Studies of P2X ATP Receptors

描述（由申请人提供）：P2X蛋白是ATP门控通道，使细胞去极化，也允许钙进入。P2X受体几乎在所有组织中表达，包括中枢和周围神经系统的神经元和胶质细胞、平滑肌、骨骼肌和心肌、耳蜗毛细胞、血小板、大多数种类的白细胞、肝细胞、肺和胃肠道的内皮细胞。P2X基因家族成员对正常生理的重要性从其缺失的表型范围中可见一斑。P2X特异性受体被敲除的小鼠表现出疼痛感知功能障碍、排尿能力、肠道运动、射精神经元控制、神经系统监测血液中氧气水平的能力、抵抗细菌感染的能力和血液凝固能力。嘌呤能受体领域的一个主要问题是可用于改变体内ATP信号的激动剂和拮抗剂的有限特异性。本实验的目的是更好地表征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特异性地作用于特定受体的药物。我们将使用电生理、生化和分子方法来研究在相邻或非相邻亚基中携带互补突变的受体。具体目的是：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。我们还将定义我们对锌促进P2X2受体通道打开的机制的理解。目的2 -测试P2X受体的ATP结合位点是否在亚基内或亚基之间，并确定暴露在ATP结合口袋中的其他残基。这些实验还将测试为了打开通道而必须结合的ATP分子的数量。目标3 -定义锌或ATP与P2X2受体结合的分子运动。由于P2X2和P2X3受体在感知损伤和向中枢神经系统发送信号方面起着至关重要的作用，这些实验对于理解和治疗与组织损伤相关的疼痛具有特殊的意义。对这些受体的结构有了更好的了解，就可以开发出治疗这种疼痛的新方法，从而极大地减轻烧伤和其他产生持续性疼痛的损伤患者的痛苦。

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.