Switching the ion selectivity of an ion channel on demand

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.

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.

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.