S-NITROSYLATION OF THE NMDA SUBTYPE OF GLUTAMATE RECEPTOR FOR NEUROPROTECTION

在许多神经系统疾病中，可能导致神经元损伤， 至少部分是由于兴奋性氨基酸受体的过度刺激 酸，包括谷氨酸、天冬氨酸和相关同系物。 这些 神经病症的范围从急性损伤如中风， 从低血糖、创伤和癫痫到慢性神经退行性疾病 诸如亨廷顿氏病、艾滋病痴呆综合症、 肌萎缩性侧索硬化症，也许还有阿尔茨海默病。 谷氨酸是大脑中主要的兴奋性神经递质， 因此，它与特定膜受体的相互作用是 负责许多正常的神经功能，包括 认知、记忆、运动和感觉。 此外，兴奋性 神经递质在塑造发育过程中很重要， 神经系统中突触连接的可塑性。 但在 各种病理状况，包括中风和各种 神经退行性疾病，谷氨酸过度活化 受体可介导神经元损伤或死亡。 奥尔尼创造了这个词 “兴奋毒性”的这种情况下，这可能构成一个最终的 不同疾病引起神经元损伤的共同途径 病理生理学 这种形式的伤害似乎主要是 介导的过度内流的Ca 2+进入神经元，通过离子 通过激活谷氨酸受体触发的通道。 一个重要 关于这些戊二酸受体亚型的一点是， N-甲基-D-天冬氨酸（NMDA）谷氨酸受体激活亚型 通道允许Ca 2+和Na +9的内流和过度刺激 这种类型的受体被认为是主要的机制 导致神经元钙超载 NMDA介导的Ca 2+内流 受体刺激激活多种酶， 氧化物合酶和随后的一氧化氮的产生。 当 NMDA受体被过度刺激，一氧化氮可能是 产量增加。 在此条件下， 02 '-可反应形成称为过氧亚硝酸盐（ONOO-）的有毒物质， 导致神经元死亡相反， 转化为一种不同的化学状态，产生相反的效果， 保护神经元免受NMDA受体过度刺激所致的损伤。 化学状态取决于去除或添加 电子转化为一氧化氮，这是一种可以受到 存在或不存在电子供体，如抗坏血酸或氨基 酸性半胱氨酸 例如，少一个电子，NO可以产生 具有NO+（亚硝基）特征的物质。 在这种情况下，NO 组似乎被转移到NMDA上的调节位点 受体，称为氧化还原调节位点。 本网站由 巯基（-SH）基团;-S-与NO+反应形成-SNO（a 称为亚硝基化的过程）导致 NMDA受体，从而提供保护免受过度刺激。 因此，取决于其化学状态，NO部分可导致 神经破坏或神经保护。 这些发现导致 降低NMDA受体过度活性的治疗方法， 具有NO+特征的药物，如硝酸甘油。 NMDA氧化还原 调节性网站这里提出的工作背后的动力是 为了表征包含氧化还原调节剂的半胱氨酸残基， NMDA受体的一个或多个位点与它们与 NO组。 我们实验室对重组NMDA受体的研究 NMDARI亚基上的两个半胱氨酸残基， NMDA受体参与氧化还原调节（半胱氨酸残基 744和798;下面的图1）。 与Chait合作 实验室，我们建议使用电喷雾电离质量 光谱法以显现NMDARI亚基的S-亚硝基妥英， 确定分子内或分子间二硫键的形成 随后发生，并将这些发现与已知的 这些反应对我们的NMDA受体的调节活性 膜片钳电生理研究。

In many neurological disorders, injury to neurons may be caused, at least in part, by overstimulation of receptors for excitatory amino acids, including glutamate, aspartate and related congeners. These neurological conditions range from acute insults such as stroke, hypoglycemia, trauma, and epilepsy, to chronic neurodegenerative states such as Huntington's disease, AIDS dementia complex, amyotrophic lateral sclerosis, and perhaps Alzheimer's disease. Glutamate is the major excitatory neurotransinitter in the brain and as such its interactions with specific membrane receptors are responsible for many normal neurological functions including cognition, memory, movement, and sensation. In addition, excitatory neurotransmitters are important in shaping the developmental plasticity of synaptic connections in the nervous system. However, in a variety of pathological conditions, including stroke and various neurodegenerative disorders, excessive activation of glutamate receptors may mediate neuronal injury or death. Olney coined the term 'excitotoxicity' for this condition, which may constitute a final common pathway for neuronal injury from diseases of diverse pathophysiology. This form of injury appears to be predominantly mediated by excessive influx of Ca2+ into neurons through ionic channels triggered by activation of glutamate receptors. An important point concerning these glutarnate receptor subtypes is that the N-methel-D-aspartate (NMDA) subtype of glutamate receptoractivated channel permits the influx of Ca2+ as well Na as+9 and overstimulation of this type of receptor is thought to be the predominant mechanism for calcium overload in neurons. Ca2+ influx triggered via NMDA receptor stimulation activates a variety of enzymes including nitric oxide synthase and the consequent production of nitric oxide. When NMDA receptors are excessively stimulated, nitric oxide may be produced in increased quantities. Under these conditions, NO- and 02'- may react to form a toxic substance called peroxynitrite (ONOO-), resulting in neuronal death In contrast, nitric oxide can be converted to a different chemical state that has just the opposite effect, protecting neurons from injury due to NMDA receptor overstimulation. The chemical state is dependent upon the removal or addition of an electron to nitric oxide, a condition that can be influenced by the presence or absence of electron donors, such as ascorbate or the amino acid cysteine. For example, with one less electron, NOmay yield a substance with NO+ (nitrosonium) character. In this form, the NO group appears to be transferred to a regulatory site on the NMDA receptor, termed the redox modulatory site. This site is comprised of sulfhydryl (-SH) groups; the reaction of -S- with NO+ to form -SNO (a process called Snitrosylation) results in decreased activity of the NMDA receptor, thus affording protection from excessive stimulation. Therefore, depending on its chemical state, the NO moiety can lead to neurodestruction or neuroprotection. These findings have lead to therapeutic approaches to decrease NMDA receptor overactivity using drugs with NO+ character, such as nitroglycerin. NMDA Redox Modulatory Site The motivating force behind the work proposed here is to characterize the cysteine residues comprising the redox modulatory site(s) of the NMDA receptor with respect to their interaction with the NO group. Work from our laboratory with recombinant NMDA receptor subunits has shown that two cysteine residues on the NMDARI subunit of the NMDA receptor are involved in redox modulation (cysteine residues 744 and 798; Fig .1, below). In collaboration with the Chait laboratory, we propose to use electrospray ionization mass spectrometry to visualize S-nitrosylatoin of the NMDARI subunit, to determine if intra- or intermolecular disulfide bond formation subsequently occurs, and to correlate these findings with the know regulatory activity of these reactions on the NMDA receptor from our patch-clamp electrophysiological studies.