The Cell Biology Of ATP Signalling At Intracellular P2X Receptors

ATP是一种能量来源，在我们体内的每个细胞内使用，但也由细胞释放，允许细胞相互通信。从细胞释放的ATP结合并打开细胞表面的受体，称为P2X受体。一旦打开，这些受体允许钠和钙流入细胞，导致进一步过程的激活。在动物中，P2X受体存在于神经，免疫细胞和肌肉的细胞表面，因此它们参与感知疼痛，炎症和控制血流。我们研究的总体目标是更好地了解P2X受体，以便在人类中开发缓解疼痛和抗炎疗法。我们最近在一种变形虫中发现了一种新的P2X受体，称为DdP2X。这是首次在单细胞生物中发现P2X受体。与动物P2X受体相反，DdP2X被发现在细胞内而不是在细胞表面。DdP2X存在于变形虫体内一种称为液泡的专门细胞器上。空泡负责泵出进入细胞的多余水分，这使得变形虫能够调节其体积。为了了解DdP2X的作用，我们通过去除产生DdP2X的基因来产生缺乏DdP2X的阿米巴。缺乏DdP2X的阿米巴不能有效地泵出水，因此当置于水中时会膨胀。这一观察结果表明，DdP2X是液泡正确发挥功能所必需的。该提案的主要目标之一是了解DdP2X如何调节变形虫中的空泡功能。这项研究的发现可能会告诉我们P2X受体如何调节人类细胞中的空泡。我们将确定钙通过DdP2X移动是否是正确的液泡功能和水的排泄所必需的。我们还将鉴定与DdP2X相关的其他蛋白质。这将有助于我们了解受体如何定位于细胞内部，并确定细胞内调节DdP2X或受DdP2X调节的分子。本研究的第二个主要重点是了解P2X受体如何工作及其进化。我们现在已经在单细胞生物如绿色藻类中鉴定出了其他几种原始的P2X受体。原始P2X受体是了解受体结构与功能关系的有用工具。这是因为原始P2X受体与人类、小鼠和大鼠受体只有很少的相似性，因此比较和操纵它们将揭示P2X受体作为ATP结合离子通道所需的关键部分。我们最终想了解P2X受体结合止痛药物的部分。总之，我们将使用阿米巴作为模型细胞来研究P2X受体在细胞内发挥的功能。使用来自单细胞生物的P2X受体的实验将用于告知我们人类P2X受体如何在健康和疾病中发挥作用。

ATP is an energy source used inside every cell in our bodies but is also released by cells allowing cells to communicate with one another. ATP released from cells binds to and opens receptors on the cell surface, called P2X receptors. Once open these receptors allow sodium and calcium to flow into the cell resulting in the activation of further processes. In animals, P2X receptors are present on the cell surface on nerves, immune cells and muscle, they consequently are involved in sensing pain, inflammation and the control of blood flow. It is the overall aim of our research to understand P2X receptors better in order to develop pain relieving and anti-inflammatory therapies in humans. We recently identified a new P2X receptor, called DdP2X, in a species of amoeba. This was the first time a P2X receptor had been found in a single celled organism. In contrast to animal P2X receptors DdP2X was found inside the cell and not at the cell surface. DdP2X was present on a specialised organelle inside the amoeba called a vacuole. The vacuole is responsible for pumping out excess water entering the cell, which allows the amoeba to regulate its volume. In order to understand the role of DdP2X, we generated amoeba lacking DdP2X by removing the gene that makes it. Amoeba lacking DdP2X could not pump water out as effectively and consequently swelled when placed in water. This observation suggested that DdP2X is required for the vacuole to function correctly. One of the major goals of this proposal is too understand how DdP2X regulates vacuole function in amoeba. This findings of this research may inform us of how P2X receptors may regulate vacuoles in human cells. We will identify whether calcium moving through DdP2X is essential for correct vacuole function and excretion of water. We will also identity other proteins that are associated with DdP2X. This will help us to understand how the receptor is localised to the inside of the cell and identify the molecules inside the cell that regulate or are regulated by DdP2X. The second major focus of this study is to understand how P2X receptors work and their evolution. We have now identified several other primitive P2X receptors in singled celled organisms like green algae. Primitive P2X receptors are useful tools for understand how receptor structure relates to function. This is because primitive P2X receptors share only few similarities with human, mouse and rat receptors and therefore comparing and manipulating them will reveal the critical parts that are required for a P2X receptor to work as an ATP-binding ion channel. We ultimately want to understand the parts of P2X receptors that bind pain relieving drugs. In summary, we will us amoeba as a model cell to investigate the functions that P2X receptors play inside the cell. Experiments using P2X receptors from single celled organisms will be used to inform us of how human P2X receptors function in health and disease.