Investigating the role of chloride in endocyticorganelle acidification

[摘要] Endocytic organelles maintain their specific, acidic pH using the V-ATPase proton pump. The accumulation of protons across the membrane generates a voltage, requiring the movement of additional ion, known as a counterion, to dissipate charge buildup. This thesis seeks to explore the role of counterions, particularly chloride, and the proteins that facilitate their movements in the acidification of clathrin-coated vesicles (CCVs), a subset of early endosomes, and lysosomes. In CCVs, we showed that isolated bovine brain vesicles acidify in the presence of external chloride, independent of the monovalent cations present, confirming previous work. While unsuccessful at identifying the mechanism of anion transport, we used a new approach to show that most brain CCVs are synaptic vesicles, complementing results from earlier studies. Secondly, acidification of isolated lysosomes has also been shown to be chloride-dependent. Given that the movement of lysosomal chloride is facilitated by ClC-7, a chloride/proton antiporter, this protein has been suggested to mediate counterion movement, allowing acidification. In contrast, previous studies show that live cell ClC-7 knockout (KO) mouse lysosomes have the same pH as WT. We generated mice with a liver-specific deletion of ClC-7 to test these results in isolated lysosomes and live cells in parallel. While isolated KO lysosomes showed a drastic decrease in chloride-facilitated acidification, hepatocytic lysosomal pH was similar in WT and KO, even during metabolic and base challenges, leaving the role of ClC-7 unclear. Lastly, we present two patients with an identical pathogenic de novo variant in ClC-7, who display hypopigmentation, developmental delay, organomegaly, and failure to thrive, but no osteopetrosis. Patient fibroblasts contain large vacuoles of unknown composition and an increased number of lysosomal-like compartments. Lysosomal pH in cultured patient fibroblasts are more acidic than neonatal controls and heterologous expression of the mutant ClC-7 in Xenopus oocytes revealed a substantial increase in transport activity. These results suggest a novel gain-of-function mutation for this antiporter, one of the first reported instances of lysosomal hyperacidification, and show that ClC-7 plays a role in lysosomal pH regulation. Overall, this work advances our understanding of ClC-7 and counterion-mediated acidification in endocytic organelles.

[发布机构] Johns Hopkins University

[效力级别] chloride

[关键词] Lysosome;chloride;counterion;acidification;organelle;clathrin-coated vesicles;Biology