The Proceedings of the American Thoracic Society 1:17-21 (2004)
© 2004 The American Thoracic Society
Revisiting Cystic Fibrosis Transmembrane Conductance Regulator Structure and Function
John W. Hanrahan and
My-Anh Wioland
Department of Physiology, McGill University, Montreal, Quebec, Canada
Correspondence and requests for reprints should be addressed to John W. Hanrahan, Ph.D., McGill University, Department of Physiology, 3655 Promenade, Sir William Osler McIntyre Medical Science Building, H3G 1Y6 Montreal, Quebec, Canada. E-mail: john.hanrahan{at}mcgill.ca
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ABSTRACT
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a channel/enzyme which mediates passive diffusion of chloride and bicarbonate through epithelial cell membranes. It is expressed in many cell types throughout the body, but in the airways it is found mainly in secretory serous cells of the submucosal glands. CFTR belongs to a large super-family of ATP binding cassette transporters that have two nucleotide binding domains with characteristic sequences or "motifs." Although most other ATP binding cassette transporters consume ATP to actively transport various substrates, in CFTR the interactions of ATP with nucleotide binding domains control opening and closing of the channel pore (i.e., channel gating). Recent high resolution structures of bacterial nucleotide binding domains combined with new biochemical and electrophysiological studies of CFTR itself have led to major advances in our understanding of CFTR gating. For example, it is now clear that the ATPase activity of CFTR is not strictly required for its channel activity. CFTR has at least two distinct gating modes; one dependent on hydrolysis and the other requiring only stable ATP binding. In this article we discuss a working hypothesis for CFTR that incorporates these recent findings and discuss some interesting implications of the paradigm shift for other aspects of CFTR function and dysfunction.
Key Words: cystic fibrosis • chloride secretion • nucleotide binding domains • channel gating
There are over 1,000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1–3) (http://www.genet.sickkids.on.ca/cftr/). Many of these reduce the number or activity of CFTR channels at the cell surface. The loss of functional CFTR chloride channels ultimately leads to the viscous secretions in the airways, pancreatic ducts, and intestine that characterize cystic fibrosis (CF) (4). CFTR has two membrane domains, two nucleotide-binding domains (NBDs), and a regulatory domain with numerous potential phosphorylation sites (see Figure 1)
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Figure 1. Cartoon of cystic fibrosis transmembrane conductance regulator (CFTR) showing glycosylation sites (illustrated as a chain of asterisks), nucleotide-binding domains (NBDs), and cytoplasmic loops. The NBDs are loosely based on those in the bacterial transporter MalK, and probably form a dimer, with ATP molecules bound to catalytic sites situated at the interface between NBD1 and NBD2 (49).
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Each membrane domain contains six membrane-spanning segments that are predominantly 
-helical, but their arrangement is not known and is probably much more complicated than depicted here. Both membrane domains are followed by NBD1 and NBD2, respectively, which are hallmarks of the ATP binding cassette transporter superfamily. This superfamily of more than 2,000 proteins transports a vast array of substrates and has members in all organisms that have been studied, from bacteria to humans. The proteins most closely related to CFTR (also called ATP binding cassette C7) are the "multidrug resistance proteins" 1–9 and the sulfonylurea receptors 1 and 2, which regulate potassium channels (see [5] for review).
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CFTR IS AN ATP-DEPENDENT CHLORIDE CHANNEL THAT IS TIGHTLY REGULATED BY PHOSPHORYLATION
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Expression of the CFTR gene generates a novel cAMP-stimulated conductance (6), which is mediated by chloride channels with relatively low unitary conductance, a nonrectifying current–voltage relationship (7), and other features typical of epithelial chloride channels (8–10). They have characteristic gating kinetics at physiological potentials (open bursts that are interrupted by rapid flickering, separated by relatively long closures) and are only weakly inhibited by external 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of many anion transporters (7, 11, 12). Strong evidence that CFTR is a chloride channel was provided by the effects of mutations in its predicted transmembrane segments, which altered the selectivity of the macroscopic anion conductance (13–15) (but see [16]). Formal proof was provided when purified CFTR was reconstituted into planar lipid bilayers and generated channels with the expected properties (17).
Activation of the CFTR channel usually requires phosphorylation of its R domain at multiple sites by protein kinase (PK) A and PKC (but see [18]); however, interactions of ATP with the NBDs are also needed for gating. These domains have several sequences, or motifs, which are diagnostic of the ATP binding cassette superfamily of transport proteins. The first is the Walker A motif (i.e., GSTGAGKTS and GRTGSGKST in NBDs 1 and 2 of CFTR, respectively, with letters indicating the single letter code for amino acids; G-glycine, S-serine, T-threonine, A-alanine, K-lysine), which has a lysine (underlined) that interacts with the terminal phosphate of ATP. The second is the Walker B motif (LYLLDSPFG, ILLLDEPSA in NBDs 1 and 2 of CFTR, respectively, with L-leucine, Y-tyrosine, P-proline, F-phenylalanine, and I-isoleucine), which has an aspartate (D) that helps coordinate magnesium or another divalent cation during hydrolysis (19). The Walker B residue immediately distal to the conserved D is also important for catalysis and is interesting because it is a negatively charged glutamate or aspartate in NBD2 of CFTR, and in the NBDs of all other members of the superfamily, yet it is a neutral serine in NBD1 of CFTR, which may allow ATP to bind stably without being hydrolyzed (20). A third characteristic sequence in the NBDs is the "signature" motif (LSGGQ and LSHGH in NBD1 and NBD2 of CFTR, respectively, where Q stands for glutamine and H for histidine). Based on comparisons with bacterial domains that have been crystallized with either ATP (21), MgADP, or have no nucleotide bound at this site (22), this signature motif is situated in a set of three helices, which are thought to rotate when ATP binds (22). The importance of this motif is highlighted by the severe effects of mutations in this region. One of these is G551D, the second most common CF mutation. This mutant is expressed normally at the plasma membrane but does not open in response to phosphorylation by PKA.
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THE ROLES OF NBD1 AND NBD2 IN CHANNEL GATING
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Initial studies of phosphorylated CFTR channels revealed that hydrolyzable nucleotides are essential for channel activity (relative potency ATP > GTP > ITP = UTP > CTP) (23, 24). This was taken as evidence for obligatory ATP hydrolysis, and further support for this view came from the apparent dependence of gating on Mg2+ (23, 25), which is an essential cofactor for ATPases. Opening and closing of the channel was proposed to result from hydrolysis cycles at one of the NBDs, with strong phosphorylation allowing a second ATP to bind at the other NBD, thereby stabilizing the open state (26, 27, 28). Strong evidence that CFTR has at least two functionally distinct nucleotide binding sites (as suggested by the presence of two NBDs) came from single channel recordings made during exposure to mixtures of ATP and the nonhydrolyzable adenosine 5'-(ß-
-imido)triphosphate (AMP-PNP), which caused channels to become "locked" open (26). Because both ATP and AMP-PNP had to be present simultaneously for this locking to occur, hydrolysis at one NBD was presumed to open the channel and allow AMP-PNP binding at the other NBD. Indeed, full length CFTR and fusion proteins that include the NBDs of CFTR do hydrolyze ATP at low rates (29–33). Long-lived openings that resemble "locked open" channels were observed in studies of mutant channels bearing an amino acid substitution in NBD2 that should abolish its hydrolysis of ATP, consistent with the locking effect of AMP-PNP being exerted at NBD2 (27). The evidence for this model, in which NBD1 opens the channel and hydrolysis at NBD2 allows it to close, has been reviewed in detail elsewhere (28, 34).
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ATP HYDROLYSIS IS NOT STRICTLY REQUIRED FOR OPENING OR CLOSING THE CFTR CHANNEL
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The above model, in which NBD1 and NBD2 have opposite functions in opening and closing the pore, accounted nicely for the effects of ATP + AMP-PNP mixtures, but does not explain more recent findings. For example, the effects of temperature on channels that have been reconstituted into planar bilayers are not consistent with strict coupling between gating and ATP hydrolysis. The duration of channel openings was only weakly temperature-dependent, providing evidence that the rate of closing is limited by diffusion rather than by the rate of ATP hydrolysis (35). Loose coupling between ATP hydrolysis and gating was further suggested when mutating the conserved Walker A lysine in NBD1 was found to reduced ATPase activity by roughly 50% (measured biochemically) in electrophysiologic experiments, but had no effect on channel activity (36). ATP hydrolysis by CFTR protein strictly depends on Mg2+ or another divalent cation in biochemical assays (36), yet large CFTR currents were observed when CFTR channels were exposed to magnesium-free solution containing ATP (37, 38) (but see [25]). In one report, the non-hydrolyzable analog AMP-PNP was found to support some channel activity at high concentrations (38). Recently, CFTR channels have been shown to open and close indefinitely after ATP is removed from the bath under some conditions, indicating that nonhydrolytic gating can occur without cycles of ATP hydrolysis or nucleotide binding/release if nucleotide remains bound to the channel (M-A Wioland and J. W. Hanrahan, unpublished observation). These findings are difficult to reconcile with the early model for CFTR gating described above.
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A REVISED WORKING MODEL FOR NUCLEOTIDE-DEPENDENT CFTR GATING
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Photoaffinity labeling has provided important new information concerning the sites of nucleotide binding within the molecule and the relative rates of hydrolysis. In this method, membranes containing CFTR are incubated with radioactive 8-azido analogs of ATP, ADP, or AMP-PNP and exposed to ultra violet light so that bound nucleotide becomes covalently attached to CFTR. Such labeling is useful because it can indicate where nucleotide interactions occur (NBD1 or NBD2), and allows inferences to be made concerning relative affinities and rates of hydrolysis within the intact molecule. Most labeling by azido-ATP occurs at NBD1 under control conditions (39). Azido-ATP binds tightly at NBD1, but is hydrolyzed there slowly if at all (20, 40), consistent with an "induced-fit" conformational change that prevents dissociation (38), as demonstrated recently for bacterial NBDs (22). Azido-ATP also interacts (with lower affinity) at NBD2; however, photolabeling is only observed there when the triphosphate of azido-ATP is labeled at the rather than the position, and experiments are performed in the presence of the general ATPase inhibitor vanadate (20). Vanadate is a transition-state analog that binds in place of phosphate immediately after hydrolysis and causes ADP to become trapped in the NBD (39). In summary, the ATPase activity of NBD2 appears to be much higher than at NBD1 (20, 40) and this rapid turnover probably accounts for the hydrolytic gating that is recorded during most electrophysiologic experiments. The precise role of NBD1 is less clear, however recent patch-clamp results in the presence and absence of Mg2+ are consistent with a model in which stable ATP binding at NBD1 is sufficient to sustain nonhydrolytic gating.
To incorporate these results and our own recent data obtained under nominally ATP-free conditions into a coherent scheme, we must assume that CFTR can operate in two distinct modes: a Mg2+-dependent hydrolytic mode, which can be locked open by the binding of AMP-PNP; and a nonhydrolytic mode that requires bound ATP but is independent of Mg2+ and unaffected by AMP-PNP (Figure 2)
. Contradictory conclusions reached by different groups in the past may simply indicate that channels were operating predominantly in different modes when the experiments were performed. For example, hydrolytic gating would account for the temperature-dependent closing rate during patch-clamp experiments (41, 42), whereas nonhydrolytic gating in planar bilayers would explain the weak dependence on temperature that was observed under those conditions (35).
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Figure 2. Hypothetical scheme for CFTR gating based on recent electrophysiology from several laboratories and -32P-N3ATP photoaffinity labeling (20, 50). According to this model, nonhydrolytic gating of the phosphorylated channel occurs spontaneously in the presence or absence of Mg2+ if nucleotide is bound at NBD1. Cycles of hydrolysis can also occur when MgATP binds at NBD2. Distinct conformations are depicted by different shapes, and rapid ATP turnover is predominantly at NBD2 (20).
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SOME IMPLICATIONS OF THE REVISED CFTR MODEL
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The new scheme for gating impacts several aspects of CFTR structure and function. For example, a popular hypothesis to explain PKA stimulation of the channel is that phosphorylation increases hydrolysis through enhanced ATP binding. Three lines of evidence have been used to support this notion: (1) PKA increases the activity of wild-type channels more at low ATP concentrations than at high ATP concentrations (43); (2) the Michaelis constant (KM) for ATP is modestly reduced when purified CFTR protein is prephosphorylated by PKA, suggesting increased affinity (29); and (3) the ATP dependence of gating shifts to lower ATP concentrations when PKA sites are removed by mutagenesis (44). However, photoaffinity experiments have not yielded any evidence for phosphorylation-stimulated binding or hydrolysis of ATP (40). Indeed, the new model implies that phosphorylation must act through some other mechanism, because PKA also stimulates nonhydrolytic gating, which obviously cannot be explained by acceleration of its ATPase activity. Improved functional coupling between the NBDs and membrane domains is a more likely mechanism of control. How this enhanced coupling is achieved is uncertain; however, recent studies from this laboratory indicate that phosphorylation of the R domain enhances its interaction with other domains, particularly those in the N-terminal half of CFTR (45).
What factors determine whether a CFTR channel operates in the hydrolytic or nonhydrolytic mode under physiologic conditions? In addition to stable ATP binding, the possibilities include Mg2+ concentration, temperature, phosphorylation, and interactions with other proteins. For example, CFTR is associated with the metabolic sensing enzyme AMP kinase, which becomes activated during metabolic stress (46). Phosphorylation by this kinase might switch CFTR into the less metabolically expensive nonhydrolytic mode. Channels bearing CF mutations need to be reexamined functionally in light of the new paradigm to see if disease mutations disrupt both gating mechanisms, or only one. Gating modes could also be an important consideration in the development of pharmacotherapies to treat CF. Indeed, the complex actions of CFTR activators, such as genistein (47) and phloxine B (48), may reflect, in part, effects on the hydrolytic and nonhydrolytic modes. Targeting the nucleotide dependence of CFTR could be a useful tool for drug design. Regardless, the new model raises many interesting new questions that will need to be addressed by converging structural, biochemical, and electrophysiologic approaches.
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ACKNOWLEDGMENTS
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The authors thank Dr. Valerie Chappe of McGill University and Drs. John R. Riordan, Luba Aleksandrov, Xiu-bao Chang, and Andrei Aleksandrov of S.C. Johnson Medical Research Center, Mayo Clinic, Scottsdale, Arizona for stimulating discussions.
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FOOTNOTES
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Supported by the Canadian Institutes of Health Research (CIHR), Canadian Cystic Fibrosis Foundation, and the National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases). M-A.W received a fellowship from Vaincre la Mucoviscidose (France). J.W.H. is a senior scientist of the CIHR.
(Received in original form June 19, 2003; accepted in final form September 26, 2003)
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ABSTRACT
CFTR IS AN ATP-DEPENDENT...
