TARP modulation of AMPA receptors

AMPA 受体的 TARP 调节

• 批准号: 8810076
• 负责人: Vasanthi Jayaraman
• 金额: $ 30.81万
• 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/8810076
• 关键词: AMPA Receptors; Accounting; Address; Affect; Agonist; Amino Acids; 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; 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; public health relevance; receptor; receptor function; response; tandem mass spectrometry; trafficking

 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（由适用提供）：AMPA受体介导哺乳动物中枢神经系统中的快速兴奋反应，并最终控制运动和认知功能。在神经元AMPA受体中，与辅助亚基共组装。一个成员是跨膜AMPA受体调节蛋白（TARPS），这对于这些受体的正常生理功能至关重要。防水布对AMPA受体门控的戏剧性影响之一是效率大大提高，即使是某些拮抗剂，例如6-甲状体-7-硝基喹啉-2,3-二酮作为部分激动剂。此外，当消除激动剂并慢慢降低激动剂时，tarps慢慢地停用了AMPA受体。这些性质的这些变化有望决定突触传播的动力学和形状。除了快速调制外，TARP的某些亚型的存在还会导致较长的时间尺度上的敏感性，从数十毫秒到几秒钟。在这些长的敏化过程中，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，我们建议对结构动态变化有基本的了解，以控制TARPS在TARPS存在下使用生物物理学和电生理学方法的结合，在TARPS存在下的效率和增密。对于拟议的研究，我们将使用发光共振能量转移来研究蛋白质的会议变化。对于具有荧光问题的蛋白质的特异性标记，我们将在蛋白质上的特定部位引入不自然的氨基酸。这样观察到的会议变化将通过突变蛋白的功能投资进一步验证，并在影响观察到的会议变化或特定会议状态的稳定性的战略场所引入突变。最后，使用质谱法将TARP ECTO结构域的位置映射到AMPA接收器细胞外域。为了研究两种蛋白质之间的相互作用位点，将在各个部位引入非天然氨基酸P-苯甲酰苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯苯胺，并将使用高分辨率的串联质谱法分析导致交联的那些位点。拟议的功能和结构研究将对TARPS调节AMPA接收器功能的机制提供全面的理解。