Electrodiffusional ATP movement through the cystic fibrosis transmembrane conductance regulator

Expression of thecystic fibrosis transmembrane conductance regulator (CFTR), and of atleast one other member of the ATP-binding cassette family of transportproteins, P-glycoprotein, is associated with the electrodiffusionalmovement of the nucleotide ATP. Evidence directly implicating CFTRexpression with ATP channel activity, however, is still missing. Hereit is reported that reconstitution into a lipid bilayer of highlypurified CFTR of human epithelial origin enables the permeation of bothCl and ATP. Similar topreviously reported data for in vivo ATP currents of CFTR-expressingcells, the reconstituted channels displayed competition betweenCl and ATP and had multipleconductance states in the presence of Cl and ATP. PurifiedCFTR-mediated ATP currents were activated by protein kinase A and ATP(1 mM) from the "intracellular" side of the molecule and wereinhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTRantibodies. The absence of CFTR-mediated electrodiffusional ATPmovement may thus be a relevant component of the pleiotropic cysticfibrosis phenotype.

     

Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.     

Glycosylation and the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.     

Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and cGMP-regulated chloride channel critical to the regulation of intestinal fluid, chloride, and bicarbonate secretion. In cystic fibrosis (CF), mutations in CFTR result in downregulation of CFTR function and small intestinal obstruction. Unlike the human CF intestine, severe gastrointestinal disease and lethal obstruction is common in transgenic mice deficient in CFTR. The relevance of the physiology of CFTR and pathophysiology of CF in genetically altered mice to that of human CF disease remains incompletely understood. We hypothesized that the expression and distribution of CFTR in mouse intestine may differ from that of human and may contribute to the variation in disease expression between the two species. Using immunocytochemical and immunoblot techniques and well-characterized anti-rodent anti-CFTR antibodies, we examined the cellular distribution of CFTR in the mouse intestinal tract. We identified significant differences in villus distribution for CFTR in the mouse proximal small intestine compared to those previously reported for human and rat. These observations are important to the understanding of CFTR pathophysiology in transgenic CF mouse model systems and bear relevance to the different phenotypic expression of disease in mice compared to human.     

Domain-domain associations in cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is caused by mutations inthe cystic fibrosis transmembrane conductance regulator (CFTR) gene.CFTR is a chloride channel whose activity requires protein kinaseA-dependent phosphorylation of an intracellular regulatory domain(R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs).To identify potential sites of domain-domain interaction within CFTR,we expressed, purified, and refolded histidine (His)- andglutathione-S-transferase (GST)-tagged cytoplasmic domainsof CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated bymeasuring tryptophan fluorescence quenching. Trypticdigestion of in vitro phosphorylated his-NBD1-R and in situphosphorylated CFTR generated the same phosphopeptides. An interactionbetween NBD1-R and NBD2 was assayed by tryptophan fluorescencequenching. Binding among all pairwise combinations of R-domain, NBD1,and NBD2 was demonstrated with an overlay assay. To identifyspecific sites of interaction between domains of CFTR, an overlay assaywas used to probe an overlapping peptide library spanning allintracellular regions of CFTR with his-NBD1, his-NBD2, andGST-R-domain. By mapping peptides from NBD1 and NBD2 that bound toother intracellular domains onto crystal structures for HisP, MalK, andRad50, probable sites of interaction between NBD1 and NBD2 wereidentified. Our data support a model where NBDs form dimers with theATP-binding sites at the domain-domain interface.

     

An unstable transmembrane segment in the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel with 12 membrane-spanning sequences, undergoes inefficient maturation in the endoplasmic reticulum (ER). Potentially charged residues in transmembrane segments may contribute to this defect in biogenesis. We demonstrate that transmembrane segment 6 of CFTR, which contains three basic amino acids, is extremely unstable in the lipid bilayer upon membrane insertion in vitro and in vivo. However, two distinct mechanisms counteract this anchoring deficiency: (i) the ribosome and the ER translocon co-operate to prevent transmembrane segment 6 from passing through the membrane co- translationally; and (ii) cytosolic domains of the ion channel post-translationally maintain this segment of CFTR in a membrane-spanning topology. Although these mechanisms are essential for successful completion of CFTR biogenesis, inefficiencies in their function retard the maturation of the protein. It seems possible that some of the disease-causing mutations in CFTR may reduce the efficiency of proper membrane anchoring of the protein.     

Phosphorylation of the cystic fibrosis transmembrane conductance regulator.

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.     

Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione

We investigated the mechanisms by which S-nitrosoglutathione (GSNO) alters cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride (Cl(-)) secretion across Calu-3 cells, an extensively used model of human airway gland serous cells. Confluent monolayers of Calu-3 cells, grown under an air-liquid interface, were mounted in Ussing chambers for the measurements of chloride short circuit current (I(sc)) and trans-epithelial resistance (R(t)). Addition of GSNO into the apical compartment of these chambers resulted in significant and sustained increase of I(sc) with an IC(50) of 3.2 +/- 1 mum (mean +/- 1 S.E.; n = 6). Addition of either glibenclamide or pre-treatment of Calu-3 cells with the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one totally prevented the GSNO-induced increase of I(sc). Conversely, BAY 41-2272, a sGC stimulator, increased I(sc) in a dose-response fashion. The GSNO increase of I(sc) was reversed by addition of two phosphatases (PP2A1, PP2A2) into the apical compartment of Ussing chambers containing Calu-3 monolayers. Oxy-myoglobin (oxy-Mb, 300 mum) added into the apical compartment of Ussing chambers either prior or after GSNO either completely prevented or immediately reversed the increase of I(sc). However, smaller concentrations of oxy-Mb (1-10 mum), sufficient to scavenge NO in the medium (as assessed by direct measurement of NO in the Ussing chamber using an ISO-NO meter) decreased I(sc) partially. Oxy-Mb did not reverse the increase of I(sc) following addition of GSNO and cysteine (50 mum). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.     

Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator

Mismanaged protein trafficking by the proteostasis network contributes to several conformational diseases, including cystic fibrosis, the most frequent lethal inherited disease in Caucasians. Proteostasis regulators, as cystamine, enable the beneficial action of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators in ΔF508-CFTR airways beyond drug washout. Here we tested the hypothesis that functional CFTR protein can sustain its own plasma membrane (PM) stability. Depletion or inhibition of wild-type CFTR present in bronchial epithelial cells reduced the availability of the small GTPase Rab5 by causing Rab5 sequestration within the detergent-insoluble protein fraction together with its accumulation in aggresomes. CFTR depletion decreased the recruitment of the Rab5 effector early endosome antigen 1 to endosomes, thus reducing the local generation of phosphatidylinositol-3-phosphate. This diverts recycling of surface proteins, including transferrin receptor and CFTR itself. Inhibiting CFTR function also resulted in its ubiquitination and interaction with SQSTM1/p62 at the PM, favoring its disposal. Addition of cystamine prevented the recycling defect of CFTR by enhancing BECN1 expression and reducing SQSTM1 accumulation. Our results unravel an unexpected link between CFTR protein and function, the latter regulating the levels of CFTR surface expression in a positive feed-forward loop, and highlight CFTR as a pivot of proteostasis in bronchial epithelial cells.     

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described. Received: 25 August 1997 / Accepted: 20 January 1998     

Partial purification of the cystic fibrosis transmembrane conductance regulator.

We have investigated several purification strategies for the cystic fibrosis transmembrane regulator (CFTR) based on its structural similarity to other proteins of the traffic ATPase/ABC transporter family. Recombinant CFTR expressed in heterologous cells was readily solubilized by digitonin and initially separated from the majority of other cellular proteins by sucrose density gradient centrifugation. CFTR, with two predicted nucleotide binding domains, bound avidly to several triazine dye columns, although elution with MgATP, MgCl2, or high ionic strength buffers was inefficient. CFTR did not bind to either ATP or ADP coupled to agarose. Because CFTR is a glycoprotein we investigated its binding to lectin columns. CFTR bound readily to wheat germ agglutinin, but poorly to Lens culinaris agglutinin. CFTR was enriched 9-10 times when eluted from wheat germ agglutinin with N-acetylglucosamine. This enrichment was tripled if lectin chromatography followed sucrose gradient centrifugation. Our results suggest the combination of sucrose density gradient centrifugation and lectin chromatography would be a satisfactory approach to initial purification of CFTR expressed in heterologous cells.     

Misfolding of the cystic fibrosis transmembrane conductance regulator and disease

Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.     

Control of cystic fibrosis transmembrane conductance regulator expression by BAP31

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is stringently controlled by molecular chaperones participating in formation of the quality control system. It has been shown that about 75% of all CFTR protein and close to 100% of the [DeltaPhe(508)] CFTR variant are rapidly degraded before leaving the endoplasmic reticulum (ER). B cell antigen receptor-associated proteins (BAPs) are ubiquitously expressed integral membrane proteins that may control association with the cytoskeleton, vesicular transport, or retrograde transport from the cis Golgi to the ER. The present study delivers evidence for cytosolic co-localization of both BAP31 and CFTR and for the control of expression of recombinant CFTR in Chinese hamster ovary (CHO) cells and Xenopus oocytes by BAP31. Antisense inhibition of BAP31 in various cell types increased expression of both wild-type CFTR and [DeltaPhe(508)]CFTR and enabled cAMP-activated Cl(-) currents in [DeltaPhe(508)]CFTR-expressing CHO cells. Coexpression of CFTR together with BAP31 attenuated cAMP-activated Cl(-) currents in Xenopus oocytes. These data therefore suggest association of BAP31 with CFTR that may control maturation or trafficking of CFTR and thus expression in the plasma membrane.     

Targeted therapy for cystic fibrosis: cystic fibrosis transmembrane conductance regulator mutation-specific pharmacologic strategies

Cystic fibrosis (CF) results from the absence or dysfunction of a single protein, the CF transmembrane conductance regulator (CFTR). CFTR plays a critical role in the regulation of ion transport in a number of exocrine epithelia. Improvement or restoration of CFTR function, where it is deficient, should improve the CF phenotype. There are >1000 reported disease-causing mutations of the CFTR gene. Recent investigations have afforded a better understanding of the mechanism of dysfunction of many of these mutant CFTRs, and have allowed them to be classified according to their mechanism of dysfunction. These data, as well as an enhanced understanding of the role of CFTR in regulating epithelial ion transport, have led to the development of therapeutic strategies based on pharmacologic enhancement or repair of mutant CFTR dysfunction. The strategy, termed 'protein repair therapy', is aimed at improving the regulation of epithelial ion transport by mutant CFTRs in a mutation-specific fashion. The grouping of CFTR gene mutations, according to mechanism of dysfunction, yields some guidance as to which pharmacologic repair agents may be useful for specific CFTR mutations. Recent data has suggested that combinations of pharmacologic repair agents may be necessary to obtain clinically meaningful CFTR repair. Nevertheless, such strategies to improve mutant CFTR function hold great promise for the development of novel therapies aimed at correcting the underlying pathophysiology of CF.     

Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity

Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels.