Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.     

Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH(2)-terminal nucleotide binding domain

The cystic fibrosis transmembrane conductance regulator is a Cl(-) channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590-830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH(2)- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH(2) terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH(2) termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH(2)-terminal segment representing aa's 3-622 (Flag3-622) or 3-633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634-1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.     

R-domain interactions with distal regions of CFTR lead to phosphorylation and activation

Cystic fibrosis is caused by the aberrant function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. We examined whether intramolecular binding interactions involving the regulatory (R) domain contributed to CFTR regulation and function. When the R-domain (amino acids 596-836) was coexpressed with Delta1-836 CFTR (a carboxyl hemi-CFTR beginning immediately after the R-domain), strong binding between the two polypeptides was exhibited. The R-domain that co-immunoprecipitated with Delta1-836 exhibited a slower mobility on SDS-PAGE that resulted from phosphorylation of the protein. A larger CFTR polypeptide that included the R-domain (M837X) also exhibited a phosphorylation-dependent mobility shift when coexpressed with Delta1-836. Moreover, coexpression of M837X and Delta1-836 led to enhanced halide permeability in living cells. The activity, unlike in full-length CFTR, was present without forskolin activation, but still sensitive to the PKA inhibitor, Rp-8-CPT-cAMPS. This PKA inhibition of activity was found to be dependent on the carboxy region of the R-domain, amino acids 723-836. Our results indicate that the R-domain binds CFTR residues after amino acid 836 and that this binding facilitates phosphorylation and CFTR activation. We have also characterized a subdomain within CFTR (residues 723-837) that is necessary for PKA-dependent constitutive activation. Finally, these experiments demonstrate that constitutive CFTR activity can be accomplished by at least two mechanisms: (1) direct modulation of the R-domain to abrogate PKA regulation and (2) modifications that increase R-domain susceptibility to steady-state phosphorylation through PKA.     

Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.     

CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR)

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.     

PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called DeltaR-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with DeltaR-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of DeltaR-Split/7CA-RD channels was similar to that of DeltaR-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.     

Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.     

Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR), when phosphorylated, undergoes conformational change, and the chloride channel opens. We investigated the contribution of R domain conformation, apart from the changes induced by phosphorylation, to channel opening, by testing the effect of the peptidyl-prolyl isomerase, cyclophilin A, on the CFTR channel. When it was applied after the channel had been opened by PKA phosphorylation, cyclophilin A increased the open probability of wild-type CFTR (from P(o) = 0.197 +/- 0.010 to P(o) = 0.436 +/- 0. 029) by increasing the number of channel openings, not open time. Three highly conserved proline residues in the R domain, at positions 740, 750, and 759, were considered as candidate targets for cyclophilin A. Mutations of these prolines to alanines (P3A mutant) resulted in a channel unresponsive to cyclophilin A but with pore properties similar to the wild type, under strict control of PKA and ATP, but with significantly increased open probability (P(o) = 0.577 +/- 0.090) compared to wild-type CFTR, again due to an increase in the number of channel openings and not open time. Mutation of each of the proline residues separately and in pairs demonstrated that all three proline mutations are required for maximal P(o). When P3A was expressed in 293 HEK cells and tested by SPQ assay, chloride efflux was significantly increased compared to cells transfected with wild-type CFTR. Thus, treatments favoring the trans-peptidyl conformation about conserved proline residues in the R domain of CFTR affect openings of CFTR, above and beyond the effect of PKA phosphorylation.     

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.     

Potential sites of CFTR activation by tyrosine kinases

The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation.     

Phosphorylation-induced conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total reflection-Fourier transform IR spectroscopy and fluorescence spectroscopy

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1H/2H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site.     

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.     

The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.     

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.     

CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.     

Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating

CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.     

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35 degrees C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(DeltaH(double dagger)) were determined from Eyring plots. DeltaH(double dagger) was 117 +/- 6 and 69 +/- 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 +/- 6 and 73 +/- 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. DeltaH(double dagger) for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 +/- 2, 39 +/- 4, and 37 +/- 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (DeltaS(double dagger)), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. DeltaS(double dagger) appears large for opening but small for normal closure. The large DeltaH(double dagger) and DeltaS(double dagger) (TDeltaS(double dagger) >/= 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small DeltaS(double dagger) for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.     

Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.