Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

Abstract

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

Staphylococcus aureus Survives in Cystic Fibrosis Macrophages, Forming a Reservoir for Chronic Pneumonia

“…Thus, Barasch et al demonstrated a regulation of the pH in trans-Golgi vesicles by CFTR and an alkalinization of these vesicles in cells lacking CFTR (28). Several more recent studies revealed that Cftr also contributes to acidification of phagolysosomes in macrophages (20,29,(34)(35)(36). These studies showed that Cftr determines the influx of chloride ions into lysosomes (29).…”

“…The negatively charged chloride ions serve as counterions for protons pumped into phagolysosomes by the lysosomal V-type H ϩ -ATPase (37,38). A lack of Cftr thereby prevents the accumulation of protons in phagolysosomes and therefore impairs acidification of these vesicles, resulting in alkalinization of phagolysosomes (28,29,(34)(35)(36). Since the acidification of phagolysosomes is required for killing pathogens and the full activity of lysosomal enzymes, these studies concluded that the defect of Cftr-deficient alveolar macrophages to kill pathogens is caused by the increased pH in lysosomes in Cftr-deficient cells (29,36).…”

Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease

“…However, the granule pH data raise the possibility that finite amounts of CFTR are present in the secretory pathway/mucin granule membrane and play a role in granule acidification. Some studies strongly dispute the contribution of CFTR to organellar acidification, but others find that CFTR provides a counter-anion permeability that facilitates V-ATPase activity (16)(17)(18)(19)(20)(21). Estimates that minimal CFTR activity is required in the secretory pathway (66) are supported by studies showing that low-level transfection with recombinant CFTR corrects abnormal mucin sulfation without significantly increasing cAMP-stimulated Cl -permeability in CF airway epithelium (67).…”

“…organellar acidification by the vacuolar H + -ATPase (V-ATPase) (16). Others have both supported (17,18) and refuted (19)(20)(21) the contention that lysosomal/trans-Golgi pH is alkaline in CFTRdeficient cells. Recently, studies of human airway epithelium showed immunolocalization of CFTR with MUC5AC in isolated mucin granules and, further, demonstrated a CFTR-dependent Cl -conductance that enhanced granule acidification (22).…”

Rescue of Compromised Lysosomes Enhances Degradation of Photoreceptor Outer Segments and Reduces Lipofuscin-Like Autofluorescence in Retinal Pigmented Epithelial Cells

“…Lysosomal alkalinization decreased staining of Bodipy-pepstatin–A, suggesting the lysosomal enzyme cathepsin D was less effective in RPE cells with perturbed lysosomes [15, 16]. The clearance of outer segments is also decreased when the lysosomal pH is elevated with tamoxifen [17]. Thus experimental lysosomal alkalinization leads to decreased activity of degradative enzymes and accumulation of partially degraded photoreceptor outer segment debris.…”