Understanding polymodal gating of a lysosomal ion channel

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Every cell is essentially a watery bag of various small and big molecules that are encapsulated by an oily, peripheral barrier (the 'plasma membrane'). Though insulated and protected this way, cells also need to respond to frequent changes happening in their external environment. A primitive yet very popular way cells do it is by sensing these outside 'weather report' through some specialised 'receptor' proteins sitting in their periphery which is then rapidly followed by a transient rise in calcium level in cellular interior (the 'cytoplasm'). This is a special, soluble form of calcium, unlike the one that is more familiar to us as an essential component of our bones and teeth. Normally, cells keep their internal calcium level low (for safety) but when needed, they secure extra calcium from outside as well as some internal organelles (mini cells) that store calcium in high amounts. Calcium is drawn from these sources via a family of specialised proteins known as ion channels. These channels, as their name implies, have specific watery holes or pores that allow, in regulated manner, the passage of calcium and/or other chemical species (e.g. sodium, potassium etc.). Whilst some ion channels are liberal allowing more than one chemical species to pass through, many others are often very choosy - only one species can best pass through them. Our proposal builds on our track record of studying a family of ion channels known as the two-pore channels (TPCs) that tunnel calcium from lysosomes - a specialised sub-cellular organelle with an acidic core. Although traditionally viewed as cellular 'recycle bins', lysosomes are emerging as important hubs for regulating cellular function in sickness and in health. TPCs have been shown to control many important functions including the shuttling of information around the cell. And they have been implicated in a number of diseases such as Parkinson's and Ebola infection. But exactly how these proteins are turned on and how much calcium passes through them is debated. TPCs are activated by two cell-made molecules namely NAADP and PI(3,5)P2 but depending on which one between these two molecules activates them, TPCs can toggle between a 'go or no go' mode for calcium. Intriguingly, we also have found two synthetic molecules - one of them behaves like NAADP whilst the other one like PI(3,5)P2 in tweaking calcium sieving property of TPCs. Our data thus challenge the textbook view that ion channels do not negotiate about what chemical species they will allow to pass through their pore. Interesting, of the natural activators of TPCs, PI(3,5)P2 directly binds to them whilst NAADP acts indirectly through binding to different protein(s) that remain associated with TPCs. This year, two plausible candidates have emerged as the NAADP binders. How these accessory proteins bind to NAADP and how that leads to activating TPCs remain a holy grail in the field. We will address these issues focusing on a major subtype of TPCs namely TPC2 using a number of complementary and interdisciplinary techniques. Building on our proven track record, we investigate how individual as well as all TPC2s open and closes through measuring the output electrical currents. Analyses of these data will tell us how different molecules activate TPC2 and tunes its calcium permeability. Parallel to these experiments, we will use state-of-the art computer simulations to predict how TPC2 structures may differentially behave when bound to these molecules and whether we can identify critical structural element(s) governing TPC2's preference for the permeating chemical species. We will validate our computer-based predictions through making changes on the predicted-regions and evaluate the function of the mutant TPC2s. Last but not the least, we will also be focusing on how one of the NAADP binders namely LSM12 protein associate with TPC2 and activates this channel.