DIFFERENCES IN SYNAPTIC VS NONSYNAPTIC AMPA RECEPTORS

突触与非突触 AMPA 受体的差异

• 批准号: 6258709
• 负责人: AMY C ARAI
• 金额: $ 16.81万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-09 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258709
• 关键词: AMPA receptors; clathrate; concanavalin A; enzyme activity; enzyme inhibitors; exo alpha sialidase; flash photolysis; glutamates; heparin; hippocampus; laboratory rat; receptor binding; recombinant proteins; sulfhydryl reagents; synapses; thiazide; voltage /patch clamp

DESCRIPTION: AMPA-type glutamate receptors mediate most excitatory synaptic transmission in the brain. Receptor binding studies have shown that AMPA receptors exist in two distinct states with 20-fold different affinities. It is suggested that the two forms are differentially localized with synaptic receptors being of the low-affinity type and non-synaptic receptors of the high-affinity type. Thus, AMPA receptors in patches excised from the soma (and hence of non-synaptic origin) may have different kinetic properties than those in synapses. This would entail that somatic and synaptic receptors contribute differentially to neurological disorders involving excitotoxic damage to neurons. This proposition will be tested by examining if differences between synaptic and non-synaptic AMPA receptors are also detected with physiological measures and if they correspond to the high-low affinity distinction seen in binding. To facilitate this comparison, both physiological data and binding data will be collected under equivalent conditions. Aim One will determine 'physiological KD' values for equilibrium currents in patches excised from hippocampal pyramidal cells and compare them with binding affinities obtained in the same buffer and at the same temperature. The results will then be compared with similar measures from recombinant AMPA receptors stably expressed in HEK293 cells that appear to be entirely of high affinity (Aim Two) and with data from synaptic receptors in autapses of cultured neurons, which presumably are of low affinity (Aim Three). It is further known that binding to the low-affinity receptors can be modulated about two-fold by biochemical manipulations such as treatment with concanavalin A, phospholipase A2 or neuroaminidase. The consequences of some of these treatments for AMPA receptor kinetics will be examined in Aim Four using the same approach. It was also observed that high affinity receptors, unlike their low-affinity counterpart, are highly unstable at 37 degrees C and it seems likely that this is related to the fast 'run-down' of AMPA receptor currents in patch experiments. Determining which cellular factor stabilizes the low-affinity receptors will constitute the Fifth Aim of this application.

描述：AMPA型谷氨酸受体介导大多数兴奋性突触 在大脑中传播。受体结合研究表明，AMPA 受体以两种截然不同的状态存在，亲和力相差20倍。它是 提示这两种形式与突触有不同的定位 受体为低亲和力类型和非突触受体 高亲和力类型。因此，从胞体切除的斑块中的AMPA受体(和 因此非突触起源)可以具有不同于 在突触中。这将需要躯体和突触受体起作用 与涉及兴奋性毒性损害的神经功能障碍的鉴别 神经元。这一命题将通过检查以下方面的差异来检验 突触和非突触的AMPA受体也可以用生理学的 度量，以及它们是否符合 有约束力的。为了便于比较，生理数据和绑定 数据将在同等条件下收集。目标一号将决定 切除的斑块中平衡电流的‘生理Kd’值 并与所获得的结合亲和力进行比较 在相同的缓冲液和相同的温度下。然后结果将是 与稳定表达的重组AMPA受体的类似措施相比 在HEK293细胞中，似乎完全具有高亲和力(目标2)和 来自培养神经元自体突触受体的数据，推测 亲和力低(目标三)。还知道，绑定到 低亲和力受体可被生物化学调节约两倍 操作，如刀豆蛋白A，磷脂酶A2或 神经氨酸酶。其中一些治疗对AMPA受体的影响 在目标4中，将使用相同的方法来研究动力学。它也是 观察到，与低亲和力受体不同，高亲和力受体， 在37摄氏度时非常不稳定，这似乎与 斑贴实验中AMPA受体电流的快速“衰退”。确定 哪种细胞因子稳定低亲和力受体将构成 本申请的第五个目的。