TARP modulation of AMPA receptors

AMPA 受体的 TARP 调节

• 批准号: 8976615
• 负责人: Vasanthi Jayaraman
• 金额: $ 29.75万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-05 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8976615
• 关键词: AMPA Receptors; Accounting; Address; Affect; Agonist; Automobile Driving; Binding; Brain; Cataloging; Catalogs; Chimera organism; Cleaved cell; Complex; Coupling; Data; Defect; Electrophysiology (science); Exhibits; Exposure to; Extracellular Domain; Fluorescent Probes; Glutamate Receptor; Glutamates; Health; Investigation; Kinetics; Label; Laboratories; Ligands; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Mediator of activation protein; Methods; Molecular Conformation; Mutate; Mutation; Neuraxis; Neurons; Phenylalanine; Physiological; Play; Positioning Attribute; Process; Property; Protein Family; Proteins; Receptor Activation; Resolution; Role; Seizures; Shapes; Signal Transduction; Site; Specificity; Structure; Synapses; Synaptic Transmission; Tertiary Protein Structure; Testing; Time; Western Blotting; Work; base; biophysical tools; cognitive function; crosslink; desensitization; dimer; excitotoxicity; fluorophore; genetic regulatory protein; insight; luminescence resonance energy transfer; member; millisecond; motor control; mutant; receptor; receptor function; response; tandem mass spectrometry; trafficking; unnatural amino acids

 DESCRIPTION (provided by applicant): AMPA receptors mediate fast excitatory responses in the mammalian central nervous system, and ultimately control motor and cognitive functions. In neurons AMPA receptors co-assemble with auxiliary subunits. One member being transmembrane AMPA receptor regulatory proteins (TARPs), which are essential for the normal physiological functioning of these receptors. One of the dramatic effects of TARPs on AMPA receptor gating is a large increase in efficacy such that even some antagonists such as 6-cyano-7-nitroquinoxaline-2,3-dione act as partial agonists. Additionally, TARPs slow deactivation of AMPA receptor when agonist is removed and slow the rate and extent of desensitization when agonist is continually applied. These changes in properties are expected to dictate the kinetics and shape of synaptic transmission. In addition to the rapid modulations, the presence of certain subtypes of TARP's leads to resensitization on longer time scales, from the tens of milliseconds to seconds. The prolonged opening of the receptors in these long resensitization processes may play a role in glutamate mediated excitotoxicity. Here we propose to gain a fundamental understanding of the structure-dynamic changes controlling the increase in efficacy and resensitization in the AMPA receptors in the presence of TARPs using a combination of biophysical and electrophysiological methods. For the proposed study, we will use luminescence resonance energy transfer to study the conformational changes in the protein. For specific labeling of the protein with fluorescent probes we will introduce unnatural amino acids at specific sites on the protein. The conformational changes thus observed will be further verified with functional investigations of mutant proteins with mutations introduced at strategic sites that affect the observed conformational change or the stability of a specific conformational state. Finally, the position of the TARP ecto domain will be mapped to the AMPA receptor extracellular domain using mass spectrometry. To study the interaction sites between the two proteins the unnatural amino acid p-benzoyl phenylalanine will be introduced at various sites and those sites leading to crosslinking will be analyzed using high resolution tandem mass spectrometry. The proposed functional and structural investigations will provide a comprehensive understanding of the mechanism by which TARPs modulates AMPA receptor function.

 描述(申请人提供)：AMPA受体介导哺乳动物中枢神经系统的快速兴奋反应，并最终控制运动和认知功能。在神经元中，AMPA受体与辅助亚单位共同组装。其中一个成员是跨膜AMPA受体调节蛋白(TARP)，它对这些受体的正常生理功能是必不可少的。TARP对AMPA受体门控的显著作用之一是大大提高了药效，甚至一些拮抗剂，如6-氰基-7-硝基-2，3-二酮也能起到部分激动剂的作用。此外，Tarps在移除激动剂时减缓AMPA受体的失活，并在继续应用激动剂时减缓脱敏的速度和程度。这些特性的变化有望决定突触传递的动力学和形状。除了快速调节外，某些TARP亚型的存在还会导致更长时间尺度的再敏化，从几十毫秒到几秒不等。在这些长时间的再敏化过程中，受体的长时间开放可能在谷氨酸介导的兴奋性毒性中起作用。在这里，我们建议使用生物物理和电生理相结合的方法，对控制AMPA受体在TARP存在下的有效性和再增敏的结构动力学变化有一个基本的了解。在拟议的研究中，我们将使用发光共振能量转移来研究蛋白质的构象变化。为了用荧光探针对蛋白质进行特定标记，我们将在蛋白质的特定位置引入非天然氨基酸。这样观察到的构象变化将通过对突变蛋白的功能研究来进一步验证，突变蛋白在关键位置引入影响观察到的构象变化或特定构象状态的稳定性的突变。最后，利用质谱仪将TARP胞外区的位置映射到AMPA受体的胞外区。为了研究这两种蛋白质之间的相互作用部位，将在不同的部位引入非天然氨基酸对苯甲酰苯丙氨酸，并用高分辨串联质谱仪分析导致交联的部位。拟议的功能和结构研究将提供一个全面的了解机制，通过它调节AMPA受体的功能。