Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance
- National Institute of Neurological Disorders and Stroke, United States
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 PIKfyve kinase 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.
Data availability
All analyzed data are included in the manuscript
Article and author information
Author details
Funding
National Institute of Neurological Disorders and Stroke
- Xavier Leray
- Jacob K Hilton
- Kamsi Nwangwu
- Alissa Becerril
- Vedrana Mikusevic
- Gabriel Fitzgerald
- Anowarul Amin
- Mary R Weston
- Joseph A Mindell
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Metrics
-
- 2,802
- views
-
- 611
- downloads
-
- 35
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance
eLife 11:e74136.
https://doi.org/10.7554/eLife.74136
Further reading
-
- Structural Biology and Molecular Biophysics
Transition-state (TS) theory has provided the theoretical framework to explain the enormous rate accelerations of chemical reactions by enzymes. Given that proteins display large ensembles of conformations, unique TSs would pose a huge entropic bottleneck for enzyme catalysis. To shed light on this question, we studied the nature of the enzymatic TS for the phosphoryl-transfer step in adenylate kinase by quantum-mechanics/molecular-mechanics calculations. We find a structurally wide set of energetically equivalent configurations that lie along the reaction coordinate and hence a broad transition-state ensemble (TSE). A conformationally delocalized ensemble, including asymmetric TSs, is rooted in the macroscopic nature of the enzyme. The computational results are buttressed by enzyme kinetics experiments that confirm the decrease of the entropy of activation predicted from such wide TSE. TSEs as a key for efficient enzyme catalysis further boosts a unifying concept for protein folding and conformational transitions underlying protein function.
-
- Structural Biology and Molecular Biophysics
Aberrant signaling of BRAFV600E is a major cancer driver. Current FDA-approved RAF inhibitors selectively inhibit the monomeric BRAFV600E and suffer from tumor resistance. Recently, dimer-selective and equipotent RAF inhibitors have been developed; however, the mechanism of dimer selectivity is poorly understood. Here, we report extensive molecular dynamics (MD) simulations of the monomeric and dimeric BRAFV600E in the apo form or in complex with one or two dimer-selective (PHI1) or equipotent (LY3009120) inhibitor(s). The simulations uncovered the unprecedented details of the remarkable allostery in BRAFV600E dimerization and inhibitor binding. Specifically, dimerization retrains and shifts the αC helix inward and increases the flexibility of the DFG motif; dimer compatibility is due to the promotion of the αC-in conformation, which is stabilized by a hydrogen bond formation between the inhibitor and the αC Glu501. A more stable hydrogen bond further restrains and shifts the αC helix inward, which incurs a larger entropic penalty that disfavors monomer binding. This mechanism led us to propose an empirical way based on the co-crystal structure to assess the dimer selectivity of a BRAFV600E inhibitor. Simulations also revealed that the positive cooperativity of PHI1 is due to its ability to preorganize the αC and DFG conformation in the opposite protomer, priming it for binding the second inhibitor. The atomically detailed view of the interplay between BRAF dimerization and inhibitor allostery as well as cooperativity has implications for understanding kinase signaling and contributes to the design of protomer selective RAF inhibitors.
