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.     

Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.     

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.     

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.     

Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".     

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.     

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.

     

Inhibition of cystic fibrosis transmembrane conductance regulator chloride channel currents by arachidonic acid

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a number of different classes of organic anions which are able to enter and block the channel pore from its cytoplasmic end. Here I show, using patch clamp recording from CFTR-transfected baby hamster kidney cell lines, that the cis-unsaturated fatty acid arachidonic acid also inhibits CFTR Cl- currents when applied to the cytoplasmic face of excised membrane patches. This inhibition was of a relatively high affinity compared with other known CFTR inhibitors, with an apparent Kd of 6.5 +/- 0.9 microM. However, in contrast with known CFTR pore blockers, inhibition by arachidonic acid was only very weakly voltage dependent, and was insensitive to the extracellular Cl- concentration. Arachidonic acid-mediated inhibition of CFTR Cl- currents was not abrogated by inhibitors of lipoxygenases, cyclooxygenases or cytochrome P450, suggesting that arachidonic acid itself, rather than some metabolite, directly affects CFTR. Similar inhibition of CFTR Cl- currents was seen with other fatty acids, with the rank order of potency linoleic > or = arachidonic > or = oleic > elaidic > or = palmitic > or = myristic. These results identify fatty acids as novel high affinity modulators of the CFTR Cl- channel.     

State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN)(2)(-), to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.     

Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore

Immediately following exposure to thiocyanate (SCN-)-containing solutions, the cystic fibrosis conductance regulator Cl- channel exhibits high unitary SCN conductance and anomalous mole fraction behaviour, suggesting the presence of multiple anion binding sites within the channel pore. However, under steady-state conditions SCN-conductance is very low. Here I show, using patch clamp recording from CFTR-transfected mammalian cell lines, that under steady-state conditions neither SCN- conductance nor SCN- permeability show anomalous mole fraction behaviour. Instead, SCN conductance, permeability, and block of Cl- permeation can all be reproduced by a rate theory model that assumes only a single intrapore anion binding site. These results suggest that under steady-state conditions the interaction between SCN- and the CFTR channel pore can be understood by a simple model whereby SCN- ions enter the pore more easily than Cl-, and bind within the pore more tightly than Cl-. The implications of these findings for investigating and understanding the mechanism of anion permeation are discussed.     

Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations.

We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.     

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.