Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance
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Curated by eLife
Evaluation Summary:
The focus of the manuscript is the lysosomal Cl-/H+ transporter CLC-7. The main finding is the direct regulation of CLC-7 by PI(3,5)P2, which keeps CLC-7 inactive. This finding may explain the lysosomal and cellular phenotype of a newly identified gain-of-function mutation in CLC-7 that causes lysosomal hyperacidification and large vacuoles.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H + ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl - /H + antiporter responsible for lysosomal Cl - permeability, is a candidate to contribute to the acidification process as part of this ‘counterion pathway’ The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here, we demonstrate that depleting PI(3,5)P2 by inhibiting the kinase PIKfyve causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together, these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.
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Evaluation Summary:
The focus of the manuscript is the lysosomal Cl-/H+ transporter CLC-7. The main finding is the direct regulation of CLC-7 by PI(3,5)P2, which keeps CLC-7 inactive. This finding may explain the lysosomal and cellular phenotype of a newly identified gain-of-function mutation in CLC-7 that causes lysosomal hyperacidification and large vacuoles.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Joint Public Review:
Lysosome-localized PI(3,5)P2, as well as lysosomal H+ and Cl-, are known to regulate lysosomal degradation, catabolite export, membrane trafficking (fusion/fission), and osmotic swelling/condensation, but the underlying mechanisms are not clear. CLC-7/Ostm1 is a lysosomal and osteoclast resorption lacuna localized chloride / proton antiporter. In osteoclasts it is essential for acid secretion and bone resorption. In lysosomes, the antiporter has been proposed to provide counter ions for efficient acidification and/or to raise luminal chloride concentration exploiting the proton gradient. Several previous lines of evidence have suggested a role of PI(3,5)P2 in CLC-7/Ostm1 regulation: i) plant vacuolar CLCs are potently inhibited by PI(3,5)P2 in the submicromolar range (plant vacuoles are somewhat similar to …
Joint Public Review:
Lysosome-localized PI(3,5)P2, as well as lysosomal H+ and Cl-, are known to regulate lysosomal degradation, catabolite export, membrane trafficking (fusion/fission), and osmotic swelling/condensation, but the underlying mechanisms are not clear. CLC-7/Ostm1 is a lysosomal and osteoclast resorption lacuna localized chloride / proton antiporter. In osteoclasts it is essential for acid secretion and bone resorption. In lysosomes, the antiporter has been proposed to provide counter ions for efficient acidification and/or to raise luminal chloride concentration exploiting the proton gradient. Several previous lines of evidence have suggested a role of PI(3,5)P2 in CLC-7/Ostm1 regulation: i) plant vacuolar CLCs are potently inhibited by PI(3,5)P2 in the submicromolar range (plant vacuoles are somewhat similar to lsyosomes); ii) inhibition of the PI(3,5)P2 producing PIKFYVE kinase leads to lysosomal enlargement that is largely inhibited by CLC-7/Ostm1 knock-out; iii) PI3P like molecules have been found to be tightly bound to CLC-7/Ostm1 in 3D structures.
Leray et al. enhance the knowledge in these matters in three important aspects. i) In cells, they show that PIKFYVE inhibition leads to a hyperacidification of lysosomes (in addition to lysosome enlargement) in a manner that is largely dependent on the presence of CLC-7/Ostm1 (this finding is in contrast to earlier reports from another group); ii) In patch-clamp recordings of plasma-membrane targets CLC-7, they show that application of relatively high concentrations of PI(3,5)P2 (50 µM) via the measuring patch pipette leads to a ~40% reduction of currents; iii) In patch-clamp recordings, the PI(3,5)P2 inhibition is completely absent with the Y715C mutant, which was previously shown to cause hyperacidification of lysosomes; moreover, Y715 is in reasonable distance to the PIP3 binding site seen in 3D structures.
The manuscript is well written, and the results and statistical analyses are clearly shown. The results potentially provide significant and important steps forward in the understanding of the role of CLC-7/Ostm1 in lysosomal biology, with implications for basic cellular processes including autophagy, and for the role of lysosomes in neurodegenerative diseases. However, the conclusion that at baseline CLC-7 is not active because it is inhibited by PI(3,5)P2 could be an overstatement because there is no PI(3,5)P2 dose-response in the manuscript and the baseline concentration of PIP2 in lysosomes is not known. In addition, the possible role of chloride accumulation needs to be assessed.
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