An ATP receptor-operated ionic conductance in Paramecium

描述(由申请人提供)： 在脊椎动物中，三磷酸腺苷是一种神经递质、血管扩张剂和细胞间 发出拉伸和疼痛的信号。所有这些动作都可以通过激活来引发 特异膜上的ATP受体(P2X受体)面对着 牢房。编码这些P2X受体的基因产生一种基因产物 这既是受体又是离子传导通道，通常是 电导。需要补充的一对是，人们对此知之甚少 参与调节这种专门化的遗传和生化机制 膜离子电导在对三磷酸腺苷刺激的适应过程中。真核生物 单细胞草履虫表现出ATP诱导的行为反应(向后游泳)， 脊椎动物ATP受体拮抗剂(PPNDS)抑制这些反应， ATP诱导的去极化，高亲和力的外部3zP-ATP结合和 包含可被多肽抗体识别的膜蛋白 脊椎动物的P2X1受体。我的长期目标是将这两个特点 草履虫AT去极化过程中的离子电导 以及在ATP适应过程中调节其功能表达的过程。 开始这项工作，我的具体目标是：1.描述野生型 三磷酸腺苷诱导的去极化和在电压钳条件下看到的电流 它们的幅度、动力学、离子依赖性和变化的时间进程 适应，2.使用经典的行为突变体选择程序(向前 遗传学])获得对三磷酸腺苷(ATP)无反应的行为突变体或 不适应ATP，3.使用“基因沉默”(反向遗传学)产生 作为ATP功能敲除的基因改变的细胞系 受体、胞外ATPase(使ATP信号失活)和其他部分 信号转导和适应途径和4.利用细胞内 电生理、体内~(32)P-ATP结合试验和蛋白质印迹分析 显示这些基因改变的细胞是否对ATP有正常的反应， 细胞质膜上ATP受体的外源结合和表达。 与健康相关的是，由于三磷酸腺苷的接收影响神经传递， 血液流向肾脏和消化器官，拉伸信号来自空洞 器官和痛觉的接收，身体各器官的功能正常即可 由于ATP接收的改变而受到影响。因为草履虫是最简单的 真核生物展示了一种类似于脊椎动物的ATP受体， 它提供了一个独特的模型系统，可以同时使用正向遗传和反向遗传 理解三磷酸腺苷的兴奋和适应阶段的方法 回应。

DESCRIPTION (provided by applicant): In vertebrates, ATP is a neurotransmitter, vasodilator and an intercellular signal for stretch and pain. All of these actions can be elicited by activation of specific membrane ATP receptors (P2X receptors) which face the outside of the cell. The genes that code for these P2X receptors produce a gene product that is both the receptor and the ion conducting channel, typically a Ca++ conductance. The pairthat needs to be filled is that very little is known about the genetic and biochemical mechanisms involved in regulating this specialized membrane ion conductance during adaptation to an ATP stimulus. The eukaryotic unicell Paramecium shows ATP-induced behavioral responses (backward swimming), inhibition of these responses by a vertebrate ATP receptor antagonist (PPNDS), ATP-induced depolarizations, high affinity external 3zP-ATP binding and contains membrane proteins that are recognized by peptide antibodies directed to vertebrate P2X1 receptors. My long term objective is to characterize both the ion conductance associated with the AT-induced depolarization of Paramecium and the processes regulating its functional expression during ATP adaptation. To begin this work, my specific aims are to: 1. Characterize the wild type ATP-induced depolarization and currents seen under voltage clamp conditions for their amplitudes, kinetics, ion dependencies and time course of changes during adaptation, 2. Use classical behavioral mutant selection procedures (forward genetics) to obtain behavioral mutants which either don't respond to ATP or don't adapt to ATP, 3. Use "gene silencing" (reverse genetics) to produce genetically altered cell lines that are functional knockouts of the ATP receptor, the ecto-ATPase (which inactivates the ATP signal) and other parts of the signal transduction and adaptation pathway and 4. Use intracellular electrophysiology, in vivo 32p-ATP binding assays and western blot analysis to show whether these genetically-altered cells have normal responses to ATP, external ATP binding and expression of ATP receptors on their plasma membranes. The health-relatedness is that since ATP reception affects neurotransmission, blood flow to kidney and digestive organs, stretch signals coming from hollow organs and pain reception, normal functions of organs of the body can be compromised by alterations in ATP reception. Since Paramecium is the simplest eukaryote to show an ATP receptor that is similar to vertebrate ATP receptors, it offers a unique model system to use both forward and reverse genetic approaches to understand both the excitation and adaptation phases of the ATP response.