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Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel with two remarkable distinctions. First, it is the only ATP-binding cassette (ABC) transporter that is known to be an ion channel--almost all others function as transport ATPases. Second, CFTR is the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle--a consequence of its enzymatic activity as an ABC transporter. We discuss these special properties of CFTR in the context of its evolutionary history as an ABC transporter. Other topics include the mechanisms by which CFTR gating is regulated by phosphorylation of its unique regulatory domain and our current view of the CFTR permeation pathway (or pore). Understanding these basic operating principles of the CFTR channel is central to defining the mechanisms of action of prospective cystic fibrosis drugs and to the development of new, rational treatment strategies.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR chloride channel. Wild type and mutant CFTR channels can be activated by curcumin, a well tolerated dietary compound with some appeal as a prospective CF therapeutic. However, we show here that curcumin has the unexpected effect of cross-linking CFTR polypeptides into SDS-resistant oligomers. This effect occurred for CFTR channels in microsomes as well as in intact cells and at the same concentrations that are effective for promoting CFTR channel activity (5-50 mum). Both mature CFTR polypeptides at the cell surface and immature CFTR protein in the endoplasmic reticulum were cross-linked by curcumin, although the latter pool was more susceptible to this modification. Curcumin cross-linked two CF mutant channels (Delta F508 and G551D) as well as a variety of deletion constructs that lack the major cytoplasmic domains. In vitro cross-linking could be prevented by high concentrations of oxidant scavengers (i.e. reduced glutathione and sodium azide) indicating a possible oxidation reaction with the CFTR polypeptide. Importantly, cyclic derivatives of curcumin that lack the reactive beta diketone moiety had no cross-linking activity. One of these cyclic derivatives stimulated the activities of wild type CFTR channels, Delta 1198-CFTR channels, and G551D-CFTR channels in excised membrane patches. Like the parent compound, the cyclic derivative irreversibly activated CFTR channels in excised patches during prolonged exposure (>5 min). Our results raise a note of caution about secondary biochemical effects of reactive compounds like curcumin in the treatment of CF. Cyclic curcumin derivatives may have better therapeutic potential in this regard.

Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR-processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (DeltaF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented DeltaF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not DeltaF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response., Introduction Cystic fibrosis (CF) is caused by abnormalities of the CF transmembrane conductance regulator (CFTR), with the most common variant being F508del (deletion of phenylalanine at position 508). The F508del [...]

W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. [ABSTRACT FROM AUTHOR]

The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ion channel in that its gating is coupled to an intrinsic enzymatic activity (ATP hydrolysis). This enzymatic activity derives from the evolutionary origin of CFTR as an ATP-binding cassette transporter. CFTR gating is distinct from that of a typical ligand-gated channel because its ligand (ATP) is usually consumed during the gating cycle. However, recent findings indicate that CFTR gating exhibits allosteric properties that are common to conventional ligand-gated channels (e.g. unliganded openings and constitutive mutations). Here, we provide a unified view of CFTR gating that combines the allosterism of a ligand-gated channel with its unique enzymatic activity. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization. [ABSTRACT FROM AUTHOR]

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and β[sub 2] adrenergic receptor (β[sub 2]AR) can bind ezrin/radixin/moesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that β[sub 2] is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. β[sub 2]AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive, Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally, This uncoupling is specific to the β[sub 2]AR receptor and does not affect CFTR coupling to other receptors (e.g, adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-β[sub 2]AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases. [ABSTRACT FROM AUTHOR]

Studies the role of syntaxin 1A complex in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Modulation of channel gating; Interaction between syntaxin 1A and amino terminal tail; Kinetic properties of voltage-gated calcium channels.

Focuses on a study which investigated the deltaF508 mutation which leads to retention of cystic fibrosis transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems. Cell culture; Immunoprecipitation; CFTR detection; Detection of tight junction formation; Results; Conclusion.

Editorial. Highlights the major goals in cystic fibrosis research. Identification of alternate chloride channels; Anion channel activity of cystic fibrosis transmembrane conductance regulator; Symptoms of cystic fibrosis.

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. [ABSTRACT FROM AUTHOR]

High mobility group nucleosomal binding domain 2 (HMGN2) is a small and unique non-histone protein that has many functions in a variety of cellular processes, including regulation of chromatin structure, transcription, and DNA repair. In addition, it may have other roles in antimicrobial activity, cell homing, and regulating cytokine release. Although the biochemical properties of HMGN2 protein are regulated by acetylation and phosphorylation, it is not yet known whether HMGN2 activity can also be regulated by SUMOylation. In this study, we demonstrated for the first time that HMGN2 is modified by covalent attachment of small ubiquitin-related modifier 1 (SUMO1) by pro-inflammatory signal and identified the major SUMOylated lysine residues that localize to the HMGN2 nucleosome-binding domain at Lys-17 and Lys-35. SENP1 can deSUMOylate SUMOylated HMGN2, and PIAS1 is the E3 ligase responsible for SUMOylation of HMGN2. Finally, using SUMO1-conjugated HMGN2 purified from a basal SUMOylation system in Escherichia coli, we demonstrated that SUMOylated HMGN2 has decreased the binding affinity to nucleosome core particles in comparison to unSUMOylated HMGN2. These observations potentially provide new perspectives for understanding the functions of HMGN2 in inflammatory reaction. [ABSTRACT FROM AUTHOR]

Most cystic fibrosis (CF) cases are caused by the ΔF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of ΔF508-CFTR can be “rescued” by culturing cells at 26-28 °C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a ΔF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ΔF508-CFTR function. Low temperature-rescued ΔF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 °C to 36.5 °C. A panel of CFTR correctors and potentiators that increased ΔF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued ΔF508-CFTR maturation and stabilized channel activity at 36.5 °C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued ΔF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these ΔF508-CFTR constructs were stable at 36.5 °C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) ΔF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from ΔF508-induced thermal instability, a finding that may inform alternative treatment strategies. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted β-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. β-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of β-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the α-, β-, and γ-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt trans- port 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. [ABSTRACT FROM AUTHOR]

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at ∼50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ΔF508-CFTR, which inefficiently traffics to the surfaces of most cells. The ΔF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ΔF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ΔF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ΔF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ΔF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ΔF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP- dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening. [ABSTRACT FROM AUTHOR]

Reports that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) channels and syntaxin 1A is mechanistically different from syntaxin 1A-SNARE interactions. Residues in the H3 domain of this SNARE that influence CFTR binding and regulation; Analysis of pertinent topics and relevant issues; Implications on studies of lipids and lipoproteins.