TARP modulation of AMPA receptors

 描述（由申请人提供）：AMPA受体介导哺乳动物中枢神经系统中的快速兴奋性反应，并最终控制运动和认知功能。在神经元中，AMPA受体与辅助亚基共组装。一个成员是跨膜AMPA受体调节蛋白（TARP），其对于这些受体的正常生理功能是必需的。TARP对AMPA受体门控的显著作用之一是功效的大幅增加，使得甚至一些拮抗剂如6-氰基-7-硝基喹喔啉-2，3-二酮也充当部分激动剂。此外，当去除激动剂时，TARPs减缓AMPA受体的失活，并且当持续应用激动剂时，减缓脱敏的速率和程度。预期这些性质的变化将决定突触传递的动力学和形状。除了快速的调节，TARP的某些亚型的存在导致更长时间尺度上的再敏感化，从几十毫秒到几秒。在这些长时间的再敏化过程中，受体的延长开放可能在谷氨酸介导的兴奋性毒性中发挥作用。在这里，我们建议获得一个基本的了解的结构动态变化，控制在TARP的存在下，使用生物物理和电生理方法相结合的AMPA受体的疗效和再敏化的增加。对于所提出的研究，我们将使用发光共振能量转移来研究蛋白质的构象变化。为了用荧光探针特异性标记蛋白质，我们将在蛋白质上的特定位点引入非天然氨基酸。将通过在影响观察到的构象变化或特定构象状态的稳定性的战略位点引入突变的突变蛋白的功能研究来进一步验证由此观察到的构象变化。最后，使用质谱法将TARP胞外结构域的位置映射到AMPA受体胞外结构域。为了研究两种蛋白质之间的相互作用位点，将在不同位点引入非天然氨基酸对苯甲酰基苯丙氨酸，并使用高分辨率串联质谱法分析导致交联的那些位点。拟议的功能和结构调查将提供一个全面的了解的机制，其中TARP调节AMPA受体功能。

 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.