CFTR Phosphorylation Research Articles

Qingchang Wenzhong Decoction ameliorates intestinal inflammation and intestinal barrier dysfunction in ulcerative colitis via the GC-C signaling pathway

Ethnopharmacological relevanceUlcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colonic mucosa, accompanied with abdominal pain, and bloody diarrhea. Currently, clinical treatment options for UC are limited. Qingchang Wenzhong Decoction (QCWZD) is an effective prescription of traditional Chinese medicine for the treatment of UC. However, the mechanism of QCWZD in alleviating intestinal barrier dysfunction in UC has not been clearly explained. Aim of the studyTo determine the mechanism whereby QCWZD promotes the recovery of intestinal barrier dysfunction in UC. Materials and methodsA secondary analysis of colonic mucosa from UC patients acquired from a prior RCT clinical trial was performed. The effects of QCWZD on intestinal mucus and mechanical barriers in UC patients were evaluated using colon tissue paraffin-embedded sections from UC patients. The mechanism was further investigated by in vivo and in vitro experiments. UC mice were established in sterile water with 3.0% dextran sodium sulfate (DSS). Meanwhile, mice in the treatment group were dosed with QCWZD or mesalazine. In vitro, an intestinal barrier model was constructed using Caco-2 and HT29 cells in co-culture. GC-C plasmid was used to overexpress/knock down GC-C to clarify the target of QCWZD. HE, AB-PAS, ELISA, immunohistochemistry and immunofluorescence assays were used to assess the level of colonic inflammation and intestinal barrier integrity. Rt-qPCR, Western Blot were used to detect the expression of genes and proteins related to GC-C signaling pathway. Molecular docking was used to simulate the binding sites of major components of QCWZD to GC-C. ResultsIn UC patients, QCWZD increased mucus secretion, goblet cell number, and promoted MUC2 and ZO-1 expression. QCWZD accelerated the recovery of UC mice from DSS-induced inflammation, including weight gain, reduced disease activity index (DAI) scores, colon length recovery, and histological healing. QCWZD promoted mucus secretion and increased ZO-1 expression in in vivo and in vitro experiments, thereby repairing mucus mechanical barrier damage. The effects of QCWZD are mediated through regulation of the GC-C signaling pathway, which in turn affects CFTR phosphorylation and MUC2 expression to promote mucus secretion, while inhibiting the over-activation of MLCK and repairing tight junctions to maintain the integrity of the mechanical barrier. Molecular docking results demonstrate the binding of the main components of QCWZD to GC-C. ConclusionOur study demonstrated that QCWZD modulates the GC-C signaling pathway to promote remission of mucus-mechanical barrier damage in the UC. The clarification of the mechanism of QCWZD holds promise for the development of new therapies for UC.

Understanding impact of δF508 and G551D CFTR mutations on CFTR/PKA-c interaction.

The CFTR anion channel is an ATP Binding Cassette protein and consists of two pore-forming transmembrane domains, two cytosolic nucleotide binding domains that bind and hydrolyze ATP to drive gating, and a cytosolic regulatory (R) domain. The catalytic subunit of protein kinase A (PKA) binds to, and phosphorylates, the R domain, causing reversible and irreversible channel activation, respectively. CFTR chloride channel mutations cause the lethal and incurable disease cystic fibrosis (CF). The most common CF mutation, ΔF508, is known to impair CFTR protein processing and channel gating, while mutation G551D disrupts ATP-dependent channel gating. In addition, ΔF508 and G551D CFTR were also shown to be defective in PKA-dependent activation. As the stimulating effect of ivacaftor, the only FDA-approved CFTR potentiator drug, depends on CFTR phosphorylation, understanding the reason for the impaired CFTR-PKA interaction in the mutants is of great significance. The reduced rate of phosphorylation of G551D or ΔF508 channels might be a consequence of impaired PKA binding to CFTR. In the presence of ATP the contributions of PKA binding vs. phosphorylation to overall channel activation are difficult to deconvolve. Therefore, we tested reversible activation by PKA of unphosphorylated and phosphorylated mutant channels gating in P-ATP, an ATP analog that supports CFTR gating but cannot be used for phosphotransfer by PKA. Using macroscopic inside-out patch clamp recordings we compared the rates and fractional amplitudes of reversible stimulation by PKA for the mutants and wild-type CFTR. Our data show that channel activation by reversible binding of PKA is preserved in the mutant CFTR channels. Additional experiments are underway to decipher the molecular mechanism for slowed activation of the mutants.

Role of Impaired CFTR Function in Pathogenesis of Pneumonia Among Alcohol Users

INTRODUCTIONImpairment of mucociliary clearance (MCC) increases the risk of respiratory infections among excessive alcohol users. The mechanisms underlying this impact of alcohol on MCC are not currently understood. Our previous in vitro studies indicated that alcohol decreases ion transport via CFTR channels, the defective protein that causes cystic fibrosis. Here, we have attempted to validate these findings in vivo and test the hypothesis that alcohol‐induced CFTR dysfunction reduces airway surface hydration and mucus transport thereby impairing airway defense against inhaled bacterial pathogens, in particular Klebsiella pneumoniae, to which alcohol users are disproportionately susceptible. Since CFTR dysfunction correlates with reduced PKA‐dependent CFTR phosphorylation and channel gating due to increased cAMP degradation by phosphodiesterase‐4B (PDE4B) enzymes, we also tested the effects of PDE4 inhibitor, roflumilast, on CFTR function and bacterial clearance in alcohol‐fed rats.METHODSAge and gender‐matched rats (CFTR+/+) were pair‐fed with Lieber‐DeCarli alcohol diet or isocaloric control diet for 4 weeks and with K. pneumoniae (KP, ATCC 43816). Subsets of rats were randomly treated with vehicle/roflumilast (5mg/kg/day) by oral gavage starting 7 days before the end of the alcohol diet. CFTR activity was measured with nasal potential difference (NPD) and MCC was assayed with radiographic clearance of Tc99. Airway surface liquid (ASL), periciliary layer (PCL), and ciliary beat frequency (CBF) of excised trachea were quantified with micro‐optical coherence tomography (μOCT).RESULTSAlcohol administration reduced CFTR ion transport by NPD. Clearance of Tc99 label in 30 min indicated delayed MCC in alcohol‐treated rats. Trachea excised from alcohol‐treated rats exhibited significantly decreased PCL depth, but no meaningful differences in ASL or CBF. Roflumilast treatment significantly increased CFTR activity in both control and alcohol‐administered rats. Increased CFTR activation by roflumilast resulted in augmented PCL depth, reduced mucus viscosity and significantly accelerated MCT rate. Compared to controls, histopathological examination of lung sections from alcohol‐treated animals exhibited more severe evidence of multifocal pneumonia with edema, fibrin deposition and numerous bacteria in alveolar spaces. Ongoing studies are determining the impact of roflumilast treatment on bacterial clearance and severity of pneumonia by K. pneumoniae.CONCLUSIONSAlcohol‐induced defects in CFTR ion transport resulted in reduced airway surface hydration and delayed mucus clearance in rat airways that was associated with diminished ability to clear bacterial pathogens. Acquired (non‐genetic) CFTR dysfunction may be a suitable pharmacologic target to improve mucus clearance impaired by chronic alcohol use. Administration of roflumilast may boost lung host defense against bacterial pathogens and improve pneumonia outcomes in individuals affected by excessive alcohol use.Support or Funding InformationNIH 1R01AA027528

WS06.1 Stabilization of CFTR at the membrane by EPAC1 activation occurs through interaction with NHERF-1

Background CFTR-mediated ion transport is stimulated by increase in subcortical cAMP levels through protein kinase A (PKA) activation thus triggering CFTR phosphorylation and channel opening. However, besides PKA, exchange proteins (EPACs) are also directly activated by cAMP. EPACs are guanine nucleotide exchange factors (GEFs) for Rap family small GTPases and shift to the plasma membrane (PM) in response to cAMP. This suggests that activation of both CFTR and EPACs might be spatially and temporally coincident. Objectives We aimed at elucidating the impact of EPAC1 on CFTR trafficking and PM anchoring. Methods Protein biochemistry and imaging techniques. Results Our results show that in CFBE cells: i. EPAC1 co-localizes and is present in protein complexes involving wt- or F508del-CFTR and this interaction is mediated by NHERF1 but not ezrin. ii. EPAC activation leads to its increased association with CFTR and increased CFTR PM levels. iii. Increase in endogenous cAMP levels increases CFTR membrane levels. iv. EPAC1 knockdown leads to decreased of CFTR PM levels. Conclusion Our results establish that EPAC1 associates to CFTR in membrane complexes with NHERF-1 and enhances CFTR traffic to the PM, making it a novel potential therapeutic target for CF. These data indicate that cAMP signalling regulates CFTR through two distinct but complementary pathways – PKA and EPAC1. Grants from FCT (Portugal) through MCTES EXPL/BIM-MEC/1451/2013 grant and PEst-OE/BIA/UI4046/2011 BioFig centre grant and by 2012 ERS Romain Pauwels Research Award.

SLC6A14 Modifies Fluid Secretory Capacity of Cystic Fibrosis Affected Epithelium by Enhancing CFTR Channel Function

Cystic fibrosis (CF) is usually caused by F508del mutation in the CFTR gene. Normally, CFTR protein aids in salt transport across the apical surface of epithelia, keeping their surface hydrated. F508del results in decreased surface expression of CFTR, leading to dehydration of the surface. This makes the lung epithelium prone to infection and in intestine leads to obstruction. Patients homozygous for F508del, have a tremendous variation in the severity of disease. Recent Genome-wide association studies indicate that this variation is due to presence of modifier genes, with SLC6A14 as the top modifier (Sun et.al.,2012). SLC6A14 is a Na+/Cl- dependent cationic/neutral amino-acid transporter on the surface of lung and colonic epithelium. As both transporters are expressed apically, we hypothesized that SLC6A14 would modify the fluid secretory capacity of the epithelium. So in collaboration with TCP, we generated a SLC6A14 knock-out mouse. We can measure in-vivo fluid secretion in mice, using an intestinal closed-loop assay. SLC6A14 knock-out mice exhibited a decrease in cAMP stimulated fluid secretion mediated via CFTR relative to Wt control. To explore the mechanism by which this modification occurs, we utilized a BHK heterologous expression system, overexpressing CFTR and SLC6A14. Interestingly, the functional interaction can be recapitulated in this system, suggesting that it not tissue-type dependent. Preliminary biochemical and anion-flux studies support the hypothesis that SLC6A14 does not affect the processing or stability of Wt or F508del-CFTR proteins rather it augments the activity of these channel proteins once localized to the cell surface. Future studies will focus on understanding if this augmentation is related to modification of CFTR's phosphorylation dependent gating. These results show a positive impact of SLC6A14 on CFTR channel function and fluid secretion, providing an alternative drug target for CF patients.

Metabolic status changes the interregulation of AMPK and CFTR (893.26)

A better understanding of CFTR Cl‐ channel regulation could suggest new therapies for cystic fibrosis (CF). We previously found that the α1 catalytic subunit of AMP‐activated kinase (AMPK) binds to the C‐terminal tail of CFTR, and AMPK inhibits PKA‐stimulated CFTR gating via direct phosphorylation of CFTR. Both binding and phosphorylation are required for CFTR regulation by AMPK, which may tune CFTR activity to metabolic and other cellular stresses. Moreover, under basal conditions tonic AMPK activity may limit CFTR activation in the absence of PKA stimulation. Conversely, the loss of normal CFTR apical expression in primary CF human bronchial epithelial cells enhances AMPK expression and activity and disrupts AMPK apical targeting in these cells, suggesting that CFTR reciprocally regulates AMPK function. Here we more closely examined the nature of the AMPK‐CFTR interaction and its potential modulation by cellular AMPK activity and metabolic status. AMPK‐α binding to CFTR was reduced with AMPK activation or chemical ischemia in co‐immunoprecipitation studies performed on HEK‐293 and colonic epithelial T84 cells. Moreover, we found that activated AMPK (AMPK‐α‐pThr172) does not appear to bind to CFTR. Changes in cellular energy status inversely regulated AMPK activity and binding to CFTR in T84 cells. Of note, the CFTR‐binding region in AMPK‐α1 overlaps with the AMPK‐β‐binding region in α1, and AMPK‐α1 subunits bound to CFTR do not appear to be in a heterotrimeric AMPK αβγ complex, suggesting that CFTR may sequester AMPK‐α monomers. In summary, our results suggest that not only does AMPK regulate CFTR function, but that CFTR, via metabolic‐dependent interactions with AMPK, regulates AMPK function and subcellular localization in epithelia. This interregulation may represent a new target for CF therapy, as disruption of AMPK binding to CFTR may prevent untoward CFTR inhibition during treatment with AMPK activators to inhibit excessive inflammation and Na+ reabsorption in the CF airway.Grant Funding Source: Supported by NIH/NIDDK

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.

A sensitive tool to measure CFTR channel activity

cystic fibrosis transmembrane conductance regulator(CFTR, ABCC7) is a member of the human ABC protein fam-ily. The characteristic feature of ABC proteins is the similararchitecture of the ATP binding sites and the common mecha-nism of ATP binding and hydrolysis. In contrast to most ofthe ABC proteins, CFTR is not an active transporter, rather itis a phosphorylation and ATP hydrolysis gated chloride ionchannel and channel regulator (1,2). Mutations in both copiesof the CFTR gene may lead to a heritable genetic disease, i.e.cystic fibrosis (CF) with variable severity depending on thesite of the mutation. Certain mutations alter biogenesis or cel-lular processing of the CFTR protein, while others affect itschannel activity. Chemical chaperons, also called correctors(e.g., VX-809) can partially rescue the misprocessing, mostlikely by improving the folding of the protein at the endoplas-mic reticulum (2). Activators of CFTR may function throughelevating cytosolic cAMP (promoting CFTR phosphoryla-tion), inhibiting phosphatase activity (thus blocking CFTR de-phosphorylation), and/or interacting directly with the channelprotein (1). Since CFTR is expressed in the epithelial cells ofseveral organs, CF is a multi-organ disease that affects thesinopulmonary and male urogenital systems, alters pancreaticand biliary secretion, therefore the current average lifespan ofCF patients is approximately 40 years of age (1,2). In mostcases, the primary cause of early onset of death is the progres-sion of lung disease (2,3). The mechanisms by which CFTRmutations cause lung disease in CF patients are not fullyunderstood. It may include altered ion and water transportacross the airway epithelium and aberrant inflammatory andimmune responses to pathogens within the airways (3,4).Several techniques have been developed to study CFTRfunction or to search for CFTR activators or modulatorsincluding halide sensitive fluorescent dyes, electrophysiologi-cal approaches such as patch clamp, short-circuit measure-ments in Ussing chambers and influx or efflux measurementsapplying radioactive ions. In addition to the above techniquesa halide sensitive mutant form of yellow fluorescent protein(YFP) has been also utilized to probe CFTR function meas-uring the iodide-mediated quenching rate of YFP, since theopen state of CFTR is also permeable to iodide ions (5). YFPfluorescence intensity is both dependent on the intracellulariodide concentration and YFP expression level. Therefore, flu-orescence intensities measured in the presence of iodideshould be normalized to YFP fluorescence intensities meas-ured in the absence of iodide to decrease experimental vari-ability related to the differences in sensor expression levelamong individual cells.However, normalization to the un-quenched YFP fluores-cence intensity is problematic especially in flow cytometricassays. The co-expression of a halide insensitive fluorescentprotein can overcome the above problem as was demonstratedby Vijftigschild et al. published in the current issue of Cytome-try Part A (page 576). When the two fluorescent proteins wereexpressed as a fusion protein formation of aggregates wasobserved. To avoid this problem the two proteins were con-nected by an auto-cleavable polypeptide chain (6) resulting inphysically not connected proteins. Theoretically, the expres-sion level of the two proteins may change upon time due to

Role of Binding and Nucleoside Diphosphate Kinase A (NDPK‐A) in the Regulation of CFTR by AMP‐Activated Protein Kinase (AMPK)

A better understanding of CFTR Cl− channel regulation could suggest new therapies for cystic fibrosis. AMPK binds to the CFTR C‐terminal tail, phosphorylates, and inhibits PKA‐activated CFTR gating, processes that are not well understood. PKA‐induced CFTR gating was unchanged by AMPK addition in excised inside‐out patch‐clamp recordings, suggesting the importance of other factor(s) in intact cells. NDPK‐A interacts with both AMPK and CFTR in overlay blots of airway epithelia. Binding studies in Xenopus oocytes and transfected HEK‐293 cells revealed that a peptide fragment of CFTR that binds AMPK (CFTR‐1420‐57) disrupted this interaction. Transduction of this peptide into Calu‐3 cells enhanced forskolin‐induced whole‐cell CFTR conductance. Injection of CFTR‐1420‐57 into Xenopus oocytes blocked AMPK‐dependent inhibition of cAMP‐stimulated CFTR conductance, as measured by two‐electrode voltage clamp. AMPK activation inhibited CFTR conductance with co‐expression of wild‐type NDPK‐A in Xenopus oocytes, but co‐expression of a catalytically inactive H118F mutant blocked this inhibition. These results suggest that NDPK‐A exists in a cellular complex with AMPK and CFTR and is required for the AMPK‐dependent regulation of CFTR. We propose that NDPK‐A may make ATP readily available for AMPK‐mediated phosphorylation and regulation of CFTR under conditions of metabolic stress. (Supported by NIH and CFF)

CFTR Cl– channel functional regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensitive ion transporting mitochondria rich cells of euryhaline killifish

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channels are the regulated exit pathway in Cl(-) secretion by teleost mitochondria rich salt secreting (MR) cells of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry, immunogold transmission electron microscopy (TEM), and co-immunoprecipitation, using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase focal adhesion kinase (FAK) phosphorylated at Y407 (FAK pY407) are colocalized in the apical membrane and in subjacent membrane vesicles of MR cells. We showed previously that basolateral FAK pY407, unlike other FAK phosphorylation sites, is osmosensitive and dephosphorylates during hypotonic shock of epithelial cells (Marshall et al., 2008). In the present study, we found that hypotonic shock and the alpha(2)-adrenergic agonist clonidine (neither of which affects cAMP levels) rapidly and reversibly inhibit Cl(-) secretion by isolated opercular membranes, simultaneous with dephosphorylation of FAK pY407, located in the apical membrane. FAK pY407 is rephosphorylated and Cl(-) secretion rapidly restored by hypertonic shock as well as by forskolin and isoproterenol, which operate via cAMP and protein kinase A. We conclude that hormone mediated, cAMP dependent and osmotically mediated, cAMP independent pathways converge on a mechanism to activate CFTR and Cl(-) secretion, possibly through tyrosine phosphorylation of CFTR by FAK.

AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR

The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.

Mutation specific therapy in CF

CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. The mutations can be classified according to the mechanisms by which mutations disrupt CFTR function. This understanding of the different molecular mechanism of CFTR dysfunction provides the scientific basis for development of targeted drugs for mutation specific therapy of CF. Class I mutations are nonsense mutations that result in the presence of premature stop codon that leads to the production of unstable mRNA or the release from the ribosome of a short truncated protein that is not functional. The aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result the mutant CFTR is retained in the ER and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperons such as Sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or ATP binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome the affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the spicing machinery and generate both aberrantly and correctly spliced transcripts, the level of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increase of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that has to be reached in order to achieve normal function.

A quantitative description of the activation and inhibition of CFTR by potentiators: Genistein

The CFTR, encoded by the gene mutated in cystic fibrosis (CF) patients, is responsible for cAMP dependent chloride transport in epithelia. Substances that activate CFTR have been suggested as possible CF therapy. Most substances investigated so far exert a dual effect on the CFTR: low concentrations stimulate CFTR, whereas higher concentrations inhibit CFTR. Besides, the CFTR phosphorylation level determines the apparent affinity of the drug. We have studied the properties of genistein, the well known CFTR potentiator, by measuring apical membrane current on epithelia formed by cells stably transfected with CFTR and stimulated with different concentrations of CPTcAMP. We propose a quantitative model to describe the activatory and inhibitory effect of genistein, accounting also for the cAMP dependent activation.

Pharmacological induction of CFTR function in patients with cystic fibrosis: mutation-specific therapy

CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. Mutations can be classified according to the mechanisms by which they disrupt CFTR function. This understanding of the different molecular mechanisms of CFTR dysfunction provides the scientific basis for the development of targeted drugs for mutation-specific therapy of cystic fibrosis (CF). Class I mutations are nonsense mutations that result in the presence of a premature stop codon that leads to the production of unstable mRNA, or the release from the ribosome of a short, truncated protein that is not functional. Aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperones such as sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or adenosine tri-phosphate (ATP) binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively, restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the splicing machinery and generate both aberrantly and correctly spliced transcripts, the levels of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increases of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that had to be reached in order to achieve normal function.

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 [α32P]8-N3ATP or [γ32P]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°C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30°C, with extensive washing, also at 30°C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30°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.

Colonocyte Basolateral Membranes Contain Escherichia coli Heat-Stable Enterotoxin Receptors

Heat-stable enterotoxin (STa) elaborated by E. coli is a major cause of diarrhea. The transmembrane protein guanylyl cyclase C (GC-C) is the acknowledged receptor for STa and for the mammalian peptides guanylin and uroguanylin. Binding to GC-C results in generation of cGMP, activation of type II cGMP-dependent protein kinase, phosphorylation of CFTR and increased chloride and bicarbonate secretion. We had previously shown that STa receptors (GC-C) are found on the brush border membranes of small intestinal enterocytes and of colonocytes. However, since it has subsequently been shown that the endogenous ligands for these receptors, guanylin and uroguanylin, circulate in blood, we proposed the existence of STa binding sites on the basolateral membranes (BLM) of colonocytes. Specific binding of 125I-STa to rat colonocyte BLM was seen. The kinetics of binding to the BLM were similar to binding to BBM. The nature of the BLM receptor is unknown. This suggests that circulating guanylin and uroguanylin, analogues of STa, may also function via the basolateral surface.

MECHANISMS UNDERLYING REGULATED CFTR TRAFFICKING

Stimulation of membrane capacitance and cell surface labeling of epitope-tagged CFTR provide evidence of cAMP-regulated CFTR trafficking. Co-expression of syntaxin 1A inhibits cAMP-stimulated current and capacitance changes in CFTR expressing cells and blocks cAMP-induced increases in cell surface CFTR. Inhibition of CFTR trafficking by syntaxin over-expression suggests a role for SNARE proteins in this process. CFTR phosphorylation may alter physical interactions with SNARE proteins to regulate plasma membrane CFTR density.

Expression and characterization of the NBD1-R domain region of CFTR: evidence for subunit-subunit interactions.

To study interactions between the contiguous NBD1 and R domains of CFTR, wild-type and DeltaF508 NBD1-R (amino acids 404-830, in fusion with His6 tag) were expressed as single proteins in Escherichia coli. NBD1-R (10-25 mg/L culture) was purified from inclusion bodies in 8 M urea by Ni-affinity chromatography, and renatured by rapid dilution at pH 5. In vitro phosphorylation by protein kinase A increased the apparent size of NBD1-R from approximately 52 to approximately 56 kDa by SDS-PAGE. The fluorescent ATP analogue TNP-ATP bound to renatured NBD1-R with of 0.81 +/- 0.1 microM (wild-type), 0.93 +/- 0.1 microM (wild-type, phosphorylated), 0.75 +/- 0.1 microM (DeltaF508 NBD1-R), and 0.72 +/- 0.1 microM (DeltaF508 NBD1-R, phosphorylated) with a stoichiometry of approximately 1 TNP-ATP site per NBD1-R molecule; TNP-ATP binding was reversed by ATP, AMP-PCP, and AMP-PNP with KIs of approximately 3.2, 4.2, and 4.6 mM, respectively. Secondary structure analysis by circular dichroism gave 19% alpha-helix, 43% beta-sheet and turn, and 38% "other" structure. To determine if nucleotide binding to NBD1 influenced R domain phosphorylation, NBD1-R was in vitro phosphorylated with protein kinase A and [gamma-32P]ATP in the presence of AMP-PCP, AMP-PNP, or TNP-ATP. Whereas the nucleotide analogues did not affect 32P-incorporation in control proteins (Kemptide, GST-R domain), phosphorylation of NBD1-R was reduced >75% by AMP-PNP or AMP-PCP (0.25 mM) and >50% by TNP-ATP (0.25 microM). Analysis of phosphorylation sites indicated that inhibition involved multiple sites in NBD1-R, including serines 660, 712, 737, 795, and 813. These results establish the conditions for NBD1-R expression, purification, and renaturation. The inhibition of R domain phosphorylation by nucleotide binding to the NBD1 domain indicates significant domain-domain interactions and suggests a novel mechanism for regulation of CFTR phosphorylation.

Rapid regulation of electrolyte absorption in sweat duct

Even though the same Cl channel (CFTR) is common to certain fluid transport functions that are oppositely directed, i.e., secretion and absorption, only fluid secretion has clearly been shown to be acutely regulated. It is now clear that fluid secretion activated by beta-adrenergic stimulation is controlled by cAMP-mediated opening and closing of CFTR-Cl channels. Since the conductance of the human sweat duct is almost wholly due to CFTR-Cl conductance (CFTR-GCl), we sought to determine whether salt absorption via CFTR-Cl channels could also be subject to acute regulation in this purely absorptive epithelium. After alpha-toxin permeabilization, we found that addition of cAMP resulted in a large increase in Cl diffusion potentials across the apical membrane and a more than twofold increase in the average membrane conductance. Since the cAMP effects were dependent on Cl alone, not on Na, and since apical Cl conductance appears to be almost exclusively comprised of CFTR-GCl, we surmise that this form of electrolyte absorption like secretion is also subject to acute control through CFTR-GCl. Acute regulation of absorption involves both activation by phosphorylation (PKA) and inactivation by dephosphorylation (unknown endogenous phosphatase) of CFTR. Phosphorylation of CFTR was shown by the facts that CFTR-GCl could be activated by cAMP and inhibited by the kinase antagonist staurosporine, or by removal of either substrate ATP or Mg2+ cofactor. Inactivation of CFTR-GCl by endogenous phosphatase(s) was indicated by a spontaneous but reversible loss of CFTR-GCl upon removal of cAMP. Such loss of CFTR-GCl activity could be prevented either by application of phosphatase inhibitors or by using phosphatase-resistant ATP-gamma-S as substrate to phosphorylate CFTR. We surmise that absorptive function is subject to rapid regulation which can be switched "on" and "off" acutely by a control system that is common to both absorptive and secretory processes and that this control is crucial to switching between conductive and nonconductive transport mechanisms during salt absorption.