Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function
- Ludwig Maximilian University of Munich, Germany
- UCL London, United Kingdom
- Medical University of Graz, Austria
- University of Leipzig, Germany
- Roche, Switzerland
- MHH Hannover, Germany
- University College London, United Kingdom
Abstract
Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Johann Schredelseker
Funding
Mucolipidosis IV Foundation (MDBR-17-120- ML4)
- Christian Grimm
Deutsche Forschungsgemeinschaft (SFB/TRR152 P04)
- Christian Grimm
Deutsche Forschungsgemeinschaft (SFB/TRR152 P06)
- Christian Wahl-Schott
Deutsche Forschungsgemeinschaft (SFB/TRR152 P12)
- Martin Biel
Deutsche Forschungsgemeinschaft (BR 1034/7-1)
- Franz Bracher
Biotechnology and Biological Sciences Research Council (BB/N01524X/1)
- Sandip Patel
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Richard S Lewis, Stanford University School of Medicine, United States
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Bavarian Government and the European Union. All of the animals were handled according to approved institutional animal care protocols of the University of Munich. The protocol was approved by the Bavarian Government (AZ55.2-1-54-2532-170-17).
Version history
- Received: December 23, 2019
- Accepted: March 12, 2020
- Accepted Manuscript published: March 13, 2020 (version 1)
- Accepted Manuscript updated: March 16, 2020 (version 2)
- Version of Record published: March 31, 2020 (version 3)
- Version of Record updated: April 6, 2020 (version 4)
Copyright
© 2020, Gerndt et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,992
- views
-
- 873
- downloads
-
- 110
- 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)
Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function
eLife 9:e54712.
https://doi.org/10.7554/eLife.54712
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
