genetic
disease that leads to chronic lung obstruction and infection.
Our understanding of the structure and function of CFTR has
increased through the elucidation of the crystal structures of the NBDs
of the protein, the three‑dimensional structure of the intact protein at
low resolution and molecular homology modelling that has suggested
structural changes likely to occur during the normal function of the
protein.
Electrophysiological measurements have shed light on the normal func‑
tion of CFTR, its regulation by phosphorylation and nucleotide bind‑
ing, and the effects of CFTR mutations.
F508del‑CFTR is the major CF mutation, which causes the protein to
misfold and be retained in the ER.
ER retention can be ‘corrected’ by small‑molecule modulators, which
increase the amount of mutant CFTR at the cell surface, whereas
‘potentiator’ molecules increase the channel activity at that location
and are useful tools to further probe the function of CFTR.
The corrector VX‑809 and the potentiator VX‑770 are undergoing
clinical trial for the treatment of CF, with the potential for pharmaco‑
logical treatment to improve both the lifespan and the quality of life of
those suffering from CF.
The studies conducted in the Bear laboratory were supported by Operating Grants
to C.E.B. from the Canadian Institutes of Health Research (CIHR) [grant numbers
GPG‑102171 and MOP 97954], Cystic Fibrosis Canada (CFC) and the U.S. Cystic
Fibrosis Foundation (CFF). P.K.C. was supported by a Studentship Award provided by
the CIHR (training programme in the structure of membrane proteins) and P.D.W.E.
was supported by a Fellowship awarded through CFC.
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