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.     

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.     

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.     

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.

     

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.     

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.     

Identification of channel-lining residues in the M2 membrane-spanning segment of the GABA(A) receptor alpha1 subunit

The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of gamma-aminobutyric acid (GABA) to the GABA(A) receptors induces the opening of an anion-selective channel that remains open for tens of milliseconds before it closes. To understand how the structure of the GABA(A) receptor determines the functional properties such as ion conduction, ion selectivity and gating we sought to identify the amino acid residues that line the ion conducting channel. To accomplish this we mutated 26 consecutive residues (250-275), one at a time, in and flanking the M2 membrane- spanning segment of the rat alpha1 subunit to cysteine. We expressed the mutant alpha1 subunit with wild-type beta1 and gamma2 subunits in Xenopus oocytes. We probed the accessibility of the engineered cysteine to covalent modification by charged, sulfhydryl-specific reagents added extracellularly. We assume that among residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction through the channel. We infer that nine of the residues, alpha1 Val257, alpha1 Thr26l, alpha1 Thr262, alpha1 Leu264, alpha1 Thr265, alpha1 Thr268, alpha1 Ile27l, alpha1 Ser272 and alpha1 Asn275 are exposed in the channel. On a helical wheel plot, the exposed residues, except alpha1 Thr262, lie on one side of the helix in an arc of 120 degrees. We infer that the M2 segment forms an alpha helix that is interrupted in the region of alpha1 Thr262. The modification of residues as cytoplasmic as alpha1 Val257 in the closed state of the channel suggests that the gate is at least as cytoplasmic as alpha1 Val257. The ability of the positively charged reagent methanethiosulfonate ethylammonium to reach the level of alpha1 Thr261 suggests that the charge-selectivity filter is at least as cytoplasmic as this residue.     

Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator.

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.     

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.     

Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.     

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.     

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.     

Involvement of cystic fibrosis transmembrane conductance regulator in infection-induced edema

Abnormal fluid accumulation in tissues, including the life-threatening cerebral and pulmonary edema, is a severe consequence of bacteria infection. Chlamydia (C.) trachomatis is an obligate intracellular gram-negative human pathogen responsible for a spectrum of diseases, causing tissue fluid accumulation and edema in various organs. However, the underlying mechanism for tissue fluid secretion induced by C. trachomatis and most of other infectious pathogens is not known. Here, we report that in mice C. trachomatis infection models, the expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel, is up regulated together with increased cytokine release and tissue fluid accumulation that can be reversed by treatment with antibiotic specific for C. trachomatis and CFTR channel blocker. However, C. trachomatis infection cannot induce tissue edema in CFTRtm1Unc mutant mice. Administration of exogenous IL-1beta to mice mimics the C. trachomatis infection-induced CFTR upregulation, enhanced CFTR channel activity and fluid accumulation, further confirming the involvement of CFTR in infection-induced tissue fluid secretion.     

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