scholarly journals Targeted Proteomic Quantitation of the Absolute Expression and Turnover of Cystic Fibrosis Transmembrane Conductance Regulator in the Apical Plasma Membrane

2014 ◽  
Vol 13 (11) ◽  
pp. 4676-4685 ◽  
Author(s):  
Adam J. McShane ◽  
Bekim Bajrami ◽  
Alex A. Ramos ◽  
Pamela A. Diego-Limpin ◽  
Vahid Farrokhi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Apical Plasma Membrane ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Absolute Expression ◽  
The Absolute ◽  
Cystic Fibrosis Transmembrane

scholarly journals Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR

2019 ◽  
Vol 317 (1) ◽  
pp. L71-L86 ◽  
Author(s):  
Samuel J. Bose ◽  
Marcel J. C. Bijvelds ◽  
Yiting Wang ◽  
Jia Liu ◽  
Zhiwei Cai ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Membrane Expression ◽  
The Impact ◽  
Cystic Fibrosis Transmembrane ◽  
Human And Mouse

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl− channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl− channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl− channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl− channels.

scholarly journals Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation

1997 ◽  
Vol 328 (2) ◽  
pp. 353-361 ◽  
Author(s):  
L. Gergely LUKACS ◽  
Gersana SEGAL ◽  
Norbert KARTNER ◽  
Sergio GRINSTEIN ◽  
Fred ZHANG
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Protein Phosphorylation ◽  
Chinese Hamster ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hypertonic Medium ◽  
Cystic Fibrosis Transmembrane

Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by > 90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.

scholarly journals Role of calpain in the regulation of CFTR (cystic fibrosis transmembrane conductance regulator) turnover

2010 ◽  
Vol 430 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Monica Averna ◽  
Roberto Stifanese ◽  
Raffaella Grosso ◽  
Marco Pedrazzi ◽  
Roberta De Tullio ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Strong Interactions ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Intact Cells ◽  
Calpain Activation ◽  
Limited Digestion ◽  
Cystic Fibrosis Transmembrane

The level of the mature native 170 kDa form of CFTR (cystic fibrosis transmembrane conductance regulator) at the plasma membrane is under the control of a selective proteolysis catalysed by calpain. The product of this limited digestion, consisting of discrete fragments still associated by strong interactions, is removed from the plasma membrane and internalized in vesicles and subject to an additional degradation. This process can be monitored by visualizing the accumulation of a 100 kDa fragment in a proliferating human leukaemic T-cell line and in human circulating lymphocytes. In reconstructed systems, and in intact cells, the conversion of native CFTR into the 100 kDa fragment linearly correlated with calpain activation and was prevented by addition of synthetic calpain inhibitors. A reduction in Ca2+ influx, by blocking the NMDA (N-methyl-D-aspartate) receptor Ca2+ channel, inhibited the conversion of the native 170 kDa fragment into the 100 kDa fragment, whereas an endosome acidification blocker promoted accumulation of the digested 100 kDa CFTR form. An important role in calpain-mediated turnover of CFTR is exerted by HSP90 (heat-shock protein 90), which, via association with the protein channel, modulates the degradative effect of calpain through a selective protection. Taken together these results indicate that CFTR turnover is initiated by calpain activation, which is induced by an increased Ca2+ influx and, following internalization of the cleaved channel protein, and completed by the lysosomal proteases. These findings provide new insights into the molecular mechanisms responsible for the defective functions of ion channels in human pathologies.

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