Switching the ion selectivity of an ion channel on demand
Lead Research Organisation:
University College London
Department Name: Cell and Developmental Biology
Abstract
Ion channels are proteins embedded within membranes that open to allow the movement of ions when activated. Such movement is essential for life. Conventional wisdom has it that the type or types of ion that flow through any one channel - so called ion selectivity - is fixed. Thus, calcium channels will only allow calcium ions to flow. Sodium channels will only allow sodium ions to flow. And non-selective channels will allow a combination of ions to pass.
This application challenges this belief by suggesting that ion selectivity is not fixed but rather that it depends on what opens the channel.
It builds on work funded by the BBSRC that aided the discovery of the two-pore channels (TPCs). TPCs are thought to allow calcium to be released from lysosomes. Lysosomes are acid-filled structures that are usually considered the cell's recycling centre. But it is clear now that they are also important stores of calcium. TPCs have been shown to control many important functions including the trafficking of material around the cell. And they have been implicated in a number of diseases such as Parkinson's and Ebola infection. But exactly how much calcium passes through them and how these proteins are turned on is debated. And they are difficult to study given their location within cells.
The proposed work is an international collaboration stemming from the discovery of two new cell permeable drugs that open TPCs. One of them appears to allow both calcium and sodium to flow whereas the other allows only sodium flow. We further show that these drugs seem to mimic physiological activators of the channel. The ion selectivity of TPCs can therefore be changed on demand.
Our plan is to extend this highly unusual finding by better defining ion selectivity of TPCs in response to different stimuli, working out where in the channel the drugs bind and using the drugs to selectively influence cell function. We will do so using a number of experimental approaches that include computational methods.
If successful, our work will change the way we think about how ion channels work. Novel chemical tools we will provide a unique resource for many to further probe function and dysfunction of TPCs. And alterations in ion selectivity offer a simple explanation for why previous studies on TPCs came to such different conclusions.
Changes of ion selectivity on demand demands further study of TPCs.
This application challenges this belief by suggesting that ion selectivity is not fixed but rather that it depends on what opens the channel.
It builds on work funded by the BBSRC that aided the discovery of the two-pore channels (TPCs). TPCs are thought to allow calcium to be released from lysosomes. Lysosomes are acid-filled structures that are usually considered the cell's recycling centre. But it is clear now that they are also important stores of calcium. TPCs have been shown to control many important functions including the trafficking of material around the cell. And they have been implicated in a number of diseases such as Parkinson's and Ebola infection. But exactly how much calcium passes through them and how these proteins are turned on is debated. And they are difficult to study given their location within cells.
The proposed work is an international collaboration stemming from the discovery of two new cell permeable drugs that open TPCs. One of them appears to allow both calcium and sodium to flow whereas the other allows only sodium flow. We further show that these drugs seem to mimic physiological activators of the channel. The ion selectivity of TPCs can therefore be changed on demand.
Our plan is to extend this highly unusual finding by better defining ion selectivity of TPCs in response to different stimuli, working out where in the channel the drugs bind and using the drugs to selectively influence cell function. We will do so using a number of experimental approaches that include computational methods.
If successful, our work will change the way we think about how ion channels work. Novel chemical tools we will provide a unique resource for many to further probe function and dysfunction of TPCs. And alterations in ion selectivity offer a simple explanation for why previous studies on TPCs came to such different conclusions.
Changes of ion selectivity on demand demands further study of TPCs.
Technical Summary
Ion channels open to allow the flow of a single ion (in selective channels) or multiple ions (in non-selective channels). Ion selectivity is a defining feature of a given ion channel and is generally considered immutable.
This application challenges this notion.
We build on the discovery of two structurally distinct, cell-permeable small molecule agonists of the lysosomal ion channel, TPC2. TPC2 was identified through previous BBSRC-funded work as the target channel for the Ca2+ mobilizing messenger, NAADP. It has since emerged as a channel of pathophysiological relevance. But fundamental attributes relating to its ion selectivity (Ca2+ v Na+) and its activating ligand are hotly debated.
Our pilot data together with collaborator Grimm show that one of the new agonists evoked robust Ca2+ signals in live cells but modest Na+ currents in isolated lysosomes, thereby mimicking the actions of NAADP. Conversely, the other agonist stimulated modest Ca2+ signals but robust Na+ currents, thus phenocopying the effects of the phosphoinositide, PI(3,5)P2, which also activates the channel. New, cell permeable agonists thus appear to mimic intracellular physiological cues in switching the ion selectivity of TPC2.
Our aims are to pursue this unique behaviour by establishing agonist-evoked changes in permeability of TPC2 in situ, identifying agonist binding sites on TPC2 and establishing agonist-specific physiological outcomes.
We will do so through an interdisciplinary approach comprising imaging, molecular biology, modelling and via international collaboration, endo-lysosomal electrophysiology.
Successful outcome of this project will establish a novel paradigm whereby a single ion channel mediates distinct ionic signatures on demand.
This application challenges this notion.
We build on the discovery of two structurally distinct, cell-permeable small molecule agonists of the lysosomal ion channel, TPC2. TPC2 was identified through previous BBSRC-funded work as the target channel for the Ca2+ mobilizing messenger, NAADP. It has since emerged as a channel of pathophysiological relevance. But fundamental attributes relating to its ion selectivity (Ca2+ v Na+) and its activating ligand are hotly debated.
Our pilot data together with collaborator Grimm show that one of the new agonists evoked robust Ca2+ signals in live cells but modest Na+ currents in isolated lysosomes, thereby mimicking the actions of NAADP. Conversely, the other agonist stimulated modest Ca2+ signals but robust Na+ currents, thus phenocopying the effects of the phosphoinositide, PI(3,5)P2, which also activates the channel. New, cell permeable agonists thus appear to mimic intracellular physiological cues in switching the ion selectivity of TPC2.
Our aims are to pursue this unique behaviour by establishing agonist-evoked changes in permeability of TPC2 in situ, identifying agonist binding sites on TPC2 and establishing agonist-specific physiological outcomes.
We will do so through an interdisciplinary approach comprising imaging, molecular biology, modelling and via international collaboration, endo-lysosomal electrophysiology.
Successful outcome of this project will establish a novel paradigm whereby a single ion channel mediates distinct ionic signatures on demand.
Planned Impact
We identify the following beneficiaries of this collaborative multi-disciplinary project:
1. International science base.
We will provide a broad range of scientific training through the interdisciplinary nature of this project involving direct collaboration with an international partner. This will be disseminated to the wider scientific community thus adding major value to this proposal.
2. General public.
This application is a basic science proposal addressing a ubiquitous signalling pathway (calcium) that underlies probably all cellular processes. The layman is likely only aware of the role of calcium as a mineral ion. This proposal will provide a better general understanding of how calcium is controlled.
3. Clinicians, patients and the pharmaceutical industry.
The pathways we target are demonstrably relevant to disease. In particular, TPCs are emerging as therapeutic targets. The outcomes of this project are thus of potential major relevance to public health and Pharma.
1. International science base.
We will provide a broad range of scientific training through the interdisciplinary nature of this project involving direct collaboration with an international partner. This will be disseminated to the wider scientific community thus adding major value to this proposal.
2. General public.
This application is a basic science proposal addressing a ubiquitous signalling pathway (calcium) that underlies probably all cellular processes. The layman is likely only aware of the role of calcium as a mineral ion. This proposal will provide a better general understanding of how calcium is controlled.
3. Clinicians, patients and the pharmaceutical industry.
The pathways we target are demonstrably relevant to disease. In particular, TPCs are emerging as therapeutic targets. The outcomes of this project are thus of potential major relevance to public health and Pharma.
People |
ORCID iD |
Sandip Patel (Principal Investigator) |
Publications
Alonso MT
(2023)
Use of aequorin-based indicators for monitoring Ca2+ in acidic organelles.
in Biochimica et biophysica acta. Molecular cell research
Clement D
(2023)
The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.
in Journal of immunology (Baltimore, Md. : 1950)
Gerndt S
(2020)
Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function.
in eLife
Gerndt S
(2020)
Discovery of lipophilic two-pore channel agonists.
in The FEBS journal
Gunaratne G
(2023)
Progesterone receptor membrane component 1 facilitates Ca2+ signal amplification between endosomes and the endoplasmic reticulum
in Journal of Biological Chemistry
Gunaratne G
(2021)
Essential requirement for JPT2 in NAADP-evoked Ca 2+ signaling
in Science Signaling
Gunaratne GS
(2023)
Convergent activation of two-pore channels mediated by the NAADP-binding proteins JPT2 and LSM12.
in Science signaling
Jaslan D
(2022)
Cation Transporters in Plants
Jaslan D
(2023)
New insights into gating mechanisms in TPCs: Relevance for drug discovery.
in Cell calcium
Jaslan D
(2023)
PI(3,5)P2 and NAADP: Team players or lone warriors? - New insights into TPC activation modes
in Cell Calcium
Li ZH
(2021)
A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii.
in Nature communications
Marchant JS
(2022)
NAADP-binding proteins find their identity.
in Trends in biochemical sciences
Morgan AJ
(2020)
Does lysosomal rupture evoke Ca2+ release? A question of pores and stores.
in Cell calcium
Patel S
(2024)
Stretching the role of TMEM63a to gatekeeping Ca2+ release in pancreatic acinar cells.
in Cell calcium
Patel S
(2022)
Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account.
in Cells
Patel S
(2020)
Preventing a shock to the system. Two-pore channel 1 negatively regulates anaphylaxis.
in Cell calcium
Patel S
(2023)
Endo-Lysosomal Two-Pore Channels and Their Protein Partners.
in Handbook of experimental pharmacology
Patel S
(2022)
Activation of endo-lysosomal two-pore channels by NAADP and PI(3,5)P2. Five things to know.
in Cell calcium
Patel S
(2021)
Teaming with NAADP.
in Science signaling
Patel S
(2021)
NAADP receptors: A one-two.
in Cell calcium
Patel S
(2022)
Quantal leaps in understanding Ca2+ signaling: A "Taylored" approach.
in Science signaling
Rahman T
(2022)
Rendezvous with PI(3,5)P2 - A rapalog gets caught opening TRPML1.
in Cell calcium
Saito R
(2023)
Convergent activation of Ca2+ permeability in two-pore channel 2 through distinct molecular routes.
in Science signaling
Vassileva K
(2020)
Two-pore channels as master regulators of membrane trafficking and endocytic well-being.
in Current opinion in physiology
Yuan Y
(2022)
Segregated cation flux by TPC2 biases Ca2+ signaling through lysosomes.
in Nature communications
Yuan Y
(2024)
Coordinating activation of endo-lysosomal two-pore channels and TRP mucolipins.
in The Journal of physiology
Yuan Y
(2021)
The lysosomotrope GPN mobilises Ca2+ from acidic organelles.
in Journal of cell science
Description | We have discovered two new cell permeable drugs that open TPC2. TPC2 is protein embedded within membranes that opens to allow the movement of ions when activated. One of the new drugs appears to allow both calcium and sodium ions to flow whereas the other allows only sodium flow. We further show that these drugs seem to mimic physiological activators of the channel. And that each affects cell function in different ways. The ion 'selectivity' of TPC2 can therefore be changed on demand to control the cell in fundamentally different ways. Yet despite such discrete actions, the drugs work together to increase the calcium but not sodium flow. |
Exploitation Route | Development of drugs to specifcally control TPC2 activity. |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | https://www.nature.com/articles/s41467-022-31959-0 |
Description | Understanding polymodal gating of a lysosomal ion channel |
Amount | £199,081 (GBP) |
Funding ID | BB/W01551X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2023 |
End | 12/2025 |
Description | Collaboration with Christian Grimm and Franz Bracher |
Organisation | Ludwig Maximilian University of Munich (LMU Munich) |
Country | Germany |
Sector | Academic/University |
PI Contribution | We tested new chemical compounds developed by the collaborators in a Parkinson's model. |
Collaborator Contribution | The partners provided the compounds FOC. |
Impact | A paper is being prepared for publication. |
Start Year | 2021 |
Description | Collaboration with Jonathan Marchant on the regulation of two-pore channels by NAADP-binding proteins |
Organisation | Medical College of Wisconsin |
Country | United States |
Sector | Academic/University |
PI Contribution | We helped identify NAADP-binding proteins in different organisms. |
Collaborator Contribution | The Marchant lab led all the experimental work. |
Impact | NIH grant in which we will characterise the effects of NAADP-binding proteins on membrane contact sites. Paper in Science Signalling: G.S. Gunaratne, E. Brailoiu, S. He, E.M. Unterwald, S. Patel, J.T. Slama, T.F. Walseth, J.S. Marchant, Essential requirement for JPT2 in NAADP-evoked Ca(2+) signaling, Science signaling 14(675) (2021). |
Start Year | 2018 |