Understanding polymodal gating of a lysosomal ion channel

每个细胞本质上都是一个由各种大小分子组成的水袋，这些分子被一层油性的外围屏障（“质膜”）包裹着。尽管细胞以这种方式被隔离和保护，但它们也需要对外部环境中发生的频繁变化做出反应。细胞的一种原始但非常流行的方式是通过位于其外围的一些专门的“受体”蛋白质感知这些外部的“天气报告”，然后迅速跟随细胞内部（“细胞质”）钙水平的短暂上升。这是一种特殊的、可溶的钙，与我们所熟悉的钙不同，钙是我们骨骼和牙齿的重要组成部分。正常情况下，细胞保持其内部钙水平较低（为了安全），但当需要时，它们从外部获得额外的钙，以及一些内部细胞器（迷你细胞）储存大量钙。钙是通过一系列被称为离子通道的特殊蛋白质从这些来源中提取的。这些通道，顾名思义，具有特定的水孔或孔隙，以受调节的方式允许钙和/或其他化学物质（如钠、钾等）通过。虽然一些离子通道是自由的，允许不止一种化学物质通过，但许多其他离子通道通常非常挑剔——只有一种化学物质能最好地通过它们。我们的建议建立在我们研究一种称为双孔通道（TPCs）的离子通道家族的记录之上，这种离子通道从溶酶体（一种具有酸性核心的特殊亚细胞细胞器）中穿过钙通道。虽然传统上认为溶酶体是细胞的“回收箱”，但溶酶体正在成为调节疾病和健康状态下细胞功能的重要枢纽。TPCs已被证明控制许多重要的功能，包括细胞周围信息的穿梭。它们与帕金森病和埃博拉感染等许多疾病有关。但这些蛋白质究竟是如何被激活的，以及有多少钙通过它们还存在争议。tpc由两种细胞生成的分子即NAADP和PI(3,5)P2激活，但取决于这两种分子中的哪一种激活它们，tpc可以在钙的“激活”或“不激活”模式之间切换。有趣的是，我们还发现了两种合成分子，其中一种类似于NAADP，另一种类似于PI(3,5)P2，可以调节tpc的钙筛分性能。因此，我们的数据挑战了教科书上的观点，即离子通道不会协商哪些化学物质可以通过它们的孔。有趣的是，在TPCs的天然激活剂中，PI(3,5)P2直接与它们结合，而NAADP通过与TPCs相关的不同蛋白的结合间接起作用。今年，NAADP出现了两个貌似合理的候选人。这些辅助蛋白如何与NAADP结合以及如何激活tpc仍然是该领域的圣杯。我们将使用一些互补和跨学科的技术来解决这些问题，重点关注tpc的一个主要亚型，即TPC2。基于我们可靠的跟踪记录，我们通过测量输出电流来研究单个和所有tpc2如何打开和关闭。对这些数据的分析将告诉我们不同的分子是如何激活TPC2并调节其钙通透性的。与这些实验平行，我们将使用最先进的计算机模拟来预测TPC2结构在与这些分子结合时的不同行为，以及我们是否可以识别控制TPC2对渗透化学物质的偏好的关键结构元素。我们将通过改变预测区域来验证我们基于计算机的预测，并评估突变TPC2s的功能。最后但并非最不重要的是，我们还将关注NAADP结合物之一即LSM12蛋白如何与TPC2结合并激活该通道。

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.