Which organelle causes cystic fibrosis

It is believed that the first transmembrane domain TMD1 plays a role in the formation of the channel pore. These results agree with those of Oblatt-Montal et al. McDonough et al. These studies suggest that spliced forms of CFTR could contribute significantly to overall Cl - secretion in vivo. The original hypothesis that CFTR would function as a conductance regulator seems to be correct, as several ion transport abnormalities found in cystic fibrosis airway epithelial cells cannot be explained by mutations in the CFTR chloride channel.

Schwiebert et al. Figure 2 - Membrane model depicting the role of ATP release in the interaction between the cystic fibrosis transmembrane conductance regulator CFTR and other conductances. In the polarized cells of airway epithelia the chloride exit occurs through the CFTR. Once out of the cell, ATP may interact with purinergic receptors that, once activated, may stimulate ORCCs through second messenger pathways, increasing chloride transport across the cell. Recently, Schwiebert et al.

Thus, the roles of CFTR as a Cl - channel and a conductance regulator are not mutually exclusive, since one function can be eliminated while the other is preserved. In agreement with these results, Sugita et al. They also demonstrated that phosphorylation and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of the associated ATP channel, suggesting that the structural changes in CFTR that control its opening and closing mechanisms have similar effects on the ATP conduction pathway.

The fact that different domains of CFTR are responsible for its Cl - channel and ORCC regulatory functions led to an improved understanding of cystic fibrosis physiopathology. The mutations expected to cause the most severe disease are those that affect the ability of CFTR to function both as a chloride channel and as a conductance regulator. Mutations that retain at least one function or only partially reduce both functions may result in less severe pulmonary disease In agreement with this assumption, Fulmer et al.

The significance of these findings is still unclear. Affected individuals have two abnormal copies of the defective gene. Subjects that have one normal and one abnormal copy of the gene are symptom-free and are not known to be at increased risk for any disease. This deletion results in the loss of a single amino acid, the phenylalanine at codon , and is thus designated as DF The phenotype of patients bearing the DF mutation varies considerably, but this mutation is often associated with severe disease.

The alteration in the structure leads to defective processing of the DFCFTR protein through the endoplasmic reticulum, resulting in a drastically reduced level of protein to be expressed on the plasma membrane of exocrine epithelia in patients carrying the DF mutation. In this review, we will focus on only a small number of mutations in specific functional domains of the protein that have been studied in more detail. Besides the DF, both the GD and GD missense mutations have been well studied as the cause of severe CF illness, characterized by severe respiratory disease, pancreatic insufficiency and high sweat Cl - concentration Smit et al.

They showed that a severe mutation in one NBD was sufficient to critically reduce channel activity, but addition of a second severe mutation in the other NBD produced no additional defect. Fulmer et al. These findings suggest that the severity of pulmonary disease may be associated not only with the chloride function, but also and perhaps mainly with the regulatory role, rather than the channel function of CFTR.

In general, these mutations cause mild phenotypes in patients with CF Their results, taken together, indicate that the a-helices 5 and 6 may form the central pore of the CFTR Cl - channel. Moreover, the results obtained by Piazza-Carrol et al. The effects of TMD2 mutations on CFTR function have not been examined and studies performed on the regulatory domain are confusing , further studies being necessary to achieve a better understanding of the function of the RD.

The information obtained from the studies reviewed here shows the importance of the regulatory properties of CFTR at the cell plasma membrane level. The studies about the structure and function of CFTR are crucial for a better understanding of the defects involved in cystic fibrosis and for the development of alternative therapies for this complex human disease. This thicker mucus can affect many organs and body systems including:.

Sweat glands. CF makes sweat very salty. When people with CF sweat, they lose large amounts of salt. More than 30, people in the U. For each experiment, 30—50 vesicles were tracked simultaneously. Endosomes were labeled as described in B and the pH dissipation rate was measured after addition of 0. The pH dissipation rate was measured in experiments described in E. To assess the relative magnitude of CFTR-dependent counterion permeability and the passive proton leak of individual endosomes, determinants of the endosomal pH, we modified a technique developed to measure these parameters in cell suspension of mouse peritoneal macrophages and Chinese hamster ovary CHO cells Lukacs et al.

Similar results were observed in parental BHK cells, ruling out the possibility that CFTR expression is responsible for the high constitutive counterion conductance and limited proton leak of endosomes see Supplemental Figure S3E. To determine the consequence of CFTR activation on the steady-state endosomal pH, the luminal pH values were plotted before and after 3-min stimulation by PKA agonists.

The inability of CFTR functional overexpression to hyperacidify BHK endosomes suggested that the relatively high endogenous counterion permeability in the presence of a small passive proton leak cannot limit the proton accumulation by the v-ATPase in cells that have no endogenous CFTR. Figure 3. Data are means of triplicate determinations from a representative experiment. Importantly, despite full activation of wt CFTR by the agonist cocktail in 1.

Likewise, we were unable to detect significant changes in the initial acidification rate of early endosomes, after the synchronized internalization of Ab-labeled CFTR from the cell surface Figure 4 , E and F. These data strongly suggest that the CFTR-independent counterion permeability is sufficient to ensure unrestricted proton accumulation during the acidification and at steady state by the v-ATPase.

Finally, the HeLa and MDCK data suggest that the endosomal acidification is not limited by their endogenous counterion permeability, because provision of exogenous CFTR chloride conductance at an inwardly directed electrochemical chloride gradient Sonawane and Verkman, could not hyperacidify endosomes. Figure 4. The endosomal pH dissipation rates were determined as described in Figure 2 , E and F. Although these observations suggested that the CFTR-Ab complex retains significant activity, we sought an alternative assay to evaluate the CFTR-dependent endosomal pH regulation in the presence of fully functional channels.

This suggests that the anti-HA Ab binding did not significantly limit the CFTR-dependent counterion flux in early or recycling endosomes. Although there is no direct evidence available for the functional expression of CFTR in phagosomes, recent observations suggested that the phagolysosomal acidification of CFTR-deficient alveolar macrophages was severely compromised Di et al.

These results could not be confirmed by Haggie and coworkers Haggie and Verkman, Considering that the phagosomal membrane undergoes substantial compositional change during maturation Steinberg and Grinstein, , it was plausible to assume that CFTR may traverse the limiting membrane of phagosomes and facilitate acidification by provision of chloride as a counterion.

To assess CFTR localization and impact on the phagosomal proton and counterion permeability, first we used transiently transfected RAW Figure 5. Data are means of triplicate determinations. Equal amounts of cell lysates were immunoblotted. Top panels, internalized CFTR was colocalized with recycling endosomes and excluded from lysosomes. Single optical sections were obtained by fluorescence laser confocal microscopy FLCM. Lower panels, phagosomal maturation was monitored by the colocalization of synchronously ingested, FITC-conjugated P.

The labeling of recycling endosomes and lysosomes are described in Materials and Methods. Single optical sections were obtained by FLCM. To assess whether the activation of the phagocytic signaling cascade influences the subcellular targeting of CFTR, the postendocytic fate of the channel was determined in cells ingesting fluorophore-labeled P. Colocalization of bacteria with labeled Tf-R confirmed that the assay can reproduce the transient recruitment of Tf-receptors into immature phagosomes Figure 5 C, bottom panels as reported earlier Botelho et al.

Similar results were obtained upon labeling CFTR on ice for 1 h before phagocytosis and initiating both internalization and phagocytosis simultaneously data not shown. Another possibility is that the anti-HA Ab complex is susceptible to rapid degradation in the proteolytically active phagosomes. This is unlikely to be the case because the labeled CFTR-Ab complex remained detectable after min chase with comparable staining intensity, and inhibition of phagolysosomal proteases did not prevent the removal of Ab-labeled CFTR from phagosomes see Figure 6 F and data not shown.

Figure 6. Vesicular pH measurements were performed as in C. E Phagosomal acidification kinetics of RAW macrophages. MalH2 was added to the medium as in B. CFTR was labeled as described in F. The pH distribution profile of internalized wt CFTR-3HA indicated that the channels were primarily targeted to recycling endosomes in accord with immunolocalization data Figure 6 A. Next, the PKA sensitivity of the endogenous counterion permeability of Tf-labeled recycling endosomes was measured.

It has been accepted that the rapid acidification of newly formed phagosomes is mediated by the vacuolar proton ATPase in macrophages Lukacs et al. CFTR-dependent chloride uptake may promote proton accumulation by shunting the membrane potential of immature phagosomes. Both the cell surface binding of P. On the basis of the immunochemical localization and pH measurements, however, we could not rule out that a small fraction of CFTR reached the lysosomes.

CFTR was proposed to be indispensable for the normal acidification and bactericidal effect of phagolysosomal compartment in alveolar macrophages Di et al. Figure 7. Tf and dextran labeling was described in Materials and Methods. Inset, the variation of immature and mature phagosomal pH after 3 min PKA stimulation is reported as the function of the initial pH. Phagocytosis was initiated synchronously and the duration 5—45 min is indicated. G Protonophore-induced increase of the pH dissipation rate of endosomes and lysosomes relative to the passive proton efflux rate as an indicator of counterion permeability.

H Protonophore-induced increase of the pH dissipation rate relative to the passive proton efflux rate of endocytic organelles in primary macrophages.

Maturation of phagosomes, however, led to the loss of PKA-stimulated counterion permeability, measured after 30 min of phagosome formation Figure 7 , C and D. The assay pH sensitivity was preserved, as demonstrated by the rapid alkalinization of phagosomes by NH 4 Cl data not shown. Neither the initial rate of acidification nor the steady-state pH of mature phagosomes was influenced by CFTR expression in alveolar and peritoneal macrophages Figure 7 , E and F.

Because lysosomal fusion was proposed to play a pivotal role in phagosomal maturation Di et al. This could be attributed to the channel efficient recycling, inactivation, and degradation in lysosomes or a combination of these processes. These observations support the notion that the phagosomal and lysosomal acidifications are CFTR-independent processes in primary macrophages. To achieve the set point of organellar pH, the proton accumulation rate should be adjusted according to the buffer capacity and the proton leak Wu et al.

Considering that CFTR overexpression, activation, and ablation failed to change the organellar acidification in endolysosomes and phagosomes, we hypothesized that the endogenous, CFTR-independent counterion permeability is sufficiently high to dissipate the membrane potential Steinberg et al. This assumption implies that the passive proton permeability of endocytic organelles is small in comparison with their counterion permeability. To support this prediction, we assessed the counterion and passive proton permeabilities of endocytic organelles in respiratory epithelia, macrophages, and other heterologous expression systems, assuming that the passive pH dissipation rate is proportional with the proton permeabilities.

The Baf-induced proton efflux reflects the passive proton leak that is compensated by the v-ATPase activity at the steady-state pH. Similar data were obtained in primary macrophages Figure 7 H. These results strongly suggest that the counterion permeability is sufficiently high to support the acidification of organelles in CFTR-deficient respiratory epithelia and primary macrophages. Decreasing passive proton permeability along the secretory pathway was proposed as a critical determinant of the progressively increasing luminal acidification Wu et al.

An analogous role of the passive proton permeability may prevail along the endocytic pathway. To determine the passive proton permeability of endocytic organelles, first the buffer capacity of endosomes, lysosomes, and phagosomes was measured, using the ammonium chloride pulse technique Roos and Boron, ; Figure 8 , A and B.

The passive proton permeability was calculated based on the Baf-induced proton efflux rate and the assumption that the predominant driving force is the transmembrane pH gradient at a constant cytoplasmic pH 7. The passive proton permeability of lysosomes was at least twofold lower than in recycling endosomes in BHK, HeLa, IB3, and CFBE cells Figure 8 C , supporting the notion that down-regulation of the passive proton leak may contribute to the progressive acidification along the endocytic pathway.

For comparison, the passive proton permeability of the ER and Golgi compartment was also plotted, as determined in previous publications Llopis et al. Figure 8. Determination of the passive proton permeability of endocytic organelles. A Measurement of the buffer capacity of endocytic organelles.

The extent of lysosome alkalinization was measured after the addition of small amounts of NH 4 Cl e. The number indicates the final concentration of NH 4 Cl in mM. The buffer capacity was calculated from the alkalinization, induced by the 0. The early and mature phagosomal buffer capacities were measured after 5 and 30 min phagocytosis of FITC-conjugated P. C The passive proton permeability of recycling endosomes, lysosomes, and phagosomes. The passive proton efflux rate from the indicated organelles was measured in the presence of nM Baf by FRIA after their selective labeling as described in Materials and Methods.

The passive proton permeability was calculated as described in Materials and Methods. For comparison, the passive proton permeability of the Golgi compartment and the ER was indicated obtained from previous publications Llopis et al. Comparison of these studies is hampered by different cellular models and pH detection methodologies used as recently reviewed by Haggie and Verkman a. Because organellar acidification has fundamental influence on various cellular functions with proposed relevance to the hyperinflammatory CF lung infection Konstan et al.

During the past decade, three major determinants of organellar acidification have been established: the counterion conductance, the intrinsic proton leak, and the v-ATPase activity Weisz, b. The major counterion conductance of endolysosomes consists of members of two branches of the ClC gene family; ClC-3, -4, and -5, and ClC-6 and -7 Marshansky et al. Subcellular localization of the ClC-3, -4, and -5 in concert with functional data suggest that these ClC transporters are permissive for the acidification of early endosomes and synaptic vesicles by providing a significant electrical shunt pathway Stobrawa et al.

Ablation of ClC-4 and -5 impairs the acidification of endosomes Gunther et al. Impaired functional expression of ClC-3, -4, or -5, therefore, impedes the v-ATPase—mediated proton accumulation by increasing the inside positive membrane potential Marshansky et al. Although these data provide supportive evidence for the permissive role of ClC-3, -4, and -5 in endosomal acidification, they are short on demonstrating that the overall counterion permeability limits the proton pump activity in vivo, a prerequisite for CFTR to directly influence the endosomal pH regulation.

Using saturating concentration of Baf in the absence or in combination with a protonophore, we determined the relative magnitude of passive proton leak and the maximum rate of pH dissipation, an indicator of the lower limit of endosomal counterion permeability. The CFTR-independent counterion permeability was several-fold larger than the passive proton permeability in apical endosomes of polarized bronchial epithelia CFBE derived from CF patient.

The channel overexpression failed to hyperacidify the endolysosomal compartments Figure 4 , C and D, and Supplemental Figure S6C , implying that the v-ATPase activity is not limited by endogenous counterion permeability.

Similar results were obtained in IB3 respiratory epithelia and HeLa cells using FITC-Tf labeling of recycling endosomes and jointly support the notion that the endosomal pH cannot be regulated by modest alteration of the relatively large counterion conductance under physiological conditions. Identification of two ubiquitously expressing endolysosomal cation efflux pathways supports the inference that the CFTR-independent counterion permeability is significant and cannot restrict the relatively slow endosomal proton accumulation.

The TRPV2, a member of the transient receptor potential channel family, was confined to early endosomes Saito et al. Cell type differences and pH measurement methodology may explain the discordant data on lysosomal pH. Teichgraber et al.

The calibration was performed on permeabilized cells, based on the correlation between the luminal pH and the fluorescence intensity of the dye. An ion channel moves atoms or molecules that have an electrical charge from inside the cell to outside, or from outside the cell to inside.

In the lung, the CFTR ion channel moves chloride ions from inside the cell to outside the cell. To get out of the cell, the chloride ions move through the center of the tube formed by the CFTR protein.

Once the chloride ions are outside the cell, they attract a layer of water. This water layer is important because it allows tiny hairs on the surface of the lung cells, called cilia, to sweep back and forth. This sweeping motion moves mucus up and out of the airways. When any of these problems occur, the chloride ions are trapped inside the cell, and water is no longer attracted to the space outside the cell. When there is less water outside the cells, the mucus in the airways becomes dehydrated and thickens, causing it to flatten the cilia.

The cilia can't sweep properly when thick, sticky mucus weighs them down. Because the cilia can't move properly, mucus gets stuck in the airways, making it difficult to breathe. In addition, germs caught in the mucus are no longer expelled from the airway, allowing them to multiply and cause infections.