Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.     

Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl(-) channel properties regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and non-epithelial cells, whereas the presence of ENaC increases CFTR functional expression. This interregulation is reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl(-) channels are increased when CFTR is co-expressed with alphabetagamma-mouse ENaC (mENaC) and conversely when the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and alphabetagamma-mENaC. The co-expression of G551D-CFTR and alphabetagamma-mENaC resulted in a 5-fold increase in G551D-CFTR Cl(-) current compared with oocytes expressing G551D-CFTR alone. Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation.     

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.     

Cystic fibrosis transmembrane conductance regulator in teleost fish

The gills and intestinal epithelia of teleost fish express cystic fibrosis transmembrane conductance regulator (CFTR), and utilize this low conductance anion channel in the apical membrane for ion secretion in seawater gill and in the basolateral membrane for ion absorption in freshwater gill. Similarly, in the intestine CFTR is present in the basolateral membrane for intestinal absorption and also in the apical membrane of secreting intestine. The expression of CFTR and the directed trafficking of the protein to the apical or basolateral membrane is salinity-dependent. The CFTR gene has been cloned and sequenced from several teleost species and although all the major elements in the human gene are present, including two nucleotide binding domains that are common to all ATP binding cassette (ABC) transporters, the sequences are divergent compared to shark or human. In euryhaline fish adapting to seawater, CFTR, localized immunocytochemically, redistributes slowly from a basolateral location to the apical membrane while ion secretory capacity increases. The facility with which teleosts regulate CFTR expression and activation during salinity adaptation make this system an appealing model for the expression and trafficking operation of this labile gene product.     

Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.     

Cystic fibrosis transmembrane conductance regulator gating requires cytosolic electrolytes

Cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis when nonfunctional, is an anion channel and a member of the ATP binding cassette superfamily. After phosphorylation, CFTR gates by binding and hydrolyzing ATP. We show that CFTR open probability (P(o)) also depends on the electrolyte concentration of the cytosol. Inside-out patches from Calu-3 cells were transiently exposed to solutions of 160 mm salt or solutions in which up to 90% of the salt was replaced by nonionic osmolytes such as sucrose. In lowered salt solutions, CFTR P(o) declined within 1 s to a stable lower value that depended on the electrolyte concentration, (K(1/2) approximately 80 mm NaCl). P(o) was rapidly restored in normal salt concentrations without regard to the electrolyte species. Reducing external electrolytes did not affect CFTR P(o). The same results were obtained when CFTR was stably phosphorylated with adenosine 5'-O-(thiotriphosphate). The decrease in P(o) resulted entirely from an increase in mean closed time. Increasing ATP levels up to 20-fold did not counteract the effect of low electrolytes. The same effect was observed for CFTR expressed in C127 cells but not for a different species of anion channel. Cytosolic electrolytes are an unsuspected, essential cofactor for CFTR gating.     

Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms

Recent studieshave demonstrated that several compounds with diverse structures canactivate wild-type cystic fibrosis transmembrane conductance regulator(CFTR) by non-receptor-mediated mechanisms. Some of these compoundshave been shown to enhance cAMP-dependent activation of F508-CFTR.This study was undertaken to compare the mechanisms by which genistein,IBMX, milrinone, 8-cyclopentyl-1,3-dipropylxanthine (CPX), thebenzimidazolone NS004, and calyculin A increase CFTR activity. Ourstudies demonstrate that, in transfected NIH-3T3 cells, maximalenhancements of forskolin-dependent F508-CFTR activity are greatestwith genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, andcalyculin A do not increase cellular cAMP. Because forskolin andcalyculin A increase in vivo phosphorylation of cAMP binding responseelement (CREB), the inability of milrinone, genistein, CPX, and NS004to increase CREB phosphorylation suggests that they do not stimulateprotein kinase A or inhibit phosphatase activity. Our data suggest thatthe mechanisms by which genistein and NS004 activate CFTR differ. Wealso demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement ofcAMP-dependent CFTR activity is not due to an increase in cellular cAMPand may involve a mechanism like that of genistein.

     

Up-regulation of acid-gated Na(+) channels (ASICs) by cystic fibrosis transmembrane conductance regulator co-expression in Xenopus oocytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as both a chloride channel and an epithelial transport regulator, interacting with Na(+) (epithelial sodium channel), Cl(-), renal outer medullary potassium channel(+), and H(2)O channels and some exchangers (i.e. Na(+)/H(+)) and co-transporters (Na(+)-HCO(3)(minus sign), Na(+)-K(+)-2Cl(-)). Acid-sensitive ion channels (ASICs), members of the epithelial sodium channel/degenerin superfamily, were originally cloned from neuronal tissue, and recently localized in epithelia. Because CFTR has been immunocytochemically and functionally identified in rat, murine, and human brain, the regulation of ASICs by CFTR was tested in oocytes. Our observations show that the proton-gated Na(+) current formed by the heteromultimeric ASIC1a/2a channel was up-regulated by wild type but not by Delta F508-CFTR. In contrast, the acid-gated Na(+) current associated with either the homomultimeric ASIC1a or ASIC2a channel was not influenced by wild type CFTR. The apparent equilibrium dissociation constant for extracellular Na(+) for ASIC1a/2a was increased by CFTR, but CFTR had no effect on the gating behavior or acid sensitivity of ASIC1a/2a. CFTR had no effect on the pH activation of ASIC1a/2a. We conclude that wild type CFTR elevates the acid-gated Na(+) current of ASIC1a/2a in part by altering the kinetics of extracellular Na(+) interaction.     

Cystic fibrosis transmembrane conductance regulator in human and mouse red blood cell membranes and its interaction with ecto-apyrase

Elevated blood ATP and increased red blood cell (RBC) ATP transport is associated with cystic fibrosis (CF). In this report, we demonstrate the presence of the wild-type and the DeltaF508 mutant form of the CF transmembrane conductance regulator protein in RBC membranes and its putative interaction with ecto-apyrase, an ATP hydrolyzing enzyme also present in the RBC membrane. RBC membranes of control and DeltaF508 individuals and of wild-type and CF transmembrane conductance regulator-knockout mice were examined by immunoblot using several antibodies directed against different epitopes of this protein. These experiments indicated that human RBC membranes contain comparable amounts of the wild-type CF transmembrane conductance regulator protein and the DeltaF508 mutant form of the protein, respectively. CF transmembrane conductance regulator protein was also detected in wild-type mouse RBC membranes but not in the gene knockout mouse RBC membranes. Antibodies directed against ecto-apyrase co-immunoprecipitated CF transmembrane conductance regulator protein of human RBC membranes indicating a physical interaction between these two membrane proteins consistent with ATP transport and extracellular hydrolysis. We conclude that RBCs are a significant repository of CF transmembrane conductance regulator protein and should provide a novel system for evaluating its expression and function.     

Cystic fibrosis transmembrane conductance regulator trafficking is mediated by the COPI coat in epithelial cells

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 beta-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. Beta-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 beta-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 alpha-, beta-, and gamma-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.     

Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts.

We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis.     

Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous (Cftr(+/-)), knockout (Cftr(-/-)), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (P(o)): wild-type=0.21 ± 0.015: Cftr(+/-)=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr(-/-)=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the P(o) and number of channels in ATII cells of wild-type, Cftr(+/-), and ΔF508, but not in Cftr(-/-) mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr(+/-) and Cftr(-/-) mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.     

Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene

Summary Three mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were discovered in a pancreas-insufficient patient with cystic fibrosis (CF) who displayed an uncommon combination of almost normal chloride concentration in sweat tests and typical symptoms of gastrointestinal and pulmonary disease. The R553Q mutation was found on the maternal F508-CFTR gene. Codon 553 is located within a consensus motif of the ATP-binding cassette transport proteins at a less conserved position. Other members of this protein superfamily contain a glutamine instead of arginine at the homologous position, suggesting a modulating rather than disease-causing role of the R553Q mutation in CFTR. The amplification refractory mutation system did not detect the R553Q mutation in a further 65 normal, 113 F508, and 91 non-F508 CF chromosomes. The index case carried the R553X nonsense mutation on the paternal chromosome. The R553X mutation was present on a further 9 out of 86 German nonF508 CF chromosomes linked with the XV2c-KM19Mp6d9-J44-GATT haplotypes 2-2-2-1-1 and 1-1-2-1-2. The location of R553X on separate haplotypes including both alleles of the intragenic GATT repeat suggests an ancient and/or multiple origins of the R553X mutations. The association of the genotype of the CFTR mutation and the clinical phenotype was assessed for the patients carrying the related genotypes F508/F508 (n = 80), F508/R553X (n = 9) and F508-R553Q/R553X (n = 1). In compound heterozygotes, the median chloride concentration in pilocarpine iontophoresis sweat tests was significantly lower than in the F508 homozygotes (P < 0.01). The patient groups were significantly different with respect to the distributions of the centiles for height (P < 0.001) and weight (P < 0.01) as the most sensitive predictors of the course and prognosis in CF. Growth retardation was more pronounced in the compound heterozygotes.     

Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis.

Cystic fibrosis (CF) is an inherited disorder causing pancreatic, pulmonary, and sinus disease in children and young adults. Abnormal viscosity of mucous secretions is a hallmark of the disease, and is believed to be the result of altered electrolyte transport across epithelial cell membranes. The monogenic etiology of this disease has been apparent for more than 40 years, but the defective gene has only recently been identified. This was made possible because of a revolution in genetic technology, called positional cloning, which can pinpoint disease genes without previous knowledge of the abnormal protein product. The protein encoded by the gene defective in CF has been termed the CF transmembrane conductance regulator (CFTR) because of its postulated role in electrolyte transport. Studies investigating the normal function of CFTR and how mutations affect that function, thereby causing CF, have required the combined skills of clinicians, geneticists, molecular biologists, and physiologists. From this collaborative effort a greater understanding of the pathogenesis of this disorder is now emerging. It may soon be possible to introduce novel therapies derived from this new knowledge that will be aimed directly at the basic defect. An ever-increasing number of genes of unknown function will be identified by continuing advances in molecular genetic technology and the advent of the genome sequencing project. The experience in cystic fibrosis research may prove to be a paradigm for investigation of the function of genes isolated by positional cloning methods.     

Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity

Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.     

Myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl(-) channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.     

Cystic fibrosis transmembrane conductance regulator-dependent up-regulation of Kir1.1 (ROMK) renal K+ channels by the epithelial sodium channel

The epithelial sodium channel (ENaC) and the secretory potassium channel (Kir1.1/ROMK) are expressed in the apical membrane of renal collecting duct principal cells where they provide the rate-limiting steps for Na(+) absorption and K(+) secretion. The cystic fibrosis transmembrane conductance regulator (CFTR) is thought to regulate the function of both ENaC and Kir1.1. We hypothesized that CFTR may provide a regulatory link between ENaC and Kir1.1. In Xenopus laevis oocytes co-expressing both ENaC and CFTR, the CFTR currents were 3-fold larger than those in oocytes expressing CFTR alone due to an increased expression of CFTR in the plasma membrane. ENaC was also able to increase Kir1.1 currents through an increase in surface expression, but only in the presence of CFTR. In the absence of CFTR, co-expression of ENaC was without effect on Kir1.1. ENaC-mediated CFTR-dependent up-regulation of Kir1.1 was reduced with a Liddle's syndrome mutant of ENaC. Furthermore, ENaC co-expressed with CFTR was without effect on the closely related K(+) channel, Kir4.1. We conclude that ENaC up-regulates Kir1.1 in a CFTR-dependent manner. CFTR may therefore provide the mechanistic link that mediates the coordinated up-regulation of Kir1.1 during the stimulation of ENaC by hormones such as aldosterone or antidiuretic hormone.     

Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs

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 deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 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 deltaF508-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 deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-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 deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.