ENDOGENOUS REGULATORS OF GLUTAMATE-ACTIVATED CHANNELS

谷氨酸在中枢兴奋性突触中的递质作用是 现在已经很成熟了。尽管这些途径显然很重要 对于整个大脑的正常信息传输来说，它已经到来了 令人惊讶的是，谷氨酸突触发出的信号 比经典的神经肌肉研究预测的要复杂得多 交叉口。这一点在边缘系统中最为明显，在那里 学习和记忆、苯环利定(PCP)等各种现象-- 诱发精神病和兴奋性中毒性脑损伤都是密切相关的 与谷氨酸受体的活性有关。因此……的活动 谷氨酸受体可能是导致这些症状的原因 精神分裂症，亨廷顿氏症和阿尔茨海默氏症等令人眩晕的疾病， 以及因长期癫痫发作或中风而造成的脑损伤。在一些 在一些病例中，谷氨酸受体的异常可能是原因。发射机 从突触前终端释放激活两类 突触后谷氨酸激活通道，由AMPA选择性激活 (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate)和Nmda(N- 甲基-D-天冬氨酸)。尽管人们对谷氨酸有着浓厚的科学兴趣 对于受体，它们的调节作用仍然没有得到充分的描述。目的 这个项目的两个方面是研究谷氨酸受体 海马神经元的调节。I.细胞内的增加 钙会导致NMDA通道的减慢和高能 磷酸盐通过一种似乎并不是 需要直接的受体磷酸化。这种下调监管的机制 可能会限制钙离子流入树突棘，从而调节 细胞对突触刺激的反应。AIMS 1-3将审查 细胞内钙和三磷酸腺苷对NMDA受体/通道的作用二、 突触后谷氨酸受体的磷酸化被认为是 突触传递的重要调节器。然而，快速的 磷酸酶和磷酸二酯酶的作用表明，激酶可能 需要放置在膜底物附近，例如受体 才能有效。在目标4中，假设通过 AMPA的磷酸化需要特定的锚定蛋白 将测试突触后密度中的受体。多肽 锚定蛋白调节亚单位(RII)的抑制剂 CAMP依赖的蛋白激酶将被引入海马区 神经元。 全细胞膜片钳记录及细胞内电压的测定 将使用单个培养的海马神经元中的钙。这个 细胞质的组成将通过细胞内来控制 笼状化合物的灌流和闪光光解。单一谷氨酸 通道将被研究在细胞内附着和由内而外 配置。包括受体表达在内的分子方法 细胞系中的亚基将被用来探测调控位点 特定的受体亚基。这些研究的结果是意料之中的 为改变突触带来更有效的治疗策略 神经精神障碍的传播。

The role of glutamate as a transmitter at central excitatory synapses is now well established. Although these pathways are of obvious importance for the normal transfer of information throughout the brain, it has come as somewhat of a surprise that signalling at glutamate synapses is much more complex than predicted from classical studies of the neuromuscular junction. This is nowhere more apparent than in the limbic system where such diverse phenomena as learning and memory, phencyclidine (PCP)- evoked psychosis and 'excitotoxic' brain injury have all been closely linked to the activity of glutamate receptors. Thus the activity of glutamate receptors is likely to contribute to the symptoms in schizophrenia, dementing illness such as Huntington's and Alzheimer's, and to brain damage caused by prolonged seizures or stroke. In some cases, abnormalities of glutamate receptors may be causal. Transmitter released from presynaptic terminals activates two classes of postsynaptic glutamate-activated channels, selectively activated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) and NMDA (N- methyl-D-aspartate). Despite intense scientific interest in glutamate receptors, their regulation remains poorly characterized. The purpose of this project is to examine two aspects of glutamate receptor regulation in hippocampal neurons. I. Increases in intracellular calcium can lead to slow 'rundown' of the NMDA channel, and high energy phosphates counteract rundown by a mechanism that does not appear to require direct receptor phosphorylation. This downregulatory mechanism may limit calcium influx into dendritic spines and thus modulate cellular responses to synaptic stimulation. Aims 1-3 will examine the action of intracellular calcium and ATP on NMDA receptor/channels. II. Phosphorylation of postsynaptic glutamate receptors is postulated to be an important regulator of synaptic transmission. However, the rapid action of phosphatases and phosphodiesterases suggest that kinases may need to be located near a membrane substrate such as a receptor in order to be effective. In Aim 4 the hypothesis that kinase localization by specific anchoring proteins is required for phosphorylation of AMPA receptors in the postsynaptic density will be tested. Peptide inhibitors of anchoring proteins for the regulatory subunit (RII) of cAMP-dependent protein kinase will be introduced into hippocampal neurons. Whole-cell patch clamp recording and measurements of intracellular calcium in single cultured hippocampal neurons will be used. The composition of the cell cytoplasm will be controlled using intracellular perfusion and flash photolysis of "caged" compounds. Single glutamate channels will be studied in the cell-attached and inside-out configuration. Molecular methods including expression of receptor subunits in cell lines will be used to probe the regulator sites on specific receptor subunits. The results of these studies are expected to lead to more effective therapeutic strategies for altering synaptic transmission in neuropsychiatric disorders.