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

描述(申请人提供)：摘要P2X受体是在大脑中发现的三磷酸腺苷门控离子通道。一旦被激活，这些通道就会打开一个阳离子选择性孔，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的方法来追踪递质门控的P2X阳离子通道的激活。该方法利用了这样一个事实，即大多数递质门控的阳离子通道，包括P2X受体，都有明显的钙离子通量。我们设计了P2X受体，在毛孔内侧附近携带钙传感器，因此在纳米区域中。我们严格测试了这种方法对P2X2受体的影响。在细胞、神经元或网络中，这种方法允许人们成像P2X受体的位置，并确定它们何时被激活，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，并提供微米级的空间信息。这些数据表明，基于FRET的成像方法可以作为一种通用的方法来实时和在活细胞中跟踪递质门控的P2X通道在神经元中的位置、区域表达的变化、流动性和激活。这种方法将有助于揭示不同的受体在生理过程中何时、何地以及如何被激活。我们有两个具体的目标，我们寻求利用和完善我们的新方法。具体目标1：我们将利用光学记者的体内表达来成像和鉴定对照组和癫痫易感小鼠海马区不同神经元中P2X受体的区域表达、定位和激活。这是因为虽然已知ATP调节与癫痫相关的过度兴奋性，但其确切作用尚不完全清楚，很大程度上是因为还没有方法选择性地测量神经元突起上的P2X受体激活。我们的方法弥补了这一不足，我们将使用它来成像来自对照组和癫痫易感小鼠神经元突起上的ATPP2X2受体激活的位置。结合高分辨率电生理学，我们的方法将使我们能够准确地确定ATP信号如何有助于增加与癫痫相关的兴奋性。总体而言，我们将确定ATP信号在健康和癫痫患者的海马体中所起的确切作用。具体目标2：我们将产生表达P2X2受体光学报告的小鼠。这些对于ATP信号界来说将是非常宝贵的通用工具，特别是将阐明P2X受体信号如何促进癫痫。我们将设计和设计新一代P2X结构，以更快的动力学和更高的空间敏感度报告受体激活。这将使我们能够成像快速毫秒时间尺度的P2X受体介导的神经元过程中的信号，而这些信号是电生理方法无法获得的。公共卫生相关性：我们将研究决定ATP信号和P2X2受体如何在癫痫小鼠模型中起作用的机制。在这样做的过程中，我们将为理解ATP受体在神经元网络中的一般作用以及P2X2受体在癫痫中的特定作用奠定基础。这一点很重要，因为引起癫痫的机制尚不完全清楚，人类对癫痫的临床治疗需求尚未得到满足。

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.