Studies of P2X ATP Receptors

描述(申请人提供)：P2X蛋白是三磷酸腺苷门控通道，使细胞去极化，也允许钙进入。P2X受体在几乎所有组织中都有表达，包括中枢和外周神经系统的神经元和神经胶质细胞，平滑肌、骨骼肌和心肌，耳蜗毛细胞，血小板，大多数类型的白细胞，肝细胞，以及肺和胃肠道的内皮细胞。这个基因家族的成员对正常生理的重要性从它们缺失的一系列表型中可以明显看出，在特定P2X受体被敲除的小鼠中，疼痛感知、排空膀胱的能力、肠道运动、神经元控制射精的能力、神经系统监测血液中氧气水平的能力、对抗细菌感染的能力和血液凝固的能力都会出现障碍。嘌呤能受体领域的一个主要问题是可用于改变体内ATP信号转导的激动剂和拮抗剂的特异性有限。这些实验的目的是为了更好地描述允许ATP和变构调节剂打开P2X受体通道的分子机制。这些研究的结果应该有助于开发更具特异性地作用于特定受体的药物。我们将使用电生理学、生化和分子方法来研究相邻或非相邻亚基中携带互补突变的受体。具体目标是：目标1-测试调节P2X2、P2X3和P2X4受体通道活性的锌结合位点是否在亚基内或亚基之间，并确定参与这些结合位点的残基。我们还将阐述我们对锌促进P2X2受体通道开放的机制的理解。目标2-测试P2X受体的ATP结合位点是否在亚基内或亚基之间，并定义暴露在ATP结合口袋中的额外残基。这些实验还将测试打开通道必须结合的ATP分子的数量。目标3-定义锌或三磷酸腺苷与P2X2受体结合的结果的分子运动。这些实验对于理解和治疗与组织损伤相关的疼痛具有特别重要的意义，因为P2X2和P2X3受体被认为在感知损伤和向中枢神经系统发送信号方面发挥着重要作用。更好地了解这些受体的结构应该可以开发出治疗这种类型疼痛的新方法，从而极大地缓解烧伤和其他产生持续性疼痛的伤害患者的痛苦。

DESCRIPTION (provided by applicant): The P2X proteins are ATP gated channels that depolarize cells and also allow calcium to enter. P2X receptors are expressed in virtually every tissue, including neurons and glia of the central and peripheral nervous system, smooth, skeletal and cardiac muscle, cochlear hair cells, platelets, most classes of white blood cells, hepatocytes, and endothelial cells in the lung and gastrointestinal tract. The importance of members of this gene family for normal physiology is apparent from the range of phenotypes that are seen in their absence, Mice in which specific P2X receptors are knocked out show dysfunction in pain perception, ability to void the bladder, gut motility, neuronal control of ejaculation, the ability of the nervous system to monitor the oxygen level in the blood, the ability to fight bacterial infection, and blood clotting. A major problem in the purinergic receptor field is the limited specificity of agonists and antagonists that can be used to alter ATP signaling in vivo. The goal of the experiments described here is to better characterize the molecular mechanisms that allow ATP and allosteric modulators to open P2X receptor channels. The results of these studies should facilitate the development of agents that act more specifically on particular receptors. We will use electrophysiological, biochemical, and molecular approaches to study receptors bearing complementary mutations in adjacent or non-adjacent subunits. The specific aims are: Goal 1 - To test whether the zinc binding sites that modulate channel activity in P2X2, P2X3, and P2X4 receptors are within or between subunits, and to define residues that participate in these binding sites. We will also define our understanding about the mechanisms by which zinc promotes channel opening in P2X2 receptors. Goal 2 - To test whether the ATP binding site of P2X receptors is within or between subunits and to define additional residues that are exposed in the ATP binding pocket. These experiments will also test the number of molecules of ATP that must be bound in order to open a channel. Goal 3 - To define the molecular movements that are a consequence of zinc or ATP binding to P2X2 receptors. These experiments are of particular relevance to making progress in understanding and treating pain associated with tissue injury, as P2X2 and P2X3 receptors have been implicated as playing essential roles as sensing the damage and signaling the central nervous system. Having a better understanding of the structure of these receptors should allow the development of new treatments for this type of pain, and so greatly ease the suffering of individuals with burns and other injuries that produce persistent pain.