Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.

Understanding how cystic fibrosis mutations cause a loss of Cl- channel function.

Defective epithelial Cl- secretion is the hallmark of the lethal genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a regulated Cl- channel. Since the identification of the single gene encoding CFTR, several hundred disease-causing mutations, associated with a wide variety of clinical phenotypes, have been reported. To understand the relationship between genotype and clinical phenotype, researchers have investigated how mutations in CFTR disrupt its function. Here, we review the recent progress in understanding how CF-associated mutations in CFTR produce defective Cl- channels, and discuss the implications of this knowledge for the development of therapy for CF.

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.

Staphylococcus aureus alpha-toxin permeabilizes the basolateral membrane of a Cl(-)-secreting epithelium.

Apical membrane ion channels control the rate of transepithelial electrolyte transport in many epithelia. One way to study such channels in their native location, the apical membrane, is to eliminate the resistance of the basolateral membrane to ion flow. Then the opening and closing of apical channels can be measured as a transepithelial current, free from the influence of basolateral membrane transport processes. To develop a method that would permeabilize an epithelial basolateral membrane to ions and nucleotides, we examined the effect of Staphylococcus aureus alpha-toxin on the Cl(-)-secreting T84 epithelial cell line. alpha-Toxin permeabilized the basolateral, but not the apical membrane to Cl-, adenosine 3',5'-cyclic monophosphate (cAMP), and GTP. However, the integrity of signal-transduction pathways, the regulation of apical membrane Cl- channels, and the transepithelial resistance remained intact. In the course of examining the effect of ATP, we found that the basolateral membrane contained purinergic receptors that both stimulated Cl- secretion on their own and, at high concentrations, inhibited cAMP-induced Cl- secretion. These effects of extracellular ATP were eliminated after prolonged exposure to ATP, suggesting receptor downregulation. In addition, depletion of intracellular ATP following permeabilization prevented cAMP-dependent regulation of apical Cl- channels. We conclude that alpha-toxin may prove to be a useful tool for studying the regulation and properties of apical membrane ion channels.

Processing of CFTR bearing the P574H mutation differs from wild-type and deltaF508-CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) containing the deltaF508 mutation is retained in the endoplasmic reticulum (ER). This defect can be partially overcome by a reduction in temperature which allows some of the deltaF508 protein to exit the ER and move to the cell surface. Earlier studies showed that the CF-associated mutants, P574H and A455E, were also misprocessed. In this study, we found that processing of P574H and A455E was also temperature-sensitive; at 26 degrees C, some of the protein matured. In contrast to other CFTR mutants, P574H accumulated in punctate cytoplasmic bodies that colocalized with endoplasmic reticulum (ER) markers. At 26 degrees C, these bodies were no longer present. P574H showed a prolonged association with Hsp70 and also colocalized with Hsp70. We used brefeldin A (BFA) to determine which processing step(s) was altered by reduced temperature. Unlike wild-type CFTR, which was converted into an intermediate that was stable in the presence of BFA at 37 degrees C, deltaF508 and P574H produced the intermediate only when the temperature was reduced to 26 degrees C. Furthermore the wild-type intermediate was not associated with Hsp70. These data suggest that formation of the stable intermediate is a key temperature-sensitive step and appears to be coincident with release of the wild-type protein from Hsp70.

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

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

Association of domains within the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel composed of two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD), and an R domain. To understand how these domains interact, we expressed various constructs of CFTR containing membrane-spanning and/or cytosolic domains either separately or together. We then tested for functional association of these domains using the SPQ halide-efflux assay or physical association using coimmunoprecipitation experiments. Coexpression of the amino-terminal half (MSD1, NBD1, and the R domain) and the carboxy-terminal half (MSD2 and NBD2) of CFTR generated functional Cl- channel activity whereas expression of either alone did not give a signal with the SPQ assay. This result suggests that the two halves associate in the membrane. Using domain-specific antibodies, we found that either half of CFTR could coimmunoprecipitate the other, suggesting a physical association. Coimmunoprecipitation persisted between halves missing the NBDs, the R domain, or the amino-terminal tail. Moreover, constructs from MSD1 containing only the first and second transmembrane sequences and intervening extracellular loop were sufficient for interaction with MSD2. These data suggest that interactions between the two membrane-spanning domains of CFTR may mediate association between the two halves of the protein.

Dysfunction of CFTR bearing the delta F508 mutation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in patients with cystic fibrosis (CF). The most common CF-associated mutation is deletion of phenylalanine at residue 508, CFTR delta F508. When expressed in heterologous cells, CFTR bearing the delta F508 mutation fails to progress through the normal biosynthetic pathway and fails to traffic to the plasma membrane. As a result, CFTR delta F508 is mislocalized and is not present in the apical membrane of primary cultures of airway epithelia. Consequently, the apical membrane of CF airway epithelia is Cl- -impermeable, a defect that probably contributes to the pathogenesis of the disease.

Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.

Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency.

Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments.

A 45-kDa protein antigenically related to band 3 is selectively expressed in kidney mitochondria.

Anion exchange similar to that catalyzed by erythrocyte band 3 occurs across many nonerythroid cell membranes. To identify anion-exchange proteins structurally related to band 3, we immunoblotted rabbit kidney medullary membrane fractions with anti-band 3 antibodies. Immunoblots using antibodies to the cytoplasmic domain of band 3 revealed cross-reactive proteins in the plasma membrane fraction only. In contrast, two monoclonal antibodies against band 3 membrane domain labeled a 45-kDa protein; further immunoblotting and immunogold studies of membrane fractions and kidney sections using one of the anti-membrane domain antibodies showed that labeling was strongest in mitochondria of H(+)-secreting collecting duct cells. Tissue-to-tissue expression of the 45-kDa mitochondrial protein was variable: kidney medulla greater than heart greater than kidney cortex much greater than liver. We conclude that a 45-kDa protein with immunological cross-reactivity to the erythrocyte band 3 membrane domain is expressed in mitochondria in a highly cell-specific fashion and speculate that the protein may play a role in mitochondrial anion transport.

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution.

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

The amino-terminal portion of CFTR forms a regulated Cl- channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel consists of two motifs (each containing a membrane-spanning domain [MSD] and a nucleotide-binding domain [NBD]) linked by an R domain. We tested the hypothesis that one MSD-NBD motif could form a Cl- channel. The amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1, and the R domain) formed Cl- channels with conductive properties identical to those of CFTR. However, channel regulation differed. Although phosphorylation increased activity, channels opened without phosphorylation. MgATP stimulated D836X more potently than CFTR and may interact at more than one site. These data and migration of D836X on sucrose density gradients suggest that D836X may function as a multimer. Thus, the amino-terminal portion of CFTR contains all of the structures required to build a regulated Cl- channel.

Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (delta F508). We constructed STE6-CFTR chimeras with portions of the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBD1. The chimeras were functional in yeast, but mating efficiency decreased when delta F508 was introduced into NBD1. We isolated two delta F508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBD1 sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and Cl- channel gating defects caused by the delta F508 mutation. These results suggest that the NBD1s of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by delta F508 can be partially corrected by additional alterations in the protein.

Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel located in the apical membrane of epithelia. Although cystic fibrosis (CF) is caused by mutations in a single gene encoding CFTR, the disease has a variable clinical phenotype. The most common mutation associated with cystic fibrosis, deletion of a phenylalanine at position 508 (frequency, 67%), is associated with severe disease. But some missense mutations, for example ones in which arginine is replaced by histidine at residue at 117 (R117H; 0.8%), tryptophan at 334 (0.4%), or proline at 347 (0.5%), are associated with milder disease. These missense mutations affect basic residues located at the external end of the second (M2) and in the sixth (M6) putative membrane-spanning sequences. Here we report that, when expressed in heterologous epithelial cells, all three mutants were correctly processed and generated cyclic AMP-regulated apical Cl- currents. Although the macroscopic current properties were normal, the amount of current was reduced. Patch-clamp analysis revealed that all three mutants had reduced single-channel conductances. In addition, R117H showed altered sensitivity to external pH and had altered single-channel kinetics. These results explain the quantitative decrease in macroscopic Cl- current, and suggest that R117, R334 and R347 contribute to the pore of the CFTR Cl- channel. Our results also suggest why R117H, R334W and R347P produce less severe clinical disease and have implications for our understanding of cystic fibrosis.

Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.

Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte incorporation of ingested 58-iron by infants.

The least abundant stable isotope of iron, 58Fe (natural abundance 0.322 weight %), was administered orally to infants to explore the feasibility of using a stable rather than a radioisotope in studies of iron absorption. The dose of 58Fe was given between feedings at age 126 days. The mass isotope ratio, 58Fe/57Fe, was determined in blood by inductively coupled plasma mass spectroscopy and at ages 140, 168, and 196 days. The percentage of the 58Fe dose entering the circulation (3.2 to 16.0%) was inversely correlated with serum ferritin concentration (r = -0.867, p less than 0.01). For individual infants the SD of the percentage of administered dose of iron appearing in the circulation ranged from 0.22 to 1.28. We conclude that the method is likely to be suitable for within-subject comparisons of iron availability from foods. Because of the large between-subject variation, we are pessimistic for this age group about the usefulness of study designs based on group comparisons.