Calpain Mediated Cleavage of NR2 in Excitotoxicity

描述（由申请方提供）：称为N-甲基-D-天冬氨酸（NMDA）受体的谷氨酸受体是多种形式兴奋性毒性的关键介质，兴奋性毒性是神经系统疾病的病理生理机制。NMDA受体由NR 1和NR 2亚基的组合构成，每个亚基具有不同的特性。不同的亚型可能发挥不同的作用，兴奋性毒性，可能是基于其细胞内结构域的可变功能。这些结构域将受体连接到细胞内信号传导过程，并且是钙蛋白酶进行蛋白水解修饰的潜在位点。这种修饰可以在控制NMDA受体的亚型特异性特性中发挥关键作用。以前，我们已经表明，NR 2A亚基是钙蛋白酶的选择性底物，而NR 1亚基不被钙蛋白酶切割。这种切割可能产生具有新特性的受体，其可以在兴奋性毒性和缺氧期间以亚基选择性方式进行调节。在这个建议中，我们将评估钙蛋白酶切割的生理特性和本地化的不同NMDA受体亚型和兴奋性毒性的作用，这些事件的影响。我们将首先确定在体外，在异源系统和神经元中的每个NR 2亚基中的钙蛋白酶裂解位点。在这些实验之后，我们将确定钙蛋白酶切割对NMDA受体的生理特性的影响，并确定钙蛋白酶切割是否改变NMDA受体在异源表达系统和神经元中的定位。在最后的目的，我们将调查是否钙蛋白酶切割的NMDA受体兴奋性毒性和缺氧模型期间发生，以及是否钙蛋白酶切割的NMDA受体改变细胞损伤的严重程度。此外，我们将确定钙蛋白酶裂解是否在癫痫的特定模型，点燃模型中发挥作用。总体而言，目前我的建议将定义如何钙蛋白酶蛋白水解的NMDA受体调节受体的特性，使我们能够确定这个过程中的作用，在神经元功能和疾病的过程。

DESCRIPTION (provided by applicant): The glutamate receptor called the N-methyl-D-aspartate (NMDA) receptor is a crucial mediator of many forms of excitotoxicity, a pathophysiological mechanism of neurologic disease. The NMDA receptor is constructed from combinations of NR1 and NR2 subunits, with each subunit conveying distinct properties. Different subtypes may play distinct roles in excitotoxicity, possibly based on the variable features of their intracellular domains. These domains link the receptor to intracellular signaling processes, and are potential sites for proteolytic modification by the enzyme calpain. Such modifications could play a crucial role in control of the subtype specific properties of NMDA receptors. Previously we have shown that the NR2A subunit is a selective substrate for calpain while the NR1 subunit is not cleaved by calpain. This cleavage may create receptors with novel properties, which could be regulated in a subunit selective manner during excitotoxicity and hypoxia. In this proposal, we will assess the effect of calpain cleavage on the physiological properties and localization of different NMDA receptor subtypes and the role of these events in excitotoxicity. We will initially determine the sites of cleavage by calpain in each NR2 subunit in vitro, in heterologous systems and in neurons. Following these experiments, we will define the effect of calpain cleavage on physiological properties of the NMDA receptor, and determine whether cleavage by calpain alters NMDA receptor localization in heterologous expression systems and in neurons. In the final Aim, we will investigate whether calpain cleavage of NMDA receptors occurs during models of excitotoxicity and hypoxia, and whether calpain cleavage of NMDA receptors alters the severity of cellular damage. In addition, we will ascertain whether calpain cleavage plays a role in a specific model of epilepsy, the kindling model. Overall, the present I proposal will define how calpain proteolysis of the NMDA receptor modulates receptor properties, allowing us to determine the role of this process in neuronal function and disease processes.