P2X channel mobility in the plasma membrane of neurons

描述(申请人提供)：ATP门控阳离子P2X受体在神经元中广泛表达，并在海马神经元膜上发现。许多研究证实了P2X受体在海马神经传递和可塑性中的生理作用。因此，人们对天然和重组P2X通道的电生理特性进行了广泛的研究。然而，由于受体迁移率不能用电生理学方法测量，因此对P2X通道的质膜迁移率知之甚少。其他配体门控离子通道的扩散被认为是突触可塑性等重要生理过程的基础，因此，P2X受体的迁移率可能是海马生理学中的一个重要机制。我们工作的目的是开发一种成像方法来监测质膜上的P2X受体的迁移率，并揭示决定这种迁移率的分子因素。我们的初步数据显示，不同的P2X受体表现出不同的迁移率，ATP加快了受体的迁移率，而P2X2的迁移率在不同的神经元区段中是不同的。这项建议的具体目标是1)使用量子点标记的P2X2受体的荧光漂白后恢复(FRAP)和单粒子跟踪(SPT)来探索在海马神经元中发生P2X2受体迁移的细胞环境，2)使用这些迁移测量来确定控制P2X2迁移的分子机制，重点关注最近被发现与P2X2的C尾巴相互作用的蛋白质。公共卫生相关性：这项提案中的研究结果与NIH的任务直接相关，因为它们将有助于更好地理解在大脑中发现的离子通道如何有助于海马体功能，以及该通道的流动性如何受到海马体中的神经传递的影响。这与癫痫最相关，在癫痫中，P2X2通道被认为是治疗这种疾病的新药靶点。

DESCRIPTION (provided by applicant): ATP-gated cationic P2X receptors are widely expressed in neurons and are found on the membrane of hippocampal neurons. Many studies have demonstrated the physiological roles of P2X receptors in hippocampal neurotransmission and plasticity. Thus the electrophysiological properties of native and recombinant P2X channels have been extensively studied. However, little is known about the plasma membrane mobility of P2X channels since receptor mobility cannot be measured with electrophysiological methods. Diffusion of other ligand gated ion channels is known to underlie important physiological processes such as synaptic plasticity and thus P2X receptor mobility is likely an important mechanism in hippocampal physiology. The objective of our work was to develop an imaging approach to monitor P2X receptor mobility in the plasma membrane and to uncover the molecular factors that determine this mobility. Our preliminary data show that different P2X receptors exhibit different levels of mobility, that ATP quickens the rate of receptor mobility and that P2X2 mobility varies in different neuronal compartments. The specific goals of this proposal are 1) to use fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) of Quantum dot labelled P2X2 receptees to probe the cellular settings in which P2X2 receptor mobility occurs in hippocampal neurons, 2) to use these measures of mobility to determine the molecular mechanisms that govern P2X2 mobility with a focus on proteins recently shown to interact with the C tail of P2X2. PUBLIC HEALTH RELEVANCE: The results of the research in this proposal are directly relevant to the mission of the NIH since they will lead to a better understanding of how an ion channel found in the brain contributes to hippocampal function, and in turn how the mobility of the channel is affected by neurotransmission in the hippocampus. This is most relevant to epilepsy where P2X2 channels are considered as new drug targets to treat this disease.