Tracking transmitter gated P2X cation channel activation in vitro and in vivo.

摘要P2X受体是存在于大脑中的atp门控离子通道。一旦激活，这些通道打开一个阳离子选择孔，导致去极化和增加神经元的兴奋性。我们一直在开发一种非侵入性方法来跟踪发射机门控P2X阳离子通道的激活。该方法利用的事实是，大多数透射门控阳离子通道，包括P2X受体，具有可观的钙通量。我们设计了P2X受体，使其在孔的内层附近携带钙传感器，因此处于纳米结构域。我们对该方法进行了严格的P2X2受体测试。在一个细胞、神经元或网络中，这种方法允许人们对P2X受体的位置进行成像，并确定它们何时被激活，其灵敏度相当于全细胞膜片钳记录。此外，该方法是非侵入性的，并提供微米尺度的空间信息。数据表明，基于FRET的成像方法可以作为一种通用的方法来实时跟踪神经元中P2X通道的位置、区域表达变化、迁移和激活。这种方法将有助于揭示在生理过程中不同的受体何时、何地以及如何被激活。我们有两个具体目标，我们力求利用和完善我们的新方法。具体目标1：我们将利用光学报告蛋白在体内的表达来成像和识别P2X受体在对照和癫痫易感小鼠海马不同神经元中的区域表达、位置和激活。这是因为尽管已知ATP调节与癫痫相关的高兴奋性，但其确切作用尚未完全了解，主要是因为没有办法在神经元过程中选择性地测量P2X受体的激活。我们的方法弥补了这一不足，我们将用它来成像ATP P2X2受体在对照和癫痫易感小鼠神经元过程中的激活位点。结合高分辨率电生理学，我们的方法将使我们能够精确地确定ATP信号如何促进与癫痫相关的兴奋性增加。总之，我们将确定ATP信号在健康和癫痫海马中发挥的确切作用。特异性目的2：我们将产生表达P2X2受体光报告基因的小鼠。这些将是ATP信号社区的宝贵通用工具，特别是将阐明P2X受体信号如何促进癫痫。我们将设计和制造新一代的P2X结构，以更快的动力学和更高的空间灵敏度报告受体激活。这将使我们能够快速成像毫秒时间尺度的P2X受体介导的神经元过程信号，这是电生理方法无法实现的。公共卫生相关性：我们将研究ATP信号和P2X2受体在小鼠癫痫模型中的作用机制。这样，我们将为理解ATP受体在一般神经网络中的作用以及P2X2受体在癫痫中的具体作用奠定基础。这一点很重要，因为引起癫痫的机制尚不完全清楚，而且对人类临床管理的需求尚未得到满足。

DESCRIPTION (provided by applicant): Abstract P2X receptors are ATP-gated ion channels that are found in the brain. Once activated these channels open a cation selective pore, leading to depolarization and increased neuronal excitability. We have been developing a non invasive approach to track activation of transmitter-gated P2X cation channels. The method exploits the fact that most transmitter-gated cation channels, including P2X receptors, have appreciable calcium fluxes. We engineered P2X receptors to carry calcium sensors near the inner aspect of the pore, and therefore in a nanodomain. We rigorously tested the method for P2X2 receptors. Within a cell, neuron or network this method allows one to image the location of P2X receptors as well as determine when they are activated, with sensitivity equal to whole-cell patch clamp recording. Additionally, the approach is non invasive and provides micrometer scale spatial information. The data show that a FRET based imaging approach can be used as a general method to track the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in neurons, in real time and in living cells. The approach will help reveal when, where and how different receptors are activated during physiological processes. We have two specific aims with which we seek to exploit and refine our new approach. Specific Aim 1: We will employ in vivo expression of the optical reporters to image and identify the regional expression, location and activation of P2X receptors in distinct neurons of the hippocampus from control and epilepsy prone mice. This is because although ATP is known to regulate hyperexcitability associated with epilepsy its precise role is not yet fully understood, largely because there has been no way to measure P2X receptor activation selectively on neuronal processes. Our approach remedies this shortfall and we will use it to image sites of ATP P2X2 receptor activation on neuronal processes from control and epilepsy prone mice. Together with high resolution electrophysiology our approach will allow us to determine precisely how ATP signaling contributes to increased excitability associated with epilepsy. Overall, we will determine the precise roles that ATP signalling plays in the healthy and epileptic hippocampus. Specific Aim 2: We will generate mice expressing optical reporters for P2X2 receptors. These will be invaluable general tools for the ATP signaling community and specifically will shed light on how P2X receptor signaling contributes to epilepsy. We will design and engineer a new generation of P2X constructs that report receptor activation with faster kinetics and higher spatial sensitivity. This will allow us to image fast millisecond time scale P2X receptor mediated signaling in neuronal processes that are inaccessible to electrophysiological methods. PUBLIC HEALTH RELEVANCE: We will study the mechanisms that determine how ATP signaling and P2X2 receptors contribute to a mouse model of epilepsy. In so doing we will establish the basis for understanding the roles of ATP receptors within neuronal networks in general, as well as the specific roles for P2X2 receptors in epilepsy. This is important because the mechanisms that give rise to epilepsy are incompletely understood, and there is an unmet need for its clinical management in humans.