Phosphorylation of GluR1 at Threonine 840 and Synaptic Plasticity

描述（由申请人提供）：AMPA型谷氨酸受体（AMPAR）的磷酸化可能对海马中的学习和记忆很重要。AMPAR是负责大脑中大多数兴奋性神经传递的配体门控离子通道，这些受体磷酸化的变化改变突触传递，导致突触可塑性。特别是，在AMPAR GluRI亚基的C-末端上的残基的磷酸化正处于紧张的研究中。当这些残基被磷酸化时，通过AMPAR的信号传导增强;因此，靶向这些位点的治疗可以改善人类的认知缺陷。此外，改变GluR1的磷酸化可能对治疗抑郁症和双相情感障碍等情绪障碍很重要。重要的是，最近发现了GluR1上的一个新的磷酸化位点，但对其功能意义知之甚少。该残基，苏氨酸840（T840），似乎在海马突触可塑性中具有作用，并且可以调节GluRI的C末端上的其它位点的磷酸化。因此，T840可能在学习和记忆中起关键作用。为了确定T840磷酸化在突触可塑性中的重要性，我们将研究：1。T840磷酸化的分子机制：我们将用不同的药理学和分子试剂处理急性海马脑片和海马培养物，并使用免疫印迹法测量T840磷酸化的变化，以检验我们的假设，即T840是已知在突触可塑性中起作用的蛋白激酶和蛋白磷酸酶的重要靶点。2. T840磷酸化的功能意义：我们将首先在用GluRI转染的HEK细胞中使用全细胞记录来研究我们的假设，即T840磷酸化影响受体的生物物理特性。其次，我们将测试我们的假设，在这个网站上的磷酸化可能会调节磷酸化在附近的网站上的C-末端的GluRI通过使用全细胞电生理学和免疫印迹转染海马神经元和HEK细胞。公共卫生研究的相关性：本提案中概述的实验与NIMH的使命直接相关，因为它们将提供对海马体细胞过程的洞察，海马体是对学习和记忆至关重要的大脑区域。这些信息可用于确定认知和情绪障碍背后的海马功能变化，并可能导致发现更有效的靶向治疗。

DESCRIPTION (provided by applicant): Phosphorylation of AMPA-type glutamate receptors (AMPARs) may be important for learning and memory in the hippocampus. AMPARs are ligand-gated ion channels responsible for most excitatory neurotransmission in the brain, and changes in phosphorylation of these receptors alter synaptic transmission, leading to synaptic plasticity. In particular, phosphorylation of residues on the C-terminus of the AMPAR GluRI subunit is under intense study. When these residues are phosphorylated, signaling through AMPARs is enhanced; thus therapeutics targeting these sites could improve cognitive deficits in humans. Additionally, altering phosphorylation of GluR1 may be important in treating mood disorders such as depression and bipolar disease. Importantly, a novel phosphorylation site on GluR1 has been recently uncovered and little is known about its functional significance. This residue, threonine 840 (T840), appears to have a role in hippocampal synaptic plasticity and may modulate phosphorylation of other sites on the C- terminus of GluRI. Thus, T840 may have a crucial role in learning and memory. In order to determine the importance of T840 phosphorylation in synaptic plasticity, we will investigate: 1. The molecular mechanisms of T840 phosphorylation: We will treat acute hippocampal brain slices and hippocampal cultures with different pharmacological and molecular reagents, and measure changes in T840 phosphorylation using immunoblotting to test our hypothesis that T840 is an important target for protein kinases and protein phosphatases known to have roles in synaptic plasticity. 2. The functional significance of T840 phosphoryation: We will first use whole-cell recordings in HEK cells transfected with GluRI to investigate our hypothesis that T840 phosphorylation influences the biophysical properties of the receptor. Second, we will test our hypothesis that phosphorylation at this site may regulate phosphorylation at nearby sites on the C-terminus of GluRI by using both whole-cell electrophysiology and immunoblotting of transfected hippocampal neurons and HEK cells. Relevence of Research to Public Health: The experiments outlined in this proposal are directly relevent to the mission of the NIMH because they will provide insight into the cellular processes in the hippocampus, a brain region crucial to learning and memory. This information can be used to determine the changes in hippocampal function underlying cognitive and mood disorders, and possibly lead to the discovery of more effective targeted therapies.