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
-
- 5,110
- views
-
- 883
- downloads
-
- 116
- 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
- Cell Biology
Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.
-
- Biochemistry and Chemical Biology
Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3′→5′ exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5′-bond of phosphodiester from 3′ to 5′ end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3′-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.
