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Physiol. Rev. 79: 77-107, 1999;
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PHYSIOLOGICAL REVIEWS   Vol. 79 No. 1 January 1999, pp. S77-S107
Copyright ©1999 by the American Physiological Society

Control of CFTR Channel Gating by Phosphorylation and Nucleotide Hydrolysis

DAVID C. GADSBY AND ANGUS C. NAIRN

Laboratory of Cardiac/Membrane Physiology, and Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York

I. INTRODUCTION
    A. Domain Organization of the CFTR Molecule
    B. CFTR Forms an Ion Channel
    C. Overview of Regulation of CFTR Channel Function
    D. CFTR in Cardiac Myocytes
II. REGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR BY PROTEIN KINASES
    A. PKA
    B. PKC
    C. cGMP-Dependent Protein Kinases
    D. Ca2+/Calmodulin-Dependent Protein Kinases
    E. Protein Tyrosine Kinases
III. REGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR BY PROTEIN PHOSPHATASES
    A. Candidate Phosphatases for Regulating CFTR
    B. Functional Effects of Exogenous Phosphatases
    C. Findings With Phosphatase Inhibitors
    D. Differential Dephosphorylation of Multiple Sites
IV. MECHANISMS OF OPENING AND CLOSING OF PHOSPHORYLATED CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHANNELS
    A. Phosphorylation of CFTR Controls ATP Hydrolysis and Channel Gating
    B. How Does Phosphorylation by PKA Modify CFTR Function?
    C. Distinct Functions of the Two NBDs
    D. Which NBD Opens, and Which Closes, a CFTR Channel?
V. WORKING MODEL OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR'S CATALYTIC AND GATING CYCLES AND INFLUENCE OF PHOSPHORYLATION
    A. Catalytic Cycles of the Two NBDs
    B. Modulation of CFTR Channel Gating by Incremental Phosphorylation
    C. Lingering Uncertainties
VI. CONCLUDING REMARKS
REFERENCES

    ABSTRACT
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Gadsby, David C., and Angus C. Nairn. Control of CTFR Channel Gating by Phosphorylation and Nucleotide Hydrolysis. Physiol. Rev. 79, Suppl.: S77-S107, 1999. --- The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is the protein product of the gene defective in cystic fibrosis, the most common lethal genetic disease among Caucasians. Unlike any other known ion channel, CFTR belongs to the ATP-binding cassette superfamily of transporters and, like all other family members, CFTR includes two cytoplasmic nucleotide-binding domains (NBDs), both of which bind and hydrolyze ATP. It appears that in a single open-close gating cycle, an individual CFTR channel hydrolyzes one ATP molecule at the NH2-terminal NBD to open the channel, and then binds and hydrolyzes a second ATP molecule at the COOH-terminal NBD to close the channel. This complex coordinated behavior of the two NBDs is orchestrated by multiple protein kinase A-dependent phosphorylation events, at least some of which occur within the third large cytoplasmic domain, called the regulatory domain. Two or more kinds of protein phosphatases selectively dephosphorylate distinct sites. Under appropriately controlled conditions of progressive phosphorylation or dephosphorylation, three functionally different phosphoforms of a single CFTR channel can be distinguished on the basis of channel opening and closing kinetics. Recording single CFTR channel currents affords an unprecedented opportunity to reproducibly examine, and manipulate, individual ATP hydrolysis cycles in a single molecule, in its natural environment, in real time.

    I. INTRODUCTION
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A. Domain Organization of the CFTR Molecule

The cystic fibrosis transmembrane conductance regulator (CFTR) protein, the single polypeptide product of the gene defective in cystic fibrosis (CF) patients (157, 166, 204), is now known to form a Cl- channel subject to complex regulation (for reviews, see Refs. 68, 165, 216). However, this was far from obvious when the gene was first identified, because the predicted amino acid sequence of CFTR (166, 167) placed it squarely in the superfamily of ATP-binding cassette (ABC) transporters. Other well-known members of this large and still growing family include a host of bacterial periplasmic transporters, each selective for a particular substrate, the yeast a-mating factor exporter (STE6), and the mammalian P-glycoprotein (Pgp) linked to multidrug resistance. All known ABC transporters seem to require two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) to function normally (82). Often, particularly in the case of the bacterial ABC transporters, separate genes encode these individual domains, or fused pairs of domains in various combinations (i.e., TMD-TMD, NBD-NBD, or TMD-NBD). An important implication of expressing several domains of a transporter as separate gene products is that the individual polypeptides must contain the necessary information to enable them to self-assemble into a fully functional transporter. It seems likely that most of those important interactions between domains have been preserved also in ABC transporters expressed from a single gene and that the nature of those interactions may provide clues as to how all of these molecules function. The CFTR is one example in which all of the functional domains are encoded by a single gene. Accordingly, the NH2- and COOH-terminal halves of CFTR both contain a TMD comprising six putative membrane-spanning alpha -helices followed by an NBD (Fig. 1), and the two halves are linked by a cytoplasmic regulatory (R) domain (not found in other ABC transporters) that incorporates multiple sites for phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC; Ref. 166). The original definition of the domain boundaries in CFTR was guided largely by the location of intron-exon junctions and by the extent of regions of sequence similarity with other ABC transporters (166). Although the proposed topology and overall organization of these domains in CFTR have proven remarkably resilient, very recent information from homology modeling (e.g., Ref. 6) and functional studies of CFTR (e.g., Ref. 29), and from the high-resolution structure of an NBD from the Escherichia coli ribose ABC transporter (7), makes it seem likely that NBD1 extends up to 60 residues into the R domain as initially defined (166). Presumably, if this new domain boundary is correct, the COOH-terminal limit of NBD2 in CFTR will need to be similarly extended.


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  		FIG. 1.   Proposed topological model of cystic fibrosis transmembrane conductance regulator (CFTR) showing cytosolic NH2 (N) and COOH (C) termini, nucleotide binding domains (NBD1, NBD2), and regulatory (R) domain, predicted membrane-spanning alpha -helices (M1-M12), and glycosylation sites in M7-M8 extracellular loop (166). Thickened section of M2-M3 cytoplasmic loop represents 30 amino acids encoded by exon 5, believed spliced out of cardiac CFTR isoform (86). Lengths of extracellular and intracellular loops are in rough proportion to number of residues they contain. NBD1 and NBD2 are drawn with fold of p21-ras (149) to emphasize proposed broad functional, and possibly even structural, homology between CFTR's NBDs and catalytic sites of G proteins (cf. Refs. 26, 68, 129). NBDs are drawn in close proximity to each other and in contact with R domain to emphasize likelihood that two NBDs directly interact with each other and with R domain. [From Gadsby and Nairn (69).]

B. CFTR Forms an Ion Channel

The CFTR is the only ABC transporter so far established to function as an ion channel [despite an earlier suggestion of channel-like activity of Pgp (70, 136), subsequently disavowed (77)]. However, there is growing evidence that CFTR is also able to modify the activity of other ion channels, such as outwardly rectifying Cl- channels (53, 66) and amiloride-sensitive Na+ channels (190-192), but it remains unclear whether such modulation is direct, resulting from association of CFTR with other channels (110), or from a chain of such protein-protein interactions perhaps beginning at CFTR's COOH-terminal PDZ (postsynaptic density protein, disc-large, ZO-1)-domain binding motif (76, 106, 158, 185, 213,), or is indirect, as suggested for CFTR's apparent influence on outward rectifier Cl- channels (175). Interestingly, it has recently become clear that another member of the ABC family, the sulfonylurea receptor (SUR; Ref. 96), forms an oligomeric complex with inward rectifier K+ channel subunits (the stoichiometry is 4 SUR per tetrameric K+ channel) and regulates their function (36, 187). This regulation appears to depend on ATP hydrolysis at the NBDs of SUR but occurs by a mechanism that is presently unknown (e.g., Ref. 8).

The possibility that CFTR subserves other roles notwithstanding, several lines of evidence have led to the incontrovertible conclusion that CFTR itself comprises a metabolically gated Cl- pore. The most straightforward evidence is the demonstration that recombinant CFTR expressed in Sf9 cells can be purified after solubilization with detergent, and then renatured and incorporated into lipid bilayers where, upon activation by PKA catalytic subunit plus ATP, it gives rise to typical small (~10 pS in symmetrical, physiological Cl-) ohmic-conductance Cl- channels (15). Additional strong evidence is that certain mutations introduced into putative transmembrane alpha -helices M5 or M6 (Fig. 1) modify the characteristics of anion interaction with the permeation pathway (reviewed in Ref. 44). For example, charge-reversing mutations at K335 or R347 (both in M6) were found to alter both anion binding within the pore and permeation through it (4, 125, 195), and the charge-neutralizing mutation R347H rendered single-channel conductance switchable between wild-type and mutant values simply by changing cytoplasmic pH back and forth between 5.5 and 8.7 (195). Further evidence comes from experiments that probed the ability of residues in M6 to interact with water-soluble reagents that could modify pore function. The mutation S341A, for instance, altered the apparent affinity (and its voltage dependence) for block of open CFTR channels by diphenylamine-2-carboxylate (133), and a cysteine-scanning method has demonstrated the accessibility to hydrophilic cysteine-modifying reagents of residues in M6 (35). The reactive cysteines appeared to lie on one face of an alpha -helix which therefore may line the pore, and comparisons of reaction rates for anionic and cationic reagents suggested that the anion selectivity filter likely resides toward the cytoplasmic end of a wide water-filled pore (35).

One of the most fundamental questions, presently unresolved for any ABC transporter, concerns the quaternary structure of the functional unit. A thorough immunoprecipitation analysis of coexpressed CFTR mutants bearing different epitopes suggested strongly that CFTR functions as a monomer (127). Analyses of electron microscopic images of purified Pgp were similarly interpreted as suggesting that active Pgp is monomeric, even though the size of the particles was consistent with that of a Pgp dimer (168). However, recent analysis of particle sizes for a range of recombinant membrane proteins, using freeze-fracture electron microscopy of oocyte membranes, led to the conclusion that CFTR exists as a dimer (56). Moreover, preliminary electrophysiological analysis of concatenated CFTR dimers, comprising linked monomers with different gating characteristics, has now prompted Zerhusen and Ma (229) to propose that a single CFTR channel contains two CFTR polypeptides. Interestingly, complementation and reconstitution studies of the E. coli ABC transporter Ars, responsible for arsenical extrusion, had already led to a model in which the transporter functions as the equivalent of a dimer in which the NH2-terminal NBD of each monomer interacts with the COOH-terminal NBD of the other to form a total of only two catalytic sites (114).

C. Overview of Regulation of CFTR Channel Function

Although CFTR seems to be the only ABC molecule that forms an ion channel, recent biochemical measurements have demonstrated that, as previously shown for other ABC transporters (e.g., Refs. 179, 180), purified intact CFTR (113), as well as an NH2-terminal CFTR NBD (NBD1; Ref. 108) or COOH-terminal CFTR NBD (NBD2; Ref. 160) peptide, expressed in fusion with the maltose-binding protein, is able to hydrolyze ATP. Moreover, ATP hydrolysis by intact CFTR was found to be enhanced after phosphorylation by PKA (113). These biochemical results provide a satisfying corollary to a substantial body of functional data that has established that opening and closing of CFTR channels is linked to ATP hydrolysis (reviewed in Ref. 68) and that, even in the presence of millimolar MgATP, CFTR channels will not open unless they are first phosphorylated by PKA (3, 140, 194). As reviewed in sections IV and V, the details of this complex interplay between phosphorylation of CFTR, ATP hydrolysis at CFTR's NBDs, and CFTR channel gating remain incompletely understood. Nevertheless, it seems likely that, during each open-close channel gating cycle, a single phosphorylated CFTR channel hydrolyzes one molecule of ATP at NBD1 (Fig. 1) to open, and then hydrolyzes a second ATP at NBD2 to permit the channel to close (14, 25, 74, 75, 91). However, it is also evident that activation of CFTR channels by PKA-mediated phosphorylation is not simply an all-or-nothing process, because biochemical measurements show that PKA readily phosphorylates the R domain of CFTR to a stoichiometry of at least 5 mol/mol (52, 152), and electrophysiological measurements have now identified at least three functionally distinct phosphorylated states of individual CFTR channels that appear to reflect the different actions of specific protein phosphatases on phosphorylated CFTR (49, 59, 88, 120, 226). Our goal in this review is to critically evaluate the experimental evidence that has led to this complex gating mechanism for CFTR channels in which phosphorylation and dephosphorylation of multiple sites in the R domain, and perhaps other domains of the protein, orchestrates the cycles of ATP binding and hydrolysis at the two NBDs that regulate opening and closing of the anion-selective pore.

D. CFTR in Cardiac Myocytes

About a decade ago, mammalian cardiac ventricular myocytes were found to display a PKA-activated Cl- current with an approximately linear whole cell current-voltage relationship in symmetrical ~150 mM Cl- solutions (10, 78, 132). Unitary current recordings in cell-attached (54, 55) and excised (55, 140) patches subsequently confirmed that these cardiac Cl- channels possess all of the hallmark characteristics used to identify epithelial CFTR Cl- channels. Thus, in excised inside-out patches, after the required phosphorylation by PKA catalytic subunit, cardiac CFTR Cl- channels close promptly upon ATP withdrawal but can be reopened by ATP or GTP, but not by ADP or 5'-adenylylimidodiphosphate (AMP-PNP); their unitary conductance is ohmic and ~12 pS in roughly symmetrical ~150 mM Cl- solutions, their open probability (Po , the average fraction of time that a channel spends open) is approximately voltage independent, and their rates of opening and closing are very low (140). Northern blot analyses confirmed the presence of CFTR mRNA in myocytes from regions of the heart and species in which PKA-regulated Cl- currents can be recorded, but not in tissue from regions with no CFTR-like currents (112, 140). Sequencing of the amplified transcript from rabbit ventricle indicated that cardiac CFTR is an alternatively spliced isoform lacking the 30 residues at the COOH-terminal end of the first cytoplasmic loop (Fig. 1, thickened segment) that are encoded by exon 5 (86). The only functional difference between cardiac and epithelial CFTR channels so far documented is that upon activation by PKA, whether in intact cells or excised patches, the Po of cardiac CFTR channels often attains a level of 0.7-0.8 (e.g., Refs. 14, 54, 55, 91, 140), whereas epithelial CFTR channels have rarely been reported to display a Po above 0.4-0.5 (e.g., Refs. 211, 223, 224, but cf. Ref. 79). However, it seems most unlikely that the higher Po of cardiac CFTR channels can be attributed to lack of exon 5 because, on the contrary, experimental deletion of exon 5 from CFTR cRNA appears to interfere with trafficking of the channels in mammalian cells, and when the channels are incorporated into bilayers, they display a marked reduction (2- to 3-fold) of Po compared with wild-type CFTR channels (225). Although the electrocardiological role of cardiac CFTR channels remains unclear (but see Refs. 196, 198), and their very existence in human heart is controversial (148, 169, 214), studies of cardiac CFTR channels have nevertheless afforded crucial insights into the gating mechanisms of epithelial CFTR channels, as we discuss here.

    II. REGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR BY PROTEIN KINASES
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A. PKA

1. Red herring: outwardly rectifying Cl- channels

Even before the gene defective in CF was cloned, there was a strong expectation that the gene product was somehow involved in a signaling pathway that depended on phosphorylation by PKA and that ultimately regulated epithelial electrolyte transport (10, 64, 155, 171). Just before the CF gene was cloned, an influential series of papers reported that, in small patches of apical membrane excised from normal human airway epithelial cells, individual Cl- channels could be activated by purified protein kinases (PKA or PKC) applied directly to the cytoplasmic surface, but not when the cells came from CF patients (89, 115, 116, 172). The Cl- channels in question, when symmetrically exposed to ~150 mM Cl- solutions on either side of the membrane, had single-channel conductances of some 30-50 pS, depending on the precise point along the curved, outwardly rectifying, single-channel current-voltage relationship at which the conductance was measured. Much smaller conductance (~15 pS) Cl- channels with linear current-voltage relationships had also been observed in an earlier study of airway cells (64), but those channels were overlooked in the experiments examining kinase action on excised patches. When the CF gene was subsequently identified and sequenced and the large cytoplasmic R domain of its protein product, human epithelial CFTR, was predicted to contain multiple sites for phosphorylation by PKA and PKC (166), this seemed to afford a straightforward and reasonable explanation for the observed lack of kinase-mediated regulation of the 30- to 50-pS, outwardly rectifying, Cl- channels in the membranes of cells from CF patients. However, this hope was soon dashed by the observation that, no matter what cell type was chosen as the host for expressing CFTR cRNA, the Cl- channels that resulted from CFTR expression were small ohmic conductance channels, not outwardly rectifying channels (3, 20, 43, 50, 94, 104, 116). As already mentioned, the defective regulation of outwardly rectifying Cl- channels in CF airway cells is nowadays attributed to (and constitutes the initial evidence for) some kind of modulatory interaction, whose mechanism remains to be established, between CFTR and other channels (53, 66, 175; cf. Refs. 191, 192).

2. Biochemical analysis of phosphorylation by PKA

A) DIBASIC VERSUS MONOBASIC CONSENSUS MOTIFS. It is now abundantly clear that CFTR itself constitutes a small ohmic Cl- channel in which five serines (Ser-660, -700, -737, -795, and -813) appear to be readily phosphorylated when PKA is activated in cells that express either native or recombinant CFTR (34, 152). It is also clear, however, that CFTR harbors a total of at least 10 serine residues (Fig. 2) that can be phosphorylated by PKA in vitro, and it seems likely that most of these may, under appropriate conditions, also be phosphorylated by PKA in vivo. Thus a variety of biochemical methods, including direct amino acid sequencing, site-directed mutagenesis combined with two-dimensional peptide mapping, and mass spectrometry (34, 141, 144, 152, 178, 199), have established that Ser-660, -700, -712, -737, -753, -768, -795, and -813 may be phosphorylated, in vitro, by PKA applied to full-length CFTR, before or after immunoprecipitation, and to recombinant R-domain or NBD1-R-domain peptides. In addition, phosphorylation of Ser-422 in NBD1-R-domain peptide (144) and of Ser-670 in R-domain peptide (141) have been demonstrated, although neither has yet been found to be phosphorylated in intact CFTR, either in vitro or in vivo. The reason for this failure is unclear, since functional consequences have been observed upon mutation of Ser-422 in a CFTR mutant already containing nine other serine-alanine mutations (31), or when Ser-670 is mutated in wild-type CFTR (220).


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  		FIG. 2.   Diagrammatic summary of CFTR phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). Seryl residues phosphorylated by PKA are shown pointing upward; sites phosphorylated by PKC point downward. For PKA, diagram attempts to integrate relative levels of phosphorylation of individual sites observed (except for Ser-768) after brief stimulation of PKA in intact cells, with relative contribution of each site to regulation of CFTR channel activity inferred from site-directed mutagenesis. Note distinction between stimulatory (+) sites and inhibitory (-) sites. Failure to detect incorporation of 32P into Ser-768 in cells is an important issue not yet resolved, but could reflect stable basal phosphorylation at that site (e.g., Ref. 30). For PKC, diagram jointly summarizes in vitro and in vivo phosphorylation studies. [Modified from Gadsby and Nairn (68).]

As indicated in Table 1, only 2 (Ser-670 and Ser-753) of these 10 phosphorylated serines so far identified in human CFTR lie within a monobasic R-X-S sequence (X represents any amino acid), the most common (twice as frequent as the classical dibasic) consensus sequence for PKA substrates (150). Curiously, in all other species examined, an additional Arg residue confers a dibasic R-R-X-S consensus motif on the Ser-670 site. The other eight phosphorylated serines occur in classical dibasic PKA consensus sites, with an amino acid sequence R-R/K-X-S/T. Other than Ser-422, which lies just NH2 terminal to NBD1 (Fig. 2), these sites so far shown to be phosphorylated by PKA are all contained within the R domain.

 
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  		TABLE 1.   Biochemical and functional analysis of phosphorylation by PKA of sites in CFTR

B) VARIATION IN PHOSPHORYLATION KINETICS. That R-domain peptide is nevertheless phosphorylated only to a stoichiometry of ~5-6 mol/mol after a limited exposure to PKA (52, 152) likely reflects variation in the kinetics of phosphorylation of the different sites. In which case, phosphorylation with higher levels of PKA and for longer periods ought to eventually result in stoichiometric phosphorylation of all the above PKA sites. Consistent with this interpretation, when each site was incorporated into a short synthetic peptide, a >10-fold variability was found in the catalytic efficiency of PKA toward the sites, presumably attributable to intrinsic differences in the ease with which PKA can bind to and phosphorylate individual serines (152). In intact CFTR, the kinetics of phosphorylation of particular serines are likely to be further influenced by local secondary, tertiary, and possibly quaternary structure, and, in cells, also by the proximity, activity, and selectivity of specific phosphatases. For example, the fact that phosphorylation of Ser-768 has not yet been demonstrated biochemically in intact cells, although it is the best in vitro PKA site, suggests that this site is also a very good substrate for one or more protein phosphatases, and this serves to emphasize the important role played by protein dephosphorylation in cellular regulation of CFTR function. A further consideration is that the pattern of phosphorylation might be ordered, because phosphorylation of one site might alter the structure of the protein and thereby influence the rate of phosphorylation (or dephosphorylation) at another site (e.g., Refs. 23, 30, 42, 141). A conformational change of the R domain upon phosphorylation by PKA was demonstrated by analysis of circular dichroism (CD) spectra (51, 52), and several distinct conformations of variably phosphorylated R-domain peptide are suggested by the multiple bands revealed by SDS-PAGE (52, 141, 152). The largest individual mobility shift appears to attend phosphorylation of Ser-737 (23, 109, 141). Moreover, a strong interdomain interaction influencing R-domain phosphorylation is suggested by the observation that, in NBD1-R-domain peptide, occupancy of NBD1 by nucleotide seemed to impair phosphorylation of the R domain (148). Along the same lines, coimmunoprecipitation experiments suggested that phosphorylation of recombinant R domain strengthened its interaction with recombinant NBD1 peptide (73). It remains to be seen whether the same interactions occur within intact CFTR and inside cells.

3. Mutational analysis of phosphorylation sites

A major goal of current research is to understand just how phosphorylation of multiple R-domain serines by PKA effectively regulates CFTR channel function, and this encompasses a number of obvious questions. Does phosphorylation of a given site have a specific function? Or is overall phosphorylation titer the functionally important parameter? If each site does contribute to a particular function, which site is linked with what function? Site-directed mutagenesis of R-domain serines, individually or in groups, provides a means to address these questions. The essential underlying assumption is that if CFTR function is altered by mutation of a serine residue (e.g., to alanine), then that site must normally become phosphorylated. For this reason, two significant caveats must be borne in mind. First, the altered function could be merely a consequence of a change in protein structure resulting from the mutation, rather than a specific response to loss of a phosphorylation site. In this regard, Dulhanty et al. (51) found that the CD spectrum of R-domain peptide bearing Ser-Ala mutations at nine sites differed substantially from that of dephosphorylated (or phosphorylated; Ref. 53) wild-type R domain, suggesting that the mutations (at least 9 of them together) did indeed alter protein structure. Second, unless molecular function can be examined at the microscopic level and/or over a wide range of conditions, there might be little chance of discriminating among effects of the same single mutation introduced into different consensus sites one at a time.

It was presumably the latter difficulty that was responsible for the initial failure (34, 163) to detect effects on grossly measured cAMP-stimulated anion permeability of individually mutating to alanine any one of the five readily phosphorylated serines (Ser-660, -700, -737, -795, or -813), or of mutating Ser-686, -712, or -768, or even of simultaneously mutating two or three of them in several combinations. However, simultaneous mutation of four of the principal serines (660737795, and 813) to alanine (to yield the 4SA mutant) clearly reduced both cAMP-stimulated iodide efflux and CFTR channel Po , but only by approximately twofold (31, 163). Additional mutation of up to all eight dibasic R-domain serines (8SA: the 4SA mutant with Ser-686, -700, -712, and -768 also mutated to alanine), or all nine dibasic R-domain sites (9SA: the 8SA mutant with Thr-788 also mutated to alanine), further reduced PKA-activated channel function only slightly, whether assayed by iodide efflux or by channel Po (31, 163). The same assays indicated small, but measurable, functional decrements attending incorporation of additional mutations at Ser-422 to yield a 10SA mutant (31), at Ser-753 to give the 11SA mutant (178), and at four remaining R-domain serines and threonines (S670, T690, T787, and S790) mutated together to yield a (11+4)SA mutant (177). When five additional serines and threonines (S728, S742, S756, T757, and T760) were simultaneously mutated to alanine in the 11SA background to yield a 16SA mutant, there was no observable further decrease (relative to 11SA) in either phosphorylation level or iodide efflux, indicating that those five are not sites for phosphorylation by PKA (178). Corresponding levels of phosphorylation of these mutants were determined either by labeling intact cells with 32P and immunoprecipitating CFTR (31, 163) or by immunoprecipitation followed by phosphorylation in vitro (163, 178). Compared with wild-type CFTR, cAMP-stimulated phosphorylation was substantially reduced in the 4SA mutant, further reduced in the 8SA mutant, greatly diminished in the 10SA mutant, and barely detectable in the 11SA, (11+4)SA, or 16SA mutants (Table 1; Refs. 31, 163, 177, 178).

4. Do functionally significant PKA phosphorylation sites exist outside the R domain?

The majority of the phosphorylation sites identified so far occur within the R domain. The exception is Ser-422. Significantly, clear phosphorylation of Ser-422 has been demonstrated only in recombinant NBD1-R-domain peptide, and not in full-length CFTR (144). On the other hand, the clear-cut decrement in channel function seen upon adding the S422A mutation to the 9SA mutant (31) implies that Ser-422 does get phosphorylated in whole CFTR (at least, in 9SA mutant CFTR). It seems valid to question the significance of a small functional consequence of one additional point mutation made in a background in which many of the supposedly major phosphorylation sites have already been eliminated. On the other hand, it is conceivable that phosphorylation of minor, normally kinetically disfavored, sites may contribute to the regulation of wild-type CFTR channels when cellular PKA activity is sufficiently high, or when phosphatases are relatively inactive. But, it could also be that any residual phosphorylation detected in heavily mutated CFTR channels is somehow made possible by those mutations, and would not ever occur in wild-type CFTR regardless of kinase and phosphatase activity. The same kind of criticism can be leveled at the observation that PKA can still enhance activity of mutant 16SA channels that lack nearly all plausible PKA phosphorylation sites in the R domain (178). An alternative viewpoint is that the PKA-dependent activation of those heavily mutated CFTR channels (yielding Po around one-quarter of that for wild-type CFTR) is large enough to suggest that it is imprudent to ignore the role of phosphorylation sites outside the R domain. We have previously pointed out that the lack of influence of PKA on single-channel currents in mutant CFTR missing R-domain residues 708-835 (CFTRDelta R), with (163) or without (122, but cf. Ref. 164) additional mutation of Ser-660 to alanine (CFTRDelta R-S660A), does not constitute proof that all the phosphorylation sites reside in the R domain (68). It is equally possible that phosphorylation sites outside the R domain require an intact R domain for transduction of their signal. Having said that, we note that Seibert et al. (178), from comparison of autoradiographs of cyanogen bromide (CnBr) digests of R-domain peptide and of intact CFTR, favor the interpretation that all detectable phosphorylation in intact CFTR occurs within the R domain, although this once again raises the question of phosphorylation of Ser-422. Among other possibilities for as yet undetected phosphorylation sites outside the R domain is a cluster of Arg, Ser, and Lys residues near the COOH terminus of CFTR (residues 1453-1457 of human epithelial CFTR). The sequence R-X-S-S-K/R at that location is conserved across species, and recent preliminary studies have shown that a synthetic peptide including these amino acids can be phosphorylated by PKA (K. Chan and A. C. Nairn, unpublished results). The safest conclusion is that all the functionally significant PKA phosphorylation sites in CFTR have not yet been identified.

5. Roles for individual PKA phosphorylation sites

The inability to discern specific functional roles for individual PKA phosphorylation sites, whether assumed to be of major (e.g., Ser-660, -700, -737, -795, and -813) or minor (e.g., Ser-422 and -753) importance, coupled with the apparent progressive decline of channel activity as the number of Ser-Ala mutations was increased, led to the idea of functional redundancy, or degeneracy, among the phosphorylation sites (31, 163). According to this idea, only the number of phosphorylated serines, and not their specific location, governs activation of CFTR (31, 34, 163). A suggested mechanism possibly underlying such an effect is a finely graded electrostatic movement of the R domain, without any specific change in its conformation, in rough proportion to the accumulated negative charge on the phosphoserines, that movement somehow leading to a progressive facilitation of ion flux through the channel pore (163, 217). One kind of observation used to support this suggestion was the finding that insertion of six to eight negative charges in place of R-domain serines (6SD-8SD: Ref. 163; 8SE: Ref. 31) yielded channels that did not require phosphorylation to be opened by ATP (although they had substantially diminished Po). On the other hand, the finding that incorporation of only four or five negative charges left channels apparently fully dependent on phosphorylation (but also with low maximal Po) implied at least some kind of threshold phenomenon (31, 163).

Several additional arguments make the simplest, accumulated charge mechanism unlikely. As already mentioned, phosphorylation of the R domain, judged on the basis of mobility shifts of recombinant R domain on SDS-PAGE (23, 52, 141, 152), is associated with reproducible, discrete conformational changes and thus seems incremental rather than continuous. Also, strong phosphorylation by PKA alters the CD spectrum of R-domain peptide, implying changes in its secondary and tertiary structure (52). Importantly, the CD spectrum of mutant 8SE CFTR R domain differs markedly from that of phosphorylated wild-type CFTR R domain, as well as from that of dephosphorylated R domain or mutant 8SA R domain (51, 52). Perhaps most persuasive, recent results demonstrate that Ser-737 and Ser-768 both exert an inhibitory (rather than stimulatory) influence on CFTR channel activity under conditions of submaximal phosphorylation (42, 218, 220). That work examined dose-response curves for activation of CFTR channels expressed in Xenopus oocytes by IBMX [at submillimolar concentrations, most likely predominantly inhibiting cAMP phosphodiesterase (50), but at millimolar levels probably also directly stimulating CFTR channels (2, 81)] in the presence of forskolin (to activate PKA). The results showed that individual substitution of Ser-660, -670, -700, -795, or -813 with alanine increased the IBMX concentration required (K0.5) for half-maximal activation of CFTR, as expected for "stimulatory" phosphorylation sites. In general accordance with phosphorylation levels observed in intact cells, mutation of Ser-813 had the largest effect, while mutation of Ser-660 or Ser-795 had lesser effects, mutation of Ser-670 or Ser-700 had small effects, and mutation of Ser-712 or Ser-686 (a PKC site, see sect. IIB) had no measurable consequence. Significantly, mutation of Ser-737 (smaller effect) or Ser-768 (larger effect) decreased the K0.5 for IBMX, demonstrating that these are "inhibitory" sites. Recent measurements of Po of S768A CFTR channels in excised patches exposed directly to PKA catalytic subunit suggest that phosphorylation of Ser-768 somehow impedes phosphorylation of one or more serines in stimulatory sites (42). Similarly, biochemical assays have shown that phosphorylation of the other inhibitory site, Ser-737, in an R domain peptide slows phosphorylation of Ser-660, -700, -712, and -795 (109), at least three of which occur in demonstrably stimulatory sites. These findings emphasize the quantitatively and qualitatively distinct contributions made by individual phosphorylation sites, and they argue that activation of CFTR channels by phosphorylation with PKA cannot readily be explained in terms of a simple build-up of negative charges (68). On the contrary, the CFTR activating influence of phosphorylation of the prominent stimulatory site Ser-813 seems to be effectively canceled by phosphorylation of the major inhibitory site Ser-768, since the double mutant S768A/S813A had a K0.5 for IBMX comparable to that of wild-type CFTR channels (220).

This recent mutagenesis work, examining one or two residues at a time, has begun to address the question of which PKA phosphorylation sites are involved in what aspect of CFTR function, although investigation of the underlying molecular mechanisms has yet to come. The finding that several phosphoforms of CFTR can be distinguished on the basis of channel function (49, 88) strongly suggests that individual phosphorylation sites do indeed play distinct, reproducible roles in regulating channel gating, presumably by somehow controlling ATP binding and hydrolysis by the NBDs (see, e.g., Refs. 128, 224).

B. PKC

1. PKC phosphorylation sites

Although phosphorylation of CFTR by PKA is still believed to be the principal pathway for acute regulation of channel gating, it has become increasingly clear that CFTR can also be phosphorylated, and consequently regulated, by other protein kinases. Thus PKC, with or without Ca2+, phosphorylates the R domain of CFTR in vitro to a stoichiometry of ~2 mol/mol (21, 52, 152), predominantly on Ser-686 and -790 (Fig. 2), although some sites phosphorylated by PKA, like Ser-660 and -700, are also slowly phosphorylated by PKC (152). Interestingly, no change in CD spectrum (52) or in mobility on SDS-PAGE (52, 152) occurred when R-domain peptide was phosphorylated by PKC alone at moderately low levels of PKC activity. Activation of PKC by phorbol ester in intact cells resulted in phosphorylation of Ser-686 and of several PKA sites, but not of Ser-790 (152). However, it is not clear whether, in those cells, PKC directly phosphorylated PKA sites on CFTR, or simply phosphorylated specific PKC sites (e.g., Ser-686) which then somehow facilitated phosphorylation of other sites by low basal activity of PKA. In vitro, prephosphorylation of CFTR by PKC did seem to enhance subsequent phosphorylation by PKA (31, but see discussion in Ref. 99).

2. PKC influence on CFTR channel function

Protein kinase C applied directly, and in the absence of exogenous PKA, to recombinant CFTR in excised patches has been consistently reported to activate some of the channels, although weakly in many cases (21, 31, 59, 130, 194). In intact cells, stimulation of PKC with phorbol ester can activate CFTR-mediated Cl- efflux (46) as well as CFTR channels in cell-attached patches (11, 40). However, during whole cell current recording, phorbol ester alone failed to elicit CFTR current in cardiac myocytes (134) or in pancreatic duct cells (222). Nevertheless, PKC stimulation or application has invariably been found to potentiate subsequent CFTR channel activation by PKA, markedly in cell-attached or excised patches (11, 194) and moderately in intact cardiac myocytes (134) and pancreatic duct cells (222). Most surprisingly, this marked potentiation was still seen in 10SA mutant channels (31) that lack the Ser-686, -660, and -700 (but not Ser-790) sites identified as being phosphorylated by PKC in vitro. A possible implication is that PKC phosphorylation of Ser-790 enhances the functional consequence, in terms of channel activation, of PKA phosphorylation at, say, the monobasic sites Ser-753 or -670 (both spared in the 10SA mutant) or at sites not yet identified.

3. Constitutive PKC phosphorylation?

The recent conclusion (99) that constitutive phosphorylation by cellular PKC does not by itself activate CFTR channels, but is a prerequisite for their subsequent activation by PKA, is beginning to shed new light on these apparently synergistic interactions between PKC and PKA sites. After pretreatment of cells with PKC inhibitors, recombinant CFTR channels in transfected Chinese hamster ovary (CHO) or baby hamster kidney (BHK) cells (99, 117), and native CFTR channels in Calu-3 cells (117) or in cardiac myocytes (134), were almost completely, or largely, refractory to PKA stimulation by elevation of cellular cAMP. These permissive PKC sites appeared to be dephosphorylated moderately slowly by membrane-bound phosphatases upon patch excision (at least, in CHO cells), because the ability of PKA catalytic subunit to reactivate rundown channels waned steadily over an ~10-min period, although that ability could be restored by exposing the patch to PKC (99). Further analysis suggested that phosphorylation by PKC did not alter the number of active channels in a patch, but enhanced the ability of PKA to increase channel Po , primarily by reducing mean closed time (99).

4. Mechanism of PKC action

This new work strengthens the suggested modulatory role(s) for PKC in CFTR channel activation, although it is still not clear whether either form of modulation (i.e., stimulatory or permissive) reflects direct phosphorylation of CFTR by PKC, or whether such phosphorylation is essential for subsequent channel activation by PKA. It might be thought that evidence against an absolute need for PKC phosphorylation was provided by the finding that addition of only PKA and MgATP (i.e., without PKC) activated recombinant CFTR channels after their extensive purification from insect cells, reconstitution in pure phospholipids, and incorporation into a lipid bilayer (15). In fact, the same CFTR preparation was recently shown to remain partially phosphorylated [at sites sensitive to protein phosphatase (PP) 2A; see sect. IVA] throughout the purification procedure (113); although whether that basal phosphorylation occurred at PKA or PKC sites was not determined. In retrospect, a useful test might be to fully dephosphorylate the purified CFTR preparation with PP2A (perhaps together with PP2C) and then test whether PKA alone could rephosphorylate and reactivate it. Along these lines, CFTR channels in patches excised from NIH-3T3 or Calu-3 cells could be activated by either PKC alone or PKA alone, then deactivated by exposure to lambda -phosphatase (lambda -PP), and finally reactivated by application of the "other" kinase, i.e., PKA or PKC, respectively. Regardless of the sequence of phosphorylation, the kinetics of CFTR channel opening and closing were faster after phosphorylation by PKC than after phosphorylation by PKA (59). If lambda -PP could be shown to fully dephosphorylate CFTR channels under the conditions of those recordings, then the simplest interpretation of the findings would be that CFTR channels can be activated by phosphorylation either by PKC alone or by PKA alone. However, as already mentioned, PKC has previously been shown to phosphorylate some R-domain sites that are also phosphorylated by PKA (152). Also, it remains conceivable that basal activity of cellular PKC and PKA, before patch excision, was sufficient to phosphorylate key sites on CFTR which, although not themselves capable of activating CFTR channels, are not only obligatory for further phosphorylation, and hence activation, of the channels by PKA or PKC, but are also resistant to lambda -PP.

5. PKC targets other than CFTR?

Of course, we still cannot be certain whether any (or all) of these effects of PKC on CFTR channels in patches or intact cells reflect PKC phosphorylation of CFTR itself. Thus phosphorylation of Ser-686 by PKC (152) might not occur in all cell types and, even when it does occur, might not have any discernible functional consequence. Indeed, although not examined at the single-channel level, the sensitivity of mutant S686A CFTR channels in oocytes to activation by IBMX in the presence of forskolin was no different from that of wild-type CFTR (220). So it remains possible that the described effects of PKC on CFTR channel activity might be indirect, reflecting phosphorylation of other targets, such as the cytoskeleton, which could lead, via membrane retrieval (222) or insertion (11), to changes in the density of CFTR channels incorporated in the cell surface. It has recently been demonstrated that membrane-targeted syntaxin 1A and Munc-18 (two proteins involved in vesicle fusion) reciprocally regulate CFTR channel function (syntaxin 1A inhibiting and Munc-18 relieving that inhibition; Ref. 142), apparently by interacting with the NH2-terminal cytoplasmic tail of CFTR (143). New findings also show that CFTR's COOH terminus binds to PDZ domains in an anchoring protein, EBP50 [ERM(ezrin-radixin-moesin)-binding phosphoprotein 50], which in turn physically interacts with ezrin, an actin-binding protein that also anchors PKA (76, 106, 158, 185, 213). These recent demonstrations suggest other possible targets for phosphorylation by PKC and, hence, pathways for PKC-mediated modulation of CFTR channel activity.

6. PKC isoforms

Once the specific targets of phosphorylation by PKC are identified, we will need to learn which PKC isoforms are involved. Only 4 of the 11 isoforms of PKC so far identified (146) depend on Ca2+ for activity, and available evidence suggests that CFTR can be phosphorylated by PKC in both a Ca2+-dependent as well as a Ca2+-independent manner (21, 152). The first clear evidence of involvement of a specific isoform of PKC has come from the recent finding that 48-h pretreatment of Calu-3 cells with antisense oligonucleotide to PKC-epsilon (but not to PKC-delta or PKC-zeta ) prevented the normal PKA-mediated increase in 36Cl- efflux via CFTR, without affecting activity of PKA itself (117). The clear implication is that the constitutive permissive phosphorylation that facilitates activation of CFTR by PKA is carried out by Ca2+-independent PKC-epsilon . It will now be important to learn whether PKC-epsilon can directly phosphorylate CFTR in vitro and, if so, at what site(s). Also, detailed analyses of single CFTR channel responses to individual PKC isoforms will be required to learn the full range of possible mechanisms by which PKC isoforms, whether Ca2+ dependent or Ca2+ independent, and whether basally active or acutely stimulated, might regulate CFTR function in cells. Despite our present meager understanding of the underlying mechanisms, CFTR channel regulation by PKC is evidently important enough to warrant further investigation in this kind of detail.

7. PKC influence on CFTR expression

In addition to possible modulation via direct phosphorylation of CFTR, PKC appears to regulate CFTR expression and degradation. Treatment of epithelial cells with phorbol ester for several hours leads to a significant reduction in transcription of CFTR mRNA (13, 45, 200), and Ca2+ ionophores such as A-23187 or ionomycin have a similar effect (13). These effects are thought to be consequences of long-term stimulation of PKC and are mediated by phorbol ester-sensitive elements in the CFTR promoter. Prolonged exposure of epithelial cells to phorbol ester has also been found to accelerate degradation of CFTR protein (24). However, because long-term treatment of cells with phorbol ester also causes rapid downregulation of sensitive PKC isoforms, the mechanisms of these effects of phorbol esters on CFTR remain unclear.

C. cGMP-Dependent Protein Kinases

1. cGMP-dependent protein kinase isoform specificity

In vitro, both of the major cGMP-dependent protein kinase (PKG) isoforms, PKGI and PKGII, efficiently phosphorylate the R domain of CFTR (21, 63, 152) to high stoichiometry (>5 mol/mol) at sites that largely overlap those phosphorylated by PKA (63, 152). Surprisingly, however, although PKGII was found to activate CFTR channels in patches from NIH-3T3 or rat intestinal IEC-CF7 cells about as robustly as PKA did (63), the same channels could not be activated by PKGI (21, 63). The solution to this seeming paradox is that the apparent specificity of PKGII in regulating CFTR is attributable to myristoylation-dependent membrane targeting of PKGII, which gives it a crucial kinetic advantage due to its colocalization with CFTR in the plasma membrane (208-210). Thus expressed membrane-associated PKGII, but not expressed soluble PKGI, phosphorylated and activated CFTR channels when IEC-CF7 cells were stimulated with atrial natriuretic peptide, which activates guanylyl cyclase (210). Correspondingly, a chimeric PKGI incorporating the NH2-terminal membrane-anchoring domain of PKGII was able to strongly activate CFTR in stimulated IEC-CF7 cells, but activation by PKGII bearing mutations at the NH2-terminal myristoylation site was severely impaired (209).

2. Tissue-specific responses to cGMP

In intestinal epithelium, stimulation of guanylyl cyclase and resulting generation of cGMP has been suggested as the mechanism by which the hormone guanylin, and heat-stable enterotoxins secreted by pathogenic strains of E. coli, activate CFTR channels (32). In support of the proposal that PKGII selectively mediates this activation of CFTR, "knockout" mice deficient in PKGII are resistant to the influence of E. coli enterotoxin on intestinal secretion (151). Related to this, Quinton (156) has suggested that the strong activation of CFTR in intestinal epithelia, and consequent stimulation of fluid secretion, by heat-stable enterotoxins acting via cGMP (also by heat-labile toxins, like cholera toxin, that act via cAMP) provides a mechanism by which individuals heterozygous for a lethal CF mutation might have gained a genetic advantage over people harboring no mutation. Cystic fibrosis heterozygotes are likely to have been afforded some protection against life-threatening enterotoxin-induced diarrhea (32, 65), and this might explain the relatively high frequency of the Delta F508 mutation (1 in 25 Caucasians is a carrier; Ref. 204). Unlike intestinal epithelium, airway epithelium lacks the PKGII isoform (63), and cGMP does not appear to activate CFTR channels in permeabilized human airway epithelial monolayers (21).

3. More than one mechanism of cGMP action?

A rise in cellular cGMP concentration in cells expressing PKGII will likely activate CFTR channels via PKG-mediated phosphorylation (126, 209, 210). But even in cells devoid of PKGII, CFTR channels may be activated if cGMP concentrations reach sufficiently high levels to promiscuously "cross-activate" PKA (32, 61, 197). And even a moderate increase of cGMP concentration might lead to activation (or enhanced activity) of CFTR channels, also via PKA-mediated phosphorylation, in cells in which type III cGMP-inhibited phosphodiesterase (which destroys cAMP) is highly expressed (105, 147, 210). Finally, a direct activation of CFTR channels expressed in Xenopus oocytes by intracellular cGMP, via a PKG-independent pathway, has recently been suggested (193). The third cytoplasmic loop of CFTR includes a domain resembling the cyclic nucleotide-binding site(s) in proteins related to the catabolite-gene activator protein, and mutations within that domain impaired the ability of cGMP to activate CFTR channels, although their activation by cAMP seemed unaltered (193).

D. Ca2+/Calmodulin-Dependent Protein Kinases

Although purified CFTR R domain can be phosphorylated in vitro by calmodulin (CaM) kinase I (152), though not by CaM kinase II (21, 152), or CaM kinase III, or casein kinase II (152), there has been no investigation yet of possible functional consequences, either in vitro or in vivo. The highest concentrations of CaM kinase I are found in neurons of the brain (153), cells that were originally believed not to express CFTR. However, the presence of CFTR mRNA and protein has recently been demonstrated using reverse transcriptase PCR, in situ hybridization, and immunocytochemistry in human hypothalamus (138) and in several regions of rat brain, including cerebral cortex (101), hypothalamus, thalamus, and amygdaloid nuclei (137). Moreover, CaM kinase I (154) and CFTR (85) are both expressed in the choroid plexus, allowing the possibility of CFTR regulation by CaM kinase I-mediated phosphorylation at least in that tissue. Outside the brain, however, a recent study found little evidence for coexpression of CaM kinase I and CFTR in nonneuronal tissues, including the intestine (131).

E. Protein Tyrosine Kinases

1. Tyrosine phosphorylation of CFTR

Much recent work has examined the striking enhancement of CFTR channel activity caused by the tyrosine kinase inhibitor genistein (94, 95, 162, 176, 226), although only within the past year has phosphorylation of tyrosyl residues in CFTR been detected (100). Coexpression of CFTR with v-Src, which encodes oncogenic, constitutively active, tyrosine kinase, caused in vivo tyrosine phosphorylation of CFTR as detected by antiphosphotyrosine antibody, and immunoprecipitated CFTR was phosphorylated, in vitro, by the tyrosine kinase p60c-Src (100). However, concomitant functional analyses suggested that this direct tyrosine phosphorylation of CFTR increases channel activity, in contrast to the implication of the findings with the inhibitor genistein which, at first glance, would be more consistent with a tyrosine kinase-mediated reduction of CFTR channel current. Thus application of Src to excised patches increased CFTR current, while restoring fast flickery gating, in PKA-activated CFTR channels (60), and Src was even able to strongly activate CFTR channels in the presence of PKA inhibitor (100). The latter finding is strikingly corroborated by undiminished activation by Src of 15SA CFTR channels (with mutations in 15 dibasic and monobasic PKA consensus sites) despite a roughly sixfold reduction in activation of the same channels by PKA (100).

2. Influence of the tyrosine kinase inhibitor genistein

A) GENISTEIN EFFECTS. If tyrosine phosphorylation of CFTR enhances channel activity, and if tyrosine kinases were constitutively active in cells expressing CFTR, then exposing such cells to a tyrosine kinase inhibitor, like genistein, would be expected to allow endogenous tyrosine phosphatases to dephosphorylate CFTR and hence diminish channel activity. On the contrary, kinase-inhibiting concentrations of genistein have been demonstrated to increase, not decrease, CFTR channel activity in all cells tested, including NIH-3T3, IEC-CF7, HT-29/B6, HEK-293, Calu-3, and T84 cell lines, Hi-5 insect cells, and Xenopus oocytes (92, 94, 95, 162, 176, 215, 226). In these various cell types, genistein enhanced iodide efflux, macroscopic CFTR Cl- current, and time-averaged single CFTR channel current (94, 95, 162, 176, 226), as well as phosphorylation of CFTR (92, 162). It is now clear that these effects of genistein are not mediated by inhibition of tyrosine kinase and that they are absolutely dependent on prior phosphorylation of CFTR by PKA. Evidence against tyrosine kinase inhibition as the mechanism of genistein action includes the findings that its effects 1) were not mimicked by the tyrosine kinase inhibitors erbstatin, tyrphostin A23, tyrphostin A51, tyrphostin B42, or herbimycin A, in T84 and HT-29/B6 cells (94), nor by AG126, tyrphostin 25, or herbimycin, in NIH-3T3 cells (47), nor by tyrphostin 47 in Xenopus oocytes (215); 2) were not abolished or prevented by the protein tyrosine phosphatase inhibitors pervanadate or orthovanadate in patches excised from T84 or HT-29 colonocytes (47), Xenopus oocytes (215), or NIH-3T3 cells (213a); and 3) the effects were similar in direction to those resulting from direct exposure of CFTR to the active tyrosine kinase Src (100). Evidence that genistein acts on CFTR channels only after their phosphorylation by PKA includes the observations that 1) genistein had no effect in permeabilized HT-29/B6 monolayers in the absence of cAMP (94), 2) genistein enhanced CFTR channel activity in resting Hi-5 insect and NIH-3T3 cells in which the channels were already active under basal conditions (i.e., even before stimulation of PKA) but had no effect on resting cells lacking basal CFTR channel activity (62, 226), and 3) genistein's effects on CFTR phosphorylation and iodide efflux in NIH-3T3 cells were inhibited by the PKA inhibitor H-89 (162).

B) GENISTEIN SITE OF ACTION. The finding that genistein increased PKA-mediated incorporation of 32P into CFTR in NIH-3T3 cells (92, 162) prompted the suggestion that genistein might inhibit a serine/threonine phosphatase that dephosphorylates PKA sites in CFTR (92, 94, 162, 226). Because genistein's effect was additive with that of calyculin A (an inhibitor of PP1 and PP2A), a protein phosphatase distinct from PP1 or PP2A was considered a likely target of genistein, the obvious candidate being PP2C (226; cf. Ref. 162). However, the most recent studies on CFTR channels in excised patches argue strongly that genistein, in fact, interacts directly with CFTR itself. The clearest evidence is that genistein (applied in the absence of PKA) can still rapidly, and reversibly, enhance the activity of phosphorylated CFTR channels even when further PKA phosphorylation is prevented by a maximally effective concentration of peptide inhibitor (213a, 215), or after the channels have been made resistant to dephosphorylation by thiophosphorylating them, by briefly replacing ATP with adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) in the presence of PKA (62). Single-channel analysis indicates that low concentrations of genistein increase CFTR current by stabilizing the channel open state, suggesting a possible interaction with NBD2 (62, 213a, 226). At higher (>50 µM) concentrations of genistein, however, activation gives way to inhibition, indicating that genistein also binds to a lower affinity site at which it slows channel opening (213a; cf. Ref. 94). It is possible that, at high concentrations, genistein (or at least its anionic form) can also bind weakly to a site within the pore of CFTR where it may act as an open-channel blocker (111).

C) GENISTEIN MECHANISM. If genistein acts directly on CFTR channels to prolong channel opening even when no further phosphorylation is possible, how can we explain the observed genistein-induced increase in CFTR phosphorylation? The simplest explanation is that genistein acts at NBD2 to stabilize the open conformation of CFTR and that the open channel is a poorer substrate for dephosphorylation by PP2C than the closed channel (67, 92, 213a, 226). Such an action would enhance, in a PKA-dependent manner, time-averaged single-channel CFTR currents, macroscopic CFTR currents and fluxes, and the steady-state phosphorylation level of a subset of serines. An influence of channel gating on phosphorylation and/or dephosphorylation need not be surprising. Because complex interactions linking R-domain phosphorylation with NBD function are believed to underlie the regulation of CFTR channel gating, simple thermodynamic constraints require that NBD function affects phosphorylation and dephosphorylation of the R domain (84, 97, 188). Indeed, biochemical evidence for such an influence of NBD conformation on kinetics of R-domain phosphorylation by PKA has recently been obtained (145).

    III. REGULATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR BY PROTEIN PHOSPHATASES
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A. Candidate Phosphatases for Regulating CFTR

As already noted, the steady-state level of phosphorylation of a protein in vivo depends on the relative rates of phosphorylation and dephosphorylation of all of its phosphorylatable sites. In all cell types examined to date, CFTR Cl- current activated by stimulation of PKA declines rapidly upon withdrawal of the PKA agonist, indicating that highly active endogenous protein phosphatases promptly dephosphorylate and inactivate CFTR (67, 68, 165, 216). The four main types of serine/threonine protein phosphatases in eukaryotic cells are encoded by members of two gene families. The protein phosphatases PP1, PP2A, and PP2B belong to the large PPP family which ensures substrate specificity in vivo by employing a variety of regulatory subunits (also known as B subunits) to target and modulate phosphatase activity, and PP2C belongs to the PPM family of Mg2+-dependent phosphatases that lack regulatory subunits (37, 38, 184). Both PP1 and PP2A are ubiquitous components of intracellular signaling pathways, and they are both potently inhibited by naturally occurring toxins like okadaic acid, microcystin, and calyculin A. Protein phosphatase 2B, also known as calcineurin, requires Ca2+ and calmodulin for its activity and is inhibited by the immunosuppressants cyclosporin or FK-506 bound to their respective partners, cyclophilin or FKBP-12. Protein phosphatase 2C, for which no good inhibitor is known, requires millimolar Mg2+ (or Mn2+) for its activity (123). In in vitro biochemical tests using PKA-phosphorylated CFTR, incubation with purified PP2A has been shown to cause substantial (18) or nearly complete (21) dephosphorylation, whereas PP1 and PP2B were both far less effective (21). More recent studies have shown that, like PP2A, PP2Calpha can almost completely dephosphorylate PKA-phosphorylated intact CFTR as well as R-domain peptide (201). PKA-phosphorylated R-domain peptide was similarly found to be strongly, but incompletely, dephosphorylated by either purified PP2A alone or recombinant PP2Cbeta alone, although, together, PP2A plus PP2Cbeta caused complete dephosphorylation, whereas neither PP1 nor PP2B was measurably effective (141). However, little is known yet about the specificity with which these different phosphatases attack individual phosphoserines on phosphorylated CFTR.

B. Functional Effects of Exogenous Phosphatases

Purified PP2A greatly reduced the Po (from 0.31 to 0.02) of PKA-phosphorylated wild-type CFTR channels reconstituted in lipid bilayers but had no effect on the constitutive activity of CFTRDelta R (122). In patches excised from NIH-3T3 cells, direct application of purified PP2A also substantially deactivated PKA-phosphorylated epithelial CFTR channels, but PP1 and PP2B were both reported to have essentially no efffect (21). Surprisingly, however, in a more recent study, PP2B was shown to reproducibly deactivate PKA-phosphorylated CFTR channels, not only in patches excised from NIH-3T3 cells but also in patches from Calu-3 cells, and PP2B inhibitors strongly potentiated PKA-dependent activation of CFTR channels in the same NIH-3T3 cells (59). The reasons for these apparently discrepant findings with PP2B in NIH-3T3 cells remain unclear, although differences in enzyme source, and hence activity, might have contributed (59). Experiments testing all four phosphatase types on patches excised from BHK cells confirmed that PP1 is without effect, PP2B weakly inactivates, and PP2A and PP2C both strongly but incompletely deactivate PKA-phosphorylated CFTR channels (120). Because the more rapid deactivation induced by exogenous PP2C most closely resembled the pattern of channel rundown observed (1 in 4 patches showed rundown) following patch excision into PKA-free (and phosphatase-free) solution, the authors concluded that endogenous membrane-attached PP2C normally causes that rundown. Kinetic analysis confirmed that the characteristic changes in CFTR channel gating caused by purified exogenous PP2C mimicked those attending the spontaneous rundown (120). Protein kinase A-phosphorylated CFTR channels in excised patches from HeLa cells were also shown to be rapidly deactivated, although incompletely (<20% of patch current remaining), by recombinant PP2Calpha (201). In the same study, coexpression of PP2Calpha with CFTR channels in Fisher rat thyroid cells, grown as an epithelial monolayer, reduced the magnitude of short-circuit current activated by PKA agonists and accelerated its deactivation on agonist withdrawal, in comparison with monolayers expressing CFTR alone (201).

The four predominant cellular phosphatases, PP1, PP2A, PP2B, and PP2C, are not the only phosphatases capable of dephosphorylating, and deactivating, CFTR. For instance, it has been shown that exogenous alkaline phosphatase can dephosphorylate PKA-phosphorylated CFTR protein (18) and can deactivate CFTR channel currents in patches excised from CHO cells (18, 194), pancreatic cells (17), NIH-3T3 cells (21), and BHK cells (120). In addition, a variety of suggested inhibitors of alkaline phosphatase, including IBMX, theophylline, levamisole, and p-bromotetramisole, were reported to slow deactivation of CFTR channels upon patch excision (17, 18). However, there are several reasons for believing that a physiological contribution of alkaline phosphatase to CFTR regulation is most unlikely. First, and foremost, although alkaline phosphatase is localized to the apical membrane of polarized pancreatic cells, it is oriented with its catalytic domain in the extracellular milieu (17), i.e., on the opposite side of the membrane from its proposed target, the R domain. Second, such high concentrations of the various inhibitors were required (e.g., bromotetramisole was used at concentrations several orders of magnitude higher than needed to inhibit alkaline phosphatase in standard assays; Ref. 120) that their specificity must be questioned. Third, deactivation of CFTR channels by alkaline phosphatase itself occurred only at concentrations eight orders of magnitude greater than those at which PP2A and PP2C deactivated the same channels in the same patches (120), a result underscored by the known ability of alkaline phosphatase to nonspecifically dephosphorylate many protein and nonprotein substrates. Similarly, lambda -PP, the viral nonspecific serine/threonine/tyrosine phosphatase, has been shown to deactivate CFTR channels previously activated in NIH-3T3 cell patches by p60c-Src (60), PKA, or PKC (59).

C. Findings With Phosphatase Inhibitors

Evidently, test applications of exogenous protein phosphatases (or catalytic subunits of phosphatases) can tell us what these enzymes are capable of, but not necessarily what happens either in cells that naturally express native CFTR or in transfected cells expressing recombinant CFTR. Information on which cellular phosphatases actually regulate CFTR comes from use of selective inhibitors. Okadaic acid or microcystin enhanced forskolin-activated CFTR Cl- current in cardiac myocytes and slowed, and made incomplete, its deactivation on washout of forskolin (Fig. 3; Ref. 88). Okadaic acid also prevented deactivation of CFTR current in isolated sweat duct, although no enhancement of cAMP-activated Cl- current was noted (161). In NIH-3T3 cells expressing CFTR, calyculin A enhanced CFTR-dependent iodide efflux in the absence of any treatment to stimulate PKA activity, and this effect was paralleled by increased phosphorylation of CFTR measured biochemically (162). In insect cells transfected with CFTR, calyculin A had no effect by itself but increased dramatically (>17-fold) forskolin-stimulated CFTR Cl- current, which did not fully deactivate when forskolin was withdrawn in the maintained presence of calyculin A (226). A phosphorylated peptide inhibitor of PP1 was found to be ineffective in a preliminary test using cardiac myocytes, suggesting that, at least in those cells, PP2A was the target of microcystin and okadaic acid (90), although it should be noted that those agents also inhibit the novel PP2A-like protein phosphatases PP4 and PP5 (e.g., Ref. 33). The conclusion that PP1 plays no role, but PP2A plays an important role, in deactivating native CFTR channels in two of their natural environments, sweat duct epithelial cells and cardiac myocytes, corresponds well with the results of tests with exogenous phosphatases described above. However, okadaic acid did not alter the amplitude or deactivation rate of PKA-mediated short-circuit current in monolayers of human airway cells or T84 cells (201), and calyculin A similarly failed to affect PKA-mediated short-circuit current in T84 cell monolayers (120), implying a less dominant role for PP2A in airway and intestinal epithelial cells.


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  		FIG. 3.   Okadaic acid prevents full deactivation of CFTR Cl- conductance. A: whole cell current in cardiac myocyte at 0 mV showing increase of forskolin-induced Cl- conductance by 10 µM okadaic acid added to pipette (intracellular) solution and persistence of a fraction of that conductance after removal of forskolin, even after introduction of PKA inhibitor peptide (PKI). B and C, steady-state whole cell difference current-voltage relationships, obtained by appropriate subtraction of digitized records of currents elicited by 80-ms voltage pulses to ±100 mV at times indicated by letters a-h above record in A. External Cl- concentration was 150 mM; intracellular Cl- concentration was 24 mM. [From Hwang et al. (88).]

An obligatory role for PP2B in deactivation of CFTR channels in cardiac myocytes can be ruled out, because channels deactivated completely on washout of forskolin (Fig. 3, A and B, c-a) despite the fact that the intracellular solutions employed for the whole cell current recordings lacked Ca2+ and included 10 mM EGTA (88, 91). The same conclusion may be drawn from the observations that the PP2B inhibitor FK-506 neither augmented the forskolin-induced activity of epithelial CFTR channels expressed in insect cells (226) nor affected the size or deactivation rate of CFTR current in human airway, or T84, epithelia (201). In contrast, the PP2B inhibitors cyclosporin A and deltamethrin were found to strongly enhance PKA-mediated activation of CFTR current in NIH-3T3 cells, but not in Calu-3 cells or in HT-29 epithelial monolayers, despite the fact that exogenous PP2B could deactivate CFTR channels in patches excised from both NIH-3T3 cells and Calu-3 cells (59). The latter findings emphasize the danger of drawing inferences about regulatory events in intact cells by extrapolating results obtained with exogenous components, since the exogenous component, even if shown to be expressed in the cell under study, might not colocalize with the target of interest (cf. Ref. 59). The same study also demonstrated that exogenous PP2B could deactivate not only PKA-activated but also PKC-activated CFTR channels in patches from NIH-3T3 cells (59). However, the likely synergism between PKA and PKC phosphorylation of CFTR channels raises the questions of whether the PP2B deactivation of PKC-activated channels in fact reflects dephosphorylation of essential, basally phosphorylated, PKA sites and/or whether the increase in PKA-activated current by PP2B inhibition in fact reflects interference with ongoing dephosphorylation of permissive PKC sites?

D. Differential Dephosphorylation of Multiple Sites

As mentioned in the previous section, selective inhibition of PP1/PP2A in cardiac myocytes with maximally effective concentrations of okadaic acid or microcystin not only augmented CFTR current activated by forskolin and slowed deactivation following forskolin removal, as expected for inhibition of a functionally important phosphatase, but it also rendered deactivation incomplete (Fig. 3, A and C, f-d; Ref. 88). Analogous results have recently been obtained with epithelial CFTR channels expressed in insect cells (226). Because up to 50% of the CFTR current persisted indefinitely in the continued presence of those phosphatase inhibitors (although not with an inhibitor of PP1), and the persistent current was insensitive to PKA inhibition with PKI (indicating that it did not depend on continuing phosphorylation by PKA), we were able to conclude that full deactivation of native cardiac CFTR requires that certain phosphoserine residues be dephosphorylated by PP2A (88). However, the fact that partial deactivation still occurs when PP2A is fully inhibited means that some phosphatase other than PP2A can dephosphorylate additional sites on CFTR. The obvious insensitivity of that phosphatase either to the presence of PP1/PP2A inhibitors or to the absence of Ca2+ (and, hence, of PP2B activity) implies that this partial deactivation can be attributed to PP2C (68, 88, 90, 91, 226). The subsequent finding (91) that a single CFTR channel could initially open to a low Po state characterized by brief open times, but then, during continued exposure to PKA, could switch to a higher Po state with longer openings (Fig. 4), suggested an explanation for the persistent CFTR current seen following deactivation of PKA when PP2A was inhibited. The persistent current (Fig. 3, A and C, f-d) was suggested to reflect the activity of partially phosphorylated CFTR channels, phosphorylated exclusively at sites that require PP2A for their dephosphorylation and that support only limited channel activity, restricted to brief openings and, hence, characterized by a low Po . The larger current recorded in the presence of forskolin (Fig. 3, A and C, e-d) was suggested to reflect activity of the same population of CFTR channels, but additionally phosphorylated at sites which were susceptible to dephosphorylation by PP2C, and which supported longer channel openings and, hence, a higher Po (91). It is not yet clear whether, under physiological conditions (i.e., in the absence of okadaic acid or microcystin), these Po modulatory sites can also be dephosphorylated by PP2A, because no selective inhibitor of PP2C is presently available. Interestingly, rather different conclusions were reached in recent tests in which purified exogenous PP2A and PP2C were each applied to patches of membrane excised from BHK cells expressing recombinant epithelial CFTR channels (120). In those experiments PP2A and PP2C both substantially deactivated PKA-phosphorylated CFTR channels. However, in contrast to the conclusions drawn from studies of cardiac CFTR, deactivation by PP2A was associated with a reduction in the duration of channel openings, whereas PP2C seemed to cause deactivation without much effect on channel open time (120). It will be important, in future experiments, to reexamine this apparent discrepancy between phosphatase regulation of native CFTR channels in myocytes and of recombinant CFTR channels in BHK cells.


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  		FIG. 4.   Influence of reversible, partial dephosphorylation on open probability (Po) of a single CFTR Cl- channel at 0 mV in a giant patch (diameter 12-20 µm; Ref. 83) excised from ventricular myocyte. Cytoplasmic surface of patch was exposed to 2 mM MgATP with or without ~100 nM PKA catalytic subunit as indicated (solid bars). Extracellular Cl- concentration was 160 mM; cytoplasmic Cl- concentration was 4 mM. Restoration of longer duration openings (higher Po) by PKA argues that fall of Po on PKA withdrawal resulted from dephosphorylation, indicating activity of membrane-associated phosphatases. [From Hwang et al. (91).]

The above discussion has focused on dephosphorylation of channel-activating sites because, so far, little attention has been paid to the likely consequences of dephosphorylation of one of the inhibitory (220) phosphorylation sites. However, preliminary tests with a range of phosphatase inhibitors on epithelial CFTR channels in NIH-3T3 cells, and in alpha -toxin-permeabilized H29 and T84 epithelial monolayers, have suggested (58) that PP2A can dephosphorylate an inhibitory site. Additional, detailed, biochemical studies of CFTR dephosphorylation are clearly warranted. It seems particularly important to characterize the site(s) dephosphorylated by PP2A and PP2C, the two enzymes critical to the incremental activation and deactivation of CFTR that has now been established both for native cardiac CFTR channels (49, 67, 68, 90, 91) and for epithelial CFTR channels expressed in insect cells (226) and in BHK cells (120). The phosphatase(s) responsible for dephosphorylating the site(s) regulated by PKC must also be identified.

The gating pattern of cardiac CFTR channels in patches excised from myocytes can change the instant PKA catalytic subunit is washed away (e.g., Fig. 4; Ref. 91), implying near instantaneous dephosphorylation and, hence, colocalization of channels with phosphatases in the membrane patch. The same inference can be drawn from the decay of channel activity observed when a patch is excised from a stimulated BHK cell, a decay mimicked by application of exogenous phosphatases (120). In both of these cases, it was concluded that the phosphatase responsible was PP2C, and yet PP2C has generally been considered to be a soluble enzyme. However, a potential myristoylation site at the NH2 terminus of PP2C, conserved across alpha -, beta -, and gamma -isoforms, could concievably anchor PP2C at the membrane (203). New findings point to another possible mechanism by which phosphatases might be colocalized with CFTR. Several groups have recently identified a strong interaction between CFTR's highly conserved COOH-terminal three residues and one (PDZ1) of the two PDZ domains of human EBP50, or its rabbit homolog, NHERF (Na+/H+ exchanger regulatory factor) (76, 106, 158, 185, 213). Many proteins that contain, or bind to, PDZ domains are found clustered at the cell membrane in signaling complexes (e.g., Ref. 205). Cystic fibrosis transmembrane conductance regulator also binds to PDZ2 of EBP50, but with much lower affinity, raising the possibility that PDZ-motif proteins other than CFTR normally interact with PDZ2 of EBP50. Given the above evidence for colocalization of CFTR channels and phosphatases at the cell membrane, an attractive hypothesis is that one of those other PDZ-motif proteins might anchor a protein phosphatase capable of dephosphorylating CFTR (106, 185). Other domains of EBP50 are known to bind to the intermediary protein ezrin, which is both an actin-binding protein and an anchoring protein for PKA (e.g., Ref. 185). At least one other PKA-anchoring protein, AKAP79, also serves to localize a protein phosphatase, in that case PP2B (reviewed in Ref. 57).

    IV. MECHANISMS OF OPENING AND CLOSING OF PHOSPHORYLATED CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHANNELS
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A. Phosphorylation of CFTR Controls ATP Hydrolysis and Channel Gating

1. ATPase activity of intact CFTR

A series of heroic biochemical measurements has recently provided direct confirmation that purified, reconstituted, epithelial CFTR indeed hydrolyzes ATP, and does so at approximately the same rate (calculated to be on the order of 1 s-1) as that at which CFTR channels (formed, from the same purified protein, in lipid bilayers) open and close (113). This important finding supports the suggestion of a strong coupling between ATP hydrolysis and channel activity of CFTR. In keeping with the known requirement for PKA-mediated phosphorylation to see CFTR channel gating, this ATPase activity of purified CFTR was also stimulated by phosphorylation and, at 1 mM MgATP, the ATPase rate was enhanced two- to threefold by PKA. Interestingly, the ATPase activity was markedly inhibited (severalfold) by pretreating the CFTR with PP2A, implying both that CFTR was basally phosphorylated in the insect cells used for expression and that it remained so throughout the purification procedure (113). Somewhat surprisingly, this PKA-dependent stimulation of ATPase activity reflected an increased apparent affinity for ATP (with no alteration of maximal hydrolysis rate) and was accompanied by a shift of the Hill coefficient from 1 to 2 for the fully phosphorylated protein, suggested to reflect recruitment of a second ATPase site (113). By extrapolation, these results imply that even after dephosphorylation by PP2A, CFTR channels might still be capable of opening and closing at sufficiently high ATP concentration, although this has not been reported by others and, unfortunately, was not tested by Li and colleagues (113). In the same study, the CF-associated CFTR mutant G551D was shown to hydrolyze ATP at only one-tenth the rate of wild-type CFTR and, correspondingly, G551D channels opened only infrequently. A similar correspondence between CFTR's ATPase and channel gating rates was found when ATP hydrolysis was impaired by chelating Mg2+ or by adding sodium azide (113).

2. ATPase activity of isolated NBDs

The individual NBDs of CFTR, expressed as fusion proteins in E. coli, also appear capable of hydrolyzing ATP at a low rate. Slow ATPase activity has been reported for residues 433-589 of CFTR's NBD1 fused to a maltose-binding protein (estimated turnover number ~0.03 s-1; Ref. 108), as well as for NBD2 residues 1208-1399 fused to a maltose-binding protein (~0.01 s-1; Ref. 160) and also, most recently, for residues 417-830 of CFTR's NBD1 and R domain in tandem (~0.2 s-1; Ref. 87). An important source of uncertainty common to all of these estimates of ATP hydrolysis rate (including that for intact CFTR) is the lack of a reliable measure of the fraction of purified protein that is active. The rates should, therefore, probably be considered lower estimates. On the other hand, it need not be surprising if true ATPase rates of the individual NBDs in isolation should turn out to be lower than those of full-length CFTR, since important functional interactions between the two NBDs have been inferred for CFTR (e.g., Refs. 68, 91, 182) and have been clearly established for other ABC transporters such as Pgp (see, e.g., Refs. 118, 119, 181, 207) and Ars (114). However, the slower ATP hydrolysis by the NBD1 and NBD2 constructs, when compared with NBD1/R and intact CFTR, might also be attributable to inappropriate selection of domain boundaries and, hence, to use of incomplete NBD constructs possibly missing up to 60 residues from their COOH termini, according to the new crystal structure of the NH2-terminal NBD from RbsA, the ATPase of the E. coli ribose ABC transporter (7), as well as to new functional data obtained using severed CFTR molecules (29). The NBD1/R construct avoids this problem and, intuitively, seems likely to be a useful model since, in intact CFTR, PKA phosphorylation of the R domain modulates ATP hydrolysis. However, surprisingly, phosphorylation of NBD1/R (on Ser-660, -700, -712, -737, -768, -795, and -813) by PKA impaired ATP hydrolysis by increasing the half-maximal ATP concentration from 70 to 300 µM (Ref. 87), a result opposite to that obtained with intact CFTR (113). It is also not yet obvious how to reconcile these latest results with the observation that PKA phosphorylation of a slightly larger NBD1/R construct (residues 404-830) had no effect on nucleotide binding, whereas nucleotide binding markedly reduced PKA phosphorylation of R-domain Ser-660, -712, -737, -795, and -813 (145). Evidently, we still have a great deal to learn about nucleotide binding and hydrolysis at CFTR's NBDs and about the regulation of these processes by phosphorylation by PKA.

3. ATP hydrolysis is relatively slow

Although these estimated hydrolysis rates of <= 1 s-1 are relatively low for ATPases (e.g., Na+-K+-ATPase, ~100 s-1; Ref. 103), they fall well within the range of nucleotide hydrolysis by G proteins (e.g., ~0.01 s-1 for the visual G protein transducin alone, and ~1 s-1 for transducin activated by its cognate GAP, RGS9; Ref. 80), and both structural and functional considerations have led to the suggestion that the NBDs of ABC transporters might more closely resemble the catalytic sites of GTPases than of ion-motive ATPases (27, 68, 124). For instance, the extreme stabilization of the active channel-open conformation by binding of AMP-PNP shares striking analogy with the action of guanosine-5'-[beta ,gamma -imido]triphosphate (GMP-PNP) to stabilize activated G proteins. Certain key regions of amino acid sequence homology (124), and even consequences of mutations in those key regions (discussed in sect. IVD; Ref. 27), are also consistent with the idea that CFTR's NBDs might function like GTPases. However, new structural information has rendered this analogy, at least in its simplest form, no longer tenable. Thus the crystal structure of the NH2-terminal NBD of RbsA, solved at high resolution (2.5 Å), turns out to have an entirely novel fold quite unlike that of the catalytic site of G proteins or of any known GTPase or ATPase structures (7). Moreover, the ease with which NBD1 of CFTR could be threaded onto the novel structure argues that the NBDs of all ABC transporters are likely to have that same fold (7). However, by the same token, these new results imply that the catalytic mechanisms of ABC transporter NBDs may be expected to display novel features, distinct from those of other nucleoside triphosphatases.

B. How Does Phosphorylation by PKA Modify CFTR Function?

1. Stimulation of CFTR ATPase rate and channel gating

Dephosphorylated CFTR channels with an intact R domain are practically incapable of hydrolyzing ATP (113) and cannot be opened by MgATP (3, 140, 194) until they are phosphorylated by PKA, whereas mutant channels (CFTRDelta R) lacking much (residues 708-835) of the R domain can be opened by ATP without first being phosphorylated by PKA (3, 122, 163). Because 8-azido-[alpha -32P]ATP apparently labels phosphorylated and dephosphorylated CFTR channels equally well (202), this would seem to suggest that the dephosphorylated R domain acts to prevent not ATP binding but ATP hydrolysis and hence channel gating. Strictly, 8-azido-ATP labeling cannot be equated with mere binding, because the stoichiometry of labeling is unknown, and 8-azido-ATP is hydrolyzable and supports CFTR channel gating (202) and, hence, interactions between CFTR's NBDs (see sect. VA). Indeed, individual mutation of serines in certain R-domain phosphorylation sites does reduce the opening rate of single CFTR channels (224), believed governed by ATP hydrolysis (see sect. IVC). Similarly, the substantially reduced Po of 4SA and 10SA mutant channels (31, 128) was found to be associated with a reduced channel opening rate and, at least for 10SA, a reduced apparent affinity for channel activation by ATP (128). A qualitatively similar slowing of the activation of macroscopic CFTR conductance, relative to wild-type CFTR, was observed for mutant S813A channels expressed in oocytes, but because those activation rates are almost two orders of magnitude lower than channel opening rates, they must be largely governed by other processes, much slower than ATP hydrolysis (220).

2. Mechanism of R-domain action

The earliest suggestion was that the dephosphorylated R domain might itself form the gate, plugging the channel and moving aside to allow anions to permeate only upon phosphorylation (34, 104, 121, 163, 217). However, this simplest scheme is easily dismissed, because CFTRDelta R channels, from which most of the R domain has been removed, nevertheless stay closed until they are provided with hydrolyzable nucleoside triphosphate (3, 163). Moreover, even wild-type CFTR channels with highly phosphorylated R domains (i.e., in which, by hypothesis, channel block should be relieved) do not open unless they are exposed to MgATP. So, PKA-phosphorylated CFTR channels are gated open by a process that requires hydrolyzable nucleoside triphosphate, and direct measurements have now shown that phosphorylation by PKA enhances ATP hydrolysis by purified CFTR (113).

3. R-domain phosphorylation by PKA causes a conformational change

The R domain cannot be the proximal gate that opens and closes a CFTR channel. Perhaps we should consider the R domain to be a secondary, distal gate which, when dephosphorylated, inhibits opening and closing of the proximal gate, but which, when phosphorylated, is permissive of normal gating? Even if the R domain is envisioned as a secondary gate, as already discussed (sect. IIA5), two kinds of results argue against a simple graded movement of that gate driven electrostatically by buildup of negative charge due to incorporation of phosphate. First, PKA phosphorylation of the R domain results in a measurable change in its secondary structure, and hence in its conformation (52). Second, phosphorylation of the stimulatory site Ser-813 seems to cancel the effect of phosphorylation of the inhibitory site Ser-768 (42, 220), although both must be expected to contribute negative charge.

Recent results obtained with CFTRDelta R channels have challenged the idea that the dephosphorylated R domain is simply an impediment to channel gating that is removed when the R domain becomes phosphorylated. Ma et al. (122) and Winter and Welsh (224) both found that the intrinsically low Po of CFTRDelta R channels was somewhat increased by phosphorylated, but not by dephosphorylated, exogenous R-domain peptide, although only to a level still two- to threefold smaller than the Po of wild-type CFTR channels. This stimulation of CFTRDelta R channel activity led these authors to conclude that phosphorylation of the R domain might have two effects, a release of the inhibition imposed by the dephosphorylated R domain and a stimulation of the interaction between ATP and the NBDs. Caution may be warranted in interpreting the behavior of CFTRDelta R channels, however, because in generating this construct, two amino acids, 707 and 836, possibly distant in three-dimensional space in wild-type CFTR, have been brought together. It seems reasonable to worry that, as a result, the structure of the mutant CFTR protein might have been altered also in regions outside the R domain and that this structural distortion might be responsible for some of the observed effects, including the extremely low Po of the CFTRDelta R channel even after maximal activation in the presence of phosphorylated exogenous R-domain peptide.

C. Distinct Functions of the Two NBDs

1. CFTR channel opening is linked to ATP hydrolysis

It is now generally accepted that PKA-phosphorylated CFTR channels require a continuous supply of ATP or another hydrolyzable nucleoside triphosphate to keep opening and closing (Fig. 5). In patches of membrane excised from transfected NIH-3T3 and HeLa cells, for instance, after the CFTR channels had been phosphorylated by PKA, they could be opened by the nucleoside triphosphates, ATP, GTP, ITP, UTP, CTP, or adenosine 5'-[alpha ,beta -methylene]triphosphate (AMP-CPP), but not by analogs from which the gamma -phosphate cannot be released by hydrolysis, like AMP-PNP (Fig. 5), adenosine 5'-[beta ,gamma -methylene]triphosphate (AMP-PCP), or ATPgamma S, nor by analogs that lack a gamma -phosphate, like ADP, or cAMP (3). At the single-channel level, the Po was found to be somewhat lower when the channels were exposed to GTP than when the same channels were exposed to the same concentration (0.5 mM) of ATP (140), a result likely attributable to differences in apparent affinity among some of these nucleotides (228). This relative promiscuity of the catalytic sites of CFTR in accepting a range of nucleotides is comparable to, and possibly even exceeds, that of the catalytic sites of Pgp (e.g., Ref. 206). The inability of the nonhydrolyzable analog AMP-PNP to open phosphorylated CFTR channels (Fig. 5; Refs. 3, 26, 140, 174) is most instructive, because AMP-PNP closely resembles ATP structurally (even in terms of bond lengths and bond angles; Ref. 227). The inescapable conclusion is that mere binding of ATP is insufficient to cause opening of CFTR channels. Because none of the nonhydrolyzable analogs is capable of opening a CFTR channel, the minimum requirement for channel opening must be, at the very least, the attainment of the transition state for hydrolysis of the nucleoside triphosphate (cf. Ref. 1). A further demonstration that ATP binding alone is inadequate to open CFTR channels is the failure of phosphorylated channels to open in the presence of supersaturating concentrations of ATP (2 mM; nearly 2 orders of magnitude higher than the half-maximal MgATP concentration for channel opening; e.g., Ref. 211) if Mg2+ are absent (3, 48).


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  		FIG. 5.   5'-Adenylylimidodiphosphate (AMP-PNP) stabilizes open conformation of cardiac CFTR Cl- channels activated by PKA catalytic subunit plus MgATP in an excised patch containing 3 active channels. Current recording at 0 mV showing response to 500 µM MgATP alone or with 100 nM PKA or 500 µM AMP-PNP, as indicated by bars. AMP-PNP alone failed to open phosphorylated channels, but AMP-PNP plus ATP locked all 3 channels in a stable open conformation for several minutes, greatly delaying their closure after washout of all nucleotides. [From Hwang et al. (91).]

Strong evidence suggesting strict coupling between channel opening and ATP hydrolysis came from analysis of the extreme stabilization of the open conformation of CFTR channels caused by the Pi analogs orthovanadate and beryllium fluoride (14). These analogs are known to interrupt ATPase cycles, but only after the ATP has been hydrolyzed, by binding tightly in place of the released hydrolysis product Pi (28). The inferred trapping in NBD1 of the other hydrolysis product, ADP, by vanadate or beryllium fluoride (14) has now been clearly established for the NBDs of Pgp (170). Since Baukrowitz et al. (14) found that vanadate locked open CFTR channels only in the presence of ATP, that vanadate did not bind tightly to closed CFTR channels, and, in preliminary tests, that closed CFTR channels exposed to vanadate plus ADP failed to generate stabilized openings, we can conclude that the tight binding site for vanadate exists only on an open CFTR channel and, hence, that ATP must have already been hydrolyzed when the channel is open. Kinetic analysis of the interaction between vanadate and open CFTR channels over a wide range of Po values further implied that channel opening is immediately followed by Pi release (14) which, in turn, implies that ATP hydrolysis precedes, or accompanies, channel opening. Fully consistent with the conclusion that channel opening requires ATP hydrolysis is the finding that increases in MgATP concentration enhance the Po of phosphorylated CFTR channels almost exclusively by reducing the length of time the channels stay closed, i.e., by increasing the rate at which they open (48, 74, 211, 223). Moreover, the finding that, at 2 mM ATP, channels open more slowly at low (µM) than at high (mM) free Mg2+ concentrations (48) strongly argues that CFTR channel opening is rate limited by ATP hydrolysis, not ATP binding (75). The obligatory link between CFTR channel gating and ATP hydrolysis was further demonstrated by the abrogation of both activities in purified CFTR by removal of Mg2+ with 1-5 mM EDTA, or by addition of 1 mM Na azide (113).

2. Closing of CFTR channels is also mediated by ATP hydrolysis

It is now clear that ATP hydrolysis controls not only channel opening, but also channel closing. Thus, although AMP-PNP by itself cannot open CFTR channels, we were surprised to find that addition of 500 µM AMP-PNP to the 500 µM ATP bathing the patch caused first one, then a second, and finally all three CFTR channels to become "locked" in the open conformation (Fig. 5; Refs. 25, 74, 91; but cf. Ref. 26). The channels remained locked open for several minutes, even after washout of all nucleotides, in sharp contrast to the rapid closure of the same three channels a few minutes earlier following withdrawal of ATP (Fig. 5). When a locked channel eventually closes, ATP addition once again causes normal opening and closing (91), suggesting that the AMP-PNP is then no longer bound. The long dwell time of the channel in the locked open state therefore provides a measure of the dwell time of AMP-PNP on the channel, and its reciprocal gives an estimate of the dissociation rate of AMP-PNP, which is extremely low, implying tight binding. Thus AMP-PNP is apparently able to bind tightly at one NBD in CFTR, but only after ATP hydrolysis at the other NBD has opened the channel. Because its structure is similar to that of AMP-PNP (227), ATP should also bind tightly at the AMP-PNP site. The far shorter open times of CFTR channels in the presence of ATP (seconds, rather than the minutes seen with AMP-PNP) may thus be attributed to the fact that ATP can be hydrolyzed, whereas AMP-PNP cannot. This realization led to the conclusion that not only is ATP hydrolysis needed for opening a CFTR channel, but closing of the channel may also depend on hydrolysis of a second ATP (91). Similar prolongation of the channel open state by ATPgamma S (74), pyrophosphate (PPi; Refs. 25, 74), and tripolyphosphate (74), all of which require the simultaneous presence of ATP for their effect, further supports the conclusion that ATP hydrolysis at a second catalytic site normally permits closing of CFTR channels; presumably, when these poorly hydrolyzable analogs bind at that site, they preclude the conformational change that attends nucleotide hydrolysis and that prompts channel closing.

The demonstration that CFTR channels both open and close more slowly at low micromolar levels of free Mg2+ (48, 75, 173) strongly supports this picture of ATP hydrolysis at one site controlling channel opening and at a second site controlling closing. Indeed, once a CFTR channel has opened in the presence of low free Mg2+, washout of all unbound ATP and Mg2+ can leave the channel trapped in the open state for tens of seconds. Such channels could be closed by adding back solely Mg2+, and they closed at a rate that increases with free Mg2+, implying involvement of ATP hydrolysis, even though the bathing solutions contained no nucleotide. We conclude that, like AMP-PNP, ATP can remain (for over a minute) tightly bound to the second site on the open channel, even without a Mg2+ (48). The finding confirms that this second site must have a very high affinity for nucleoside triphosphates and, unless it can accommodate more than one ATP at a time, that channel closure must be stoichiometrically coupled to hydrolysis of a single ATP at that site. It appears, then, that ATP hydrolysis represents the rate-limiting step both for channel opening and for channel closure and that these two hydrolysis events occur at two different sites on a single CFTR channel.

D. Which NBD Opens, and Which Closes, a CFTR Channel?

1. Motifs for mutation

The obvious candidates for the two ATP binding and hydrolysis sites are CFTR's two NBDs, an assumption recently confirmed by site-directed mutagenesis. Each NBD contains a conserved Walker A sequence GXXXXGKT (212), the P-loop which curves around the beta - and gamma -phosphates in known nucleoside triphosphatase structures. By analogy with results from other ATPases and GTPases, substitutions at the lysine residues in these sequences in CFTR's NBD are expected to impair their ability to hydrolyze, although not necessarily to bind, ATP (e.g., Ref. 9). Each NBD also includes a consensus Walker B sequence, terminated by several hydrophobic residues and a conserved Asp believed to help coordinate the Mg2+ required for nucleotide hydrolysis. Mutation of this Asp may be anticipated to destabilize binding of the catalytic Mg2+ and hence slow ATP hydrolysis (e.g., Ref. 102). A third conserved motif, LSGGQ, which has come to be known as the ABC transporter signature sequence, occurs between the Walker A and B sequences and has been suggested to play a role in coupling ATP hydrolysis to transport (93, 186). In CFTR, naturally occurring mutations at the Gly preceding the Gln in this motif, G551 in NBD1 and G1349 in NBD2, are associated with CF disease. The crystal structure of the RbsA NBD indeed confirms that the Walker A loop cradles the phosphates and the Walker-B Asp contacts the Mg2+, and it locates the LSGGQ motif outside that catalytic site in a helical region that could interact with the transmembrane domain (7). Functional analyses of CFTR channels bearing point mutations in these three key regions have begun to clarify the roles of the two NBDs in channel gating.

2. NBD1 opens; NBD2 closes

Nucleotide-binding domain 1 was originally suggested to be the site of the ATP hydrolysis event coupled to CFTR channel opening, and NBD2 to be where ATP hydrolysis closes the channel (14, 67, 68), for several reasons. First, disease-associated NBD1 mutations are more numerous (204) and severe (71) than NBD2 mutations (although, admittedly, many NBD1 mutations interrupt trafficking, rather than gating; Ref. 217). Next, ATP was still able to open CFTR channels with a mutation, K1250M, in the NBD2 Walker A motif that was expected to impair ATP hydrolysis there (3). Finally, mutations in the conserved motifs that were anticipated to interfere with NBD function were found to favor the channel closed state when introduced in NBD1 but to favor the channel open state when introduced into NBD2 (218, 219). Detailed functional analysis of CFTR mutants with alterations at the Walker A lysines largely support that assignment of NBD1 to the crucial role in channel opening, and of NBD2 to channel closing. Thus mutations at K464 in NBD1 were found to slow opening of individual CFTR channels in patches or bilayers and activation of CFTR Cl- conductance in whole cells, whereas replacement of K1250 in NBD2 slowed channel closing and deactivation of the Cl- conductance (25, 75, 219). Kinetic analysis of consequences of mutations at other conserved NBD residues, G551 and D572 in NBD1, and G1349 and D1370 in NBD2, in terms of macroscopic rates of activation and deactivation of CFTR channels, were also qualitatively consistent with this assignment (219).

Further support has come from mutations made in the ABC signature sequence. The LSGGQ motif in ABC NBDs shares sequence similarity with the Switch II region of G proteins, which includes the consensus sequence DXXGQ (124). X-ray crystallographic studies have shown that the highly conserved Gln in this G protein sequence (Q61 in p21ras, Q204 in Gialpha , Q200 in Gtalpha , Q227 in Gsalpha ) contributes importantly to stabilization of the GDP-AlF4 bound transition state (39, 149, 189), but not to stabilization of the guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S)-bound active state. As might be expected, substitutions at this Gln impair GTP hydrolysis (e.g., Ref. 189). Intriguingly, although the ABC signature motif in NBD1 of CFTR includes Q552, the corresponding residue in NBF2 is not a Gln, but a His (H1350). Moreover, the severalfold slower GTPase rate of the elongation factor, EF-Tu, compared with p21ras has been attributed to the analogous EF-Tu residue being a His instead of a Gln (19) and that reduced GTPase rate is comparable to that of the mutant p21ras(Q61H) (98). In apparently striking concordance with these results from G proteins, introduction of the mutation Q552H into NBD1 of CFTR slowed CFTR channel opening without affecting closing, whereas making the converse mutation, H1350Q, in NBD2 accelerated channel closing without influencing the channel opening rate (27). The clear results of this bold experiment strongly support the assignment of ATP hydrolysis at NBD1 to a role in channel opening and at NBD2 to channel closing, and they simultaneously strengthened the notion that G proteins and the NBDs of CFTR share both structural and functional similarities (27, 68, 124). Unfortunately, however, as already mentioned, the crystal structure of the RbsA NBD has now revealed that the LSGGQ motif lies far (>= 15 Å) from the catalytic site (cf. Ref. 135) and that the NBD displays a novel fold (7). Although this would seem to rule out the postulated close structural relationship between G proteins and CFTR's NBDs, the assignment of their roles in channel opening (NBD1) and closing (NBD2) still seems secure. Indeed, channel opening rate and ATPase rate are both markedly reduced in purified, reconstituted, mutant CFTR bearing the mutation G551D in the same LSGGQ motif of NBD1 (113). Considered together, at face value these results imply that ATP hydrolysis at the catalytic site of NBD1 in CFTR is strictly coupled, in a reciprocal manner, to conformational changes that occur some distance from the catalytic site and that are relayed to the gate controlling the anion pore. Consistent with this interpretation, an analogous mutation (G534D) in human Pgp, in which drug interaction sites are believed to lie in the transmembrane domains (e.g., Refs. 72, 183), has been found to abolish drug-stimulated ATPase activity (12).

    V. WORKING MODEL OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR'S CATALYTIC AND GATING CYCLES AND INFLUENCE OF PHOSPHORYLATION
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A. Catalytic Cycles of the Two NBDs

1. The two NBDs interact

A simplified working model that links opening and closing of PKA-phosphorylated CFTR channels to the catalytic cycles of their NH2- and COOH-terminal NBDs, as just discussed, is presented in Figure 6 (see legend to Fig. 6 for full description). The model encapsulates the conclusions drawn from the results described above that, at least for a highly phosphorylated CFTR channel (the complex influence of phosphorylation on channel gating is discussed in sect. VB), a conformational change attending ATP hydrolysis at NBD1 rate limits channel opening, and hydrolysis of a second ATP at NBD2 rate limits channel closing (14, 68, 91). Further details of the model are based firmly on our findings with AMP-PNP (Fig. 5). Given the established roles of NBD1 and NBD2, the record in Figure 5 shows that although AMP-PNP fails to open CFTR channels and hence does not interact productively with NBD1 (see sect. VC for possible inhibitory action), AMP-PNP can interact with NBD2, locking the channels open for several minutes, but only once the channels have been opened by hydrolysis of ATP at NBD1. These prolonged openings provide evidence of protracted dwell times of AMP-PNP at NBD2, implying very tight binding of AMP-PNP to NBD2 in open channels, and this conclusion allows further analysis of the experiment in Figure 5. Thus, when the three closed channels were exposed to AMP-PNP alone they did not open, but if the high-affinity NBD2 site had been accessible on the closed channels, those sites should have bound AMP-PNP tightly and then held onto it for several minutes. That AMP-PNP should have remained bound at NBD2 throughout the following brief wash period, and so the subsequent application of MgATP alone should then have caused the channels to become locked open, just as happened when the ATP and AMP-PNP were applied concurrently a couple of minutes later. However, the channels opened and closed normally during that exposure to MgATP alone, although they did become locked open later when exposed to ATP and AMP-PNP together (Fig. 5). We can draw two important conclusions from these results. First, the high-affinity nucleotide site at NBD2 seems not to exist, or is not accessible, when the channel is closed. Second, because that tight binding site does become available once ATP has been hydrolyzed at NBD1, there must be strong coupling, direct or indirect, between CFTR's two NBDs, in this instance passing a signal from NBD1 to NBD2.


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  		FIG. 6.   Simplified model showing proposed catalytic cycles of CFTR's nucleotide binding domains (NBDs), emphasizing strict, sequential interactions between NBDs reminiscent of interactions between G proteins and their cognate dissociation inhibitors and exchange factors. Two NBDs, NBD1 (NH2 terminal) and NBD2 (COOH terminal), are represented as freely accessible (circles) for binding or release of nucleotide or closed (squares), entrapping ATP hydrolysis product ADP. In a highly phosphorylated CFTR channel, hydrolysis of ATP at NBD1 (top right) is proposed to open both Cl- pore and NBD2 catalytic site (now a circle), permitting dissociation of ADP from NBD2 and binding of more plentiful ATP. Tight binding of ATP at NBD2 is then proposed to close NBD1 catalytic site (now a square; bottom right) around its hydrolysis product ADP, so stabilizing channel open state. Cycle is completed by ATP hydrolysis at NBD2, which opens NBD1, so permitting release of ADP from NBD1 that we have postulated is a requirement for channel closure (14, 91). Closing of channel is proposed to trap newly formed ADP in NBD2. Diagonal arrow indicates proposed pathway by which poorly phosphorylated (i.e., largely dephosphorylated) channels may close, resulting in abbreviated openings (e.g., Fig. 4, after PKA withdrawal; Fig. 3Af, after forskolin withdrawal), and precluding stabilization of open state by ATP, or AMP-PNP, binding at NBD2. [Modified from Senior and Gadsby (182).]

2. ATP binding at one NBD might trap ADP in the other

The trapping of ADP by orthovanadate and beryllium fluoride, inferred for CFTR (14) and demonstrated for Pgp (170), is associated with marked stabilization of the channel open state of CFTR, suggesting that the hydrolysis product Pi dissociates from NBD1 once the channel is open, leaving ADP in the catalytic site of the still open channel. This implies that it is the eventual release of that ADP from NBD1 that causes the channel to close. If so, the extreme stabilization of the channel open state caused by tight binding of AMP-PNP to NBD2 can reasonably be interpreted as a prolonged inhibition of that dissociation of ADP from NBD1. Likewise, the very long channel openings observed at low free Mg2+ concentration, taken as evidence that ATP also binds with high affinity at NBD2 (48, 75), are interpreted as reflecting a similar prevention of ADP dissociation from NBD1. On the other hand, at high (e.g., 1 mM) free Mg2+ concentration, efficient hydrolysis of the ATP tightly bound at NBD2 is envisaged as causing a conformational change that permits the release of ADP from NBD1, so allowing the channel to close. Such a mechanism would again imply strict coupling between the two NBDs, in this case signaling from NBD2 to NBD1. We have previously pointed out that the apparent stabilization of an active (open channel) state by nucleoside triphosphate (ATP or AMP-PNP) binding at NBD2 and termination of that state by hydrolysis of the nucleotide (ATP) are characteristics reminiscent of G proteins (68). Moreover, the suggested actions of NBD2 to first inhibit, and then to promote, the release of ADP from NBD1, depending on whether NBD2 has merely bound, or hydrolyzed, its nucleoside triphosphate (ATP) invite comparisons with the actions of dissociation inhibitors (GDI) and nucleotide exchange factors (GEF), respectively, to regulate the activity of G proteins (22). Along the same lines, to account for the inability of AMP-PNP to bind tightly to the closed state of the channel, the model in Figure 6 incorporates our proposal that the conformational change associated with channel closing inhibits the dissociation of ADP from NBD2. Hydrolysis of ATP at NBD1 associated with the next opening of the channel is postulated to terminate that inhibition, leading to the exchange of the ADP that had been trapped at NBD2. Despite the recent evidence (7) that the NBDs of CFTR are unlikely to closely resemble G proteins in terms of molecular structure, we nevertheless find it instructive to consider these inferred interactions between the two NBDs in terms of the well-documented interactions among GTPases, and their GDIs and GEFs.

3. Comparing catalytic cycles of CFTR and Pgp

For comparison, recent biochemical experiments using wild-type and mutant Pgp have clearly demonstrated that both NBDs of Pgp hydrolyze ATP and that the two NBDs display mandatory positive catalytic cooperativity (reviewed in Refs. 179, 182). This ultimate in strong interaction is evident as complete abrogation of ATP hydrolysis by both NBDs in response to mutation (9, 119, 139, 207), or chemical modification (181), of the Walker A Lys in either NBD. Indeed, analysis of photolabeling by vanadate-trapped 8-azido-[alpha -32P]ADP showed that mutation or modification of either NBD prevented even a single turnover of nucleotide in the other NBD (181, 207). This and other evidence shows that Pgp's two NBDs are functionally identical and that, in intact Pgp, their interaction is so strong that neither can function as a catalytic site independently of the other. Although the molecular basis for this catalytic interaction is not yet established, coimmunoprecipitation assays have revealed direct physical association between the two NBDs of Pgp (118). Comparably tight physical and functional interactions have been demonstrated between the NBDs of the bacterial Ars ABC transporter, although in that case the functional unit is inferred to be a dimer incorporating four NBDs which pair up to form two catalytic sites for ATP hydrolysis (114). In contrast to the conclusions from studies of Pgp, present evidence suggests that although the NH2- and COOH-terminal NBDs of CFTR do interact (as described above; Fig. 6), they are structurally and functionally distinct. For example, mutations (or chemical modification; Ref. 41) in the Walker A (25, 75, 219) or LSGGQ (27, 219) motifs of NBD1 or NBD2 affect channel function differently, and a single mutation in neither motif fully abolishes ATPase activity (113, 159) or channel activity (5, 25, 75, 113, 159, 218, 219). The origins of these functional similarities and differences between CFTR and Pgp have yet to be uncovered, but they represent important subjects for future investigations.

B. Modulation of CFTR Channel Gating by Incremental Phosphorylation

1. Highly phosphorylated channels

A great oversimplification of the gating model of Figure 6 is that it ignores mounting evidence that multiple PKA-mediated phosphorylation events, currently believed to occur predominantly in the R domain, regulate the functioning of the two NBD as well as the interactions between them (e.g., Refs. 68, 88, 91). The scheme in Figure 6 is proposed to represent the gating of highly phosphorylated CFTR channels, corresponding to those believed to underlie the large whole cell CFTR current seen in the presence of forskolin (Fig. 3, A, B, b-a, and C, e-d) or to the high Po state (characterized by relatively long openings) seen during exposure of a single CFTR channel to PKA (Fig. 4). As discussed, we interpret that gating pattern as indicating that upon channel opening, caused by ATP hydrolysis at NBD1, tight binding of ATP to NBD2 stabilizes the open state until it is terminated by hydrolysis of the ATP at NBD2. Because of these dual hydrolysis events, both channel opening and channel closing are slowed if the free Mg2+ concentration is lowered (48, 75, 173), and AMP-PNP binding to NBD2 can lock the channels in the open state.

2. Partially phosphorylated channels

Figures 3 and 4 also provide evidence for a second gating pattern, that attributable to partially phosphorylated channels, corresponding to those underlying the residual whole cell CFTR current that persists after washing away PKA agonists in myocytes (Fig. 3, A and C, f-d and g-d) or insect cells (226) treated with inhibitors of PP1 and PP2A, or to the low Po state (characterized by relatively brief openings) seen after withdrawal of PKA from a single channel in an excised patch (Fig. 4). We have proposed that when those partially phosphorylated channels are opened by ATP hydrolysis at NBD1, ATP does not bind tightly to NBD2, so the channels can close quickly (presumably by release of the NBD1 hydrolysis products Pi and ADP; Fig. 6, diagonal arrow) because they do not have to wait for ATP hydrolysis at NBD2. Nor does AMP-PNP bind to NBD2 on these partially phosphorylated channels, and they do not (or do not readily) become locked open by AMP-PNP in the presence of ATP (91; cf. Ref. 130). Effectively, this abbreviated gating cycle of partially phosphorylated channels allows prompt closure of a just-opened channel, before ATP can replace the ADP at NBD2.

3. Moderately phosphorylated channels

The relationship between these partially and highly phosphorylated forms of CFTR is schematically illustrated in Figure 7, which emphasizes the functional state of the two NBDs. Figure 7 also incorporates two additional conformations of CFTR, an intermediate, moderately phosphorylated state and the dephosphorylated state. As already discussed, dephosphorylated CFTR channels hydrolyze ATP only poorly, if at all, and cannot be opened by exposure to MgATP alone. Although partially phosphorylated channels show only brief openings, and highly phosphorylated channels undergo only long openings, moderately phosphorylated channels can show a mixture of both kinds of openings. In current recordings of single channels in patches excised from myocytes, shortly after washout of PKA there is sometimes a period of quasi-stability during which both kinds of openings can be discerned (indicative of the moderately phosphorylated state), despite the progressive dephosphorylation of sensitive sites by endogenous phosphatases (microcystin resistant and Ca2+ independent, and hence presumably PP2C) (49). In terms of the gating scheme of Figure 6, this mixed behavior of a moderately phosphorylated channel can be envisaged as reflecting a condition in which opening of the channel by ATP hydrolysis at NBD1 may, or may not, be followed by tight binding of ATP at NBD2 which, in turn, will determine whether or not closing of the channel is rate limited by hydrolysis of that ATP. In other words, the rates of the two pathways leading away from the just-opened channel state are assumed to be comparable.


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  		FIG. 7.   Cartoon of proposed regulation of CFTR channel gating by incremental phosphorylation. Sequential, incremental phosphorylation of at least three sites (P-), possibly in R domain (R), progressively enhances Po of a CFTR channel by regulating likelihood of ATP binding tightly to, and being hydrolyzed by, NBD2. In dephosphorylated channel (left), neither NBD can hydrolyze ATP and channel remains closed. In a partially phosphorylated channel (2nd from left), phosphorylated at a site(s) that requires protein phosphatase (PP) 2A for dephosphorylation, NBD1 can hydrolyze ATP, whereupon channel opens, but ATP does not bind tightly to NBD2 so channel closes rapidly, yielding only brief open times and a low Po . In a moderately phosphorylated channel (3rd from left), additionally phosphorylated at a site(s) that can be dephosphorylated by an okadaic acid-insensitive phosphatase, likely PP2C, ATP hydrolysis at NBD1 opens channel whereupon ATP may bind tightly to NBD2 and be hydrolyzed there, yielding some longer openings. However, during many openings, ATP is not bound and hydrolyzed at NBD2, yielding a proportion of brief openings and an overall intermediate Po . In highly phosphorylated channels (right), further phosphorylated at a site(s) also likely dephosphorylated by PP2C, ATP hydrolysis at NBD1 opens channel whereupon ATP invariably binds tightly at, and is hydrolyzed by, NBD2, yielding exclusively long openings and a high Po . [From Gadsby and Nairn (69).]

4. Incremental phosphorylation

Viewed in this way, incremental PKA-mediated phosphorylation of a single channel controls the likelihood that a prolonged opening will occur, by modulating the probability that ATP will bind at NBD2, and so stabilize the open state, before dissociation of the hydrolysis products (Pi and ADP) from NBD1 can close the channel (Fig. 6, diagonal arrow). The phosphorylation status of at least one site (or group of sites) seems to control whether or not ATP hydrolysis can occur at NBD1, and hence whether the channel can open. The phosphorylation status of an additional site (or group of sites) seems to determine whether NBD2 will have the opportunity to tightly bind ATP, and the phosphorylation status of yet another site (or group of sites) appears to regulate whether that tight binding occurs upon every opening of the channel or follows only some of the openings. Preliminary data appear to support a prediction of this general scheme, which is that the phosphorylation status of a channel ought to influence the ability of a reduction in the free Mg2+ concentration to slow channel closing (48). Recent observations of slowed opening of CFTR channels with individual R-domain Ser-Ala mutations (224), or even of 10SA mutant channels (31, 128), imply that still other PKA phosphorylation sites modulate the rate of ATP hydrolysis at NBD1. An important goal of future work will be to identify the site (or sites) associated with each of these kinds of channel behavior. It will be interesting to learn which, if any, of these functionally distinct phosphorylated states corresponds to each of the different bands seen on acrylamide gels of phosphorylated R domain (23, 109, 141, 152). It will also be interesting to learn whether the incremental phosphorylation occurs strictly sequentially, as implied by the scheme in Figure 7, or more randomly (see Refs. 88, 226). Preliminary mass spectrometric analysis (109, 141) as well as functional analysis (42) suggest that indeed some of the phosphorylation events may be sequential, although the precise nature and mechanism of the ordering remain to be determined. In keeping with the proposed role of PKA-mediated phosphorylation, presumably largely within the R domain, in modulating the function of NBD2, it has recently been shown that neither AMP-PNP nor PPi (both of which are believed to bind tightly at NBD2) is able to lock open CFTRDelta R channels (122; but see above caveat concerning the CFTRDelta R construct; sect. IVB3).

Recent analyses of the dependence on temperature of the opening and closing rates of CFTR channels have demonstrated a somewhat stronger influence of temperature on opening than on closing (2, 130). This finding is consistent with the schemes in Figures 6 and 7, since every opening is expected to be rate limited by ATP hydrolysis, whereas only a fraction (which varies with phosphorylation status) of the closings are expected to be rate limited by ATP hydrolysis. Furthermore, that differential influence of temperature on channel opening and closing provides corroborative evidence that these two processes reflect different mechanisms and that channel closing does not occur simply by a reversal of the opening; in other words, the gating transitions are part of a cycle (130).

C. Lingering Uncertainties

1. Flies in the ointment?

Although, as discussed, the functional consequences of mutagenesis in the NBDs of CFTR support the generally accepted roles for NBD1 in channel opening, and for NBD2 in channel closing, Gunderson and Kopito (74) proposed, on the basis of two of their results, that it is ATP binding to NBD2 that actually opens the channel. First, they noted that the opening rate of mutant K464A CFTR channels was only approximately twofold lower than that of wild-type CFTR, whereas the equivalent mutation in other nucleoside triphosphatases (NTPases) reduces the hydrolysis rate by several orders of magnitude. However, it has recently been shown that the structure of an ABC transporter NBD is not like that of other NTPases (7), and direct measurements have revealed that the mutation K464A in purified CFTR slows ATP hydrolysis only less than twofold (159). On the other hand, it remains puzzling that, as already discussed, the equivalent mutation, or chemical modification, of the NBD1 Walker A Lys in Pgp does strictly abolish ATPase activity (9, 118, 139, 181, 207), although perhaps this discrepancy is consistent with other noted differences between the catalytic cycles of CFTR and of Pgp (182). Second, mutant G1247D/G1249E CFTR channels bearing negative charges in the NBD2 P loop, designed to impair nucleotide binding there, were able to open only very rarely and very briefly (75). The structural consequences of those mutations remain unclear, however, and given the mounting evidence for strong interactions between CFTR's NBDs, some functional consequence for NBD1 activity should not be ruled out.

K1250A CFTR channels bearing a mutation at the Walker A Lys of NBD2, expected to impair ATP hydrolysis there, have been reported to display slowed opening as well as the slowed closing anticipated for openings stabilized by tight binding of ATP at NBD2. The overall result is that Po of K1250A CFTR is reduced to about one-half that of wild-type CFTR channels (25, 159) and that the ATPase rate is even more markedly diminished (159). Substantially reduced ATPase activity of K1250A CFTR would be expected from the loss of ATP hydrolysis associated with wild-type CFTR channel closing, a loss inferred from detailed electrophysiological analysis of single K1250A channel records (75). If K1250A channels eventually close following dissociation, rather than hydrolysis, of the ATP at NBD2, the latter might reasonably be expected to temporarily adopt a conformation different from that attained after hydrolysis. In which case, the additional time taken for the mutant channel to return to a closed conformation competent to enter another open-close gating cycle might account for the observed reduced opening rate.

Given the structural similarity between ATP and AMP-PNP, the scheme in Figure 6 implies that AMP-PNP might compete with ATP for binding not only at NBD2, where AMP-PNP acts to lock open CFTR channels, but also at NBD1, where AMP-PNP might inhibit channel opening. Failure to observe such inhibition has been cited as evidence that AMP-PNP does not interact at all with CFTR's NBDs (174), a conclusion untenable in light of the result shown in Figure 5. Moreover, the anticipated inhibitory influence of high concentrations of AMP-PNP on channel gating was recently detected when AMP-PNP concentration was more than fourfold greater than ATP concentration (130).

2. One catalytic site per monomer, or two?

It is important to point out that, despite its relatively large size (240 amino acids), the RbsA NH2-terminal NBD in the crystal was essentially catalytically incompetent, since the structure was solved with MgATP still bound in the catalytic site (7). One implication is that some other segment of polypeptide, possibly part, or all, of the COOH-terminal NBD, or perhaps the transmembrane domain of the ribose transporter, is required to compose one complete catalytic site, or perhaps two active catalytic sites. An inference from the work on ArsA is that the four NBDs of an ArsA homodimer are required to compose just two catalytic sites (114), an arrangement that would be consistent with the recent proposal that one functional CFTR channel comprises a CFTR dimer (229). Alternatively, the NH2- and COOH-terminal NBDs of RbsA could conceivably pack together in a head-to-tail configuration, forming two catalytic sites, in each of which the nucleotide binding pocket of one NBD might be complemented by, say, the LSGGQ sequence of the other NBD. Perhaps, in that way, the functional analogy between CFTR's NBDs and G proteins (discussed in sect. IVD2) could be resurrected and reconciled with the new structural evidence. But, unfortunately, these crucial questions cannot be resolved with the information presently available, so they must await new experimental reults.

    VI. CONCLUDING REMARKS
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The correlation between genotype and phenotype in terms of CF disease remains perplexingly elusive. But, although in two-thirds of CF patients the disease stems from the same mutation in one chromosome 7 copy (causing deletion of Phe-508), other influences of nature and nurture probably make it unrealistic to expect the same progression of this pleiotropic disease in any large fraction of patients. Some of this heterogeneity possibly arises because CFTR may be multifunctional. There seems little doubt that CFTR's primary role in normal epithelia is that of a regulated Cl- channel, but evidence is steadily mounting that CFTR likely subserves other functions, such as directly, and possibly indirectly, interacting with other channels and transporters, and perhaps itself mediating transport of something other than small anions. For certain CF mutations, however, there is some correlation between their influence on CFTR's function as a Cl- channel and the severity of disease. A milder form of CF disease, for example, has been shown to be associated with mutations that reduce, but do not abolish, average Cl- flow through the channel. Examples are mutations at residues like R117, R334, and R347, believed to reside near or in the pore, which diminish channel conductance, and at residues like A455, and also R117, which reduce channel Po (e.g., Ref. 221). Severe disease is associated with mutations at residues like G551, which result in only very rare, brief, openings of the channel. Because the time-averaged magnitude of the ion flow through any channel depends on both the average frequency and the average duration of channel openings, which in CFTR appear to be controlled by separate processes, this rough correlation between macroscopic CFTR current size and the severity of cystic fibrosis disease provides a strong motivation for trying to learn precisely what mechanisms govern CFTR channel gating.

One thing is already abundantly clear, and that is that the concerted regulation of the opening and closing of CFTR channels by incremental phosphorylation of multiple sites, and by binding and hydrolysis of ATP at the NBDs, is extremely complex. One of the consequences of this complexity is the difficulty of unambiguously interpretating the macroscopic behavior of CFTR mutants. For instance, if the channel opening rate is controlled by ATP hydrolysis at NBD1 and the channel closing rate is regulated by ATP hydrolysis at NBD2, an observed reduction in Po , say caused by mutating one of the R-domain serines, could result from (among several possible mechanisms) slowed channel opening by impaired ATP hydrolysis at NBD1 or faster channel closing due to accelerated ATP hydrolysis at NBD2. Considering the proposed interactions between the two NBDs and between the NBDs and the R domain, a mutation in any one of these domains may be expected to result in altered function of another. It seems, then, that if we are to begin to unravel the complex mechanisms by which phosphorylation of the R domain at multiple sites regulates the catalytic cycles at the NBDs, and hence the gating of CFTR channels, we will need measurements of the rates of opening and closing of individual CFTR channels, each bearing just a single mutation. On the other hand, a dividend of this functional complexity is that high-resolution recording of unitary CFTR channel currents affords an opportunity, unprecedented in biology, to examine details of individual ATP hydrolysis cycles in a single protein molecule, in its natural environment, in real time.

    FOOTNOTES

   We are indebted to Kate Egnatz and Peter Hoff for invaluable assistance with the manuscript and illustrations and to P. S. I. J. G. Ault for taming the bibliography.

   Preparation of this review and our research on CFTR were supported by National Institutes of Health Grants HL-49907 and DK-51767.

  

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0031-9333/99 $15.00 Copyright ©1999 The American Physiological Society



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