Mechanism of functional modulation of glutamate receptors by their auxiliary subunits

AMPA型嗜离子谷氨酸受体(AMPAs)是一种由神经细胞激活的配基门控离子通道。 谷氨酸递质是大脑中大部分兴奋性神经传递的媒介。信号传输- 这些复合体对突触的可塑性、学习和记忆至关重要。Ampar辅助亚基 规范AMPAR的贩运和门限调制。在这项提案中，我们将研究 其辅助亚单位对Ampar的调节。海马体中的两个主要的AMPAR辅助亚基， 皮质和纹状体是油布和角质(CNIHs)。防水油布被广泛研究并具有治疗作用 针对γ-8TARP，一种富含海马体的TARP，已经有缓解癫痫的化合物可用。在……上面 另一方面，我们对CNIH的理解是有限的。在CNIH家族中，已知CNIH2/3的功能 作为AMPAR辅助亚单位。在人类中，CNIH2的N端，形成与之相互作用的界面 Ampar对错义突变不耐受，这表明CNIH2-AMPAR相互作用在HU-HO中起着重要作用。 曼斯。我们的假设是，CNIHs在调节突触中的AMPAR门控方面起着基础性作用 传递性和可塑性。为了进一步确立这一假说，我们将研究其作用机制。 以AMPAR的GluA2亚基和CNIH3为模型制成的复合体。我们的实验室最近已经解决了低温电磁 GluA2：CNIH3=4：4化学计量比高分辨下GluA2/CNIH3络合物的结构在目标1中，我们假设- GluA2/CNIH3复合体在其他化学计量学中可能存在的大小，并建议揭示其结构 在GluA2：CNIH3=4：2化学计量比使用冷冻-EM。CNIH1目前未归类为AMPAR 辅助亚单位。然而，GluA2/CNIH3复合体的冷冻EM结构告诉我们，CNIH1具有 存在于CNIH2/3中的Ampar结合基序。冷冻-EM结构也揭示了脂类的存在 围绕着建筑群。我们推测，这些脂质可能在AMPAR GAT-GAT中发挥重要作用。 ING调制。在AIM2中，我们将测试CNIH1和脂类在AMPAR门控调制中的作用。最后，我们- 要揭示CNIH3的变构门控调制机制，需要获得Snap- 通道关闭、开放和脱敏状态下脂质包埋的GluA2/CNIH3复合体的快照。在目标3中，我们 提出了解决GluA2/CNIH3复合物包埋在脂质双层膜中的高分辨冷冻EM结构的建议。 并比较它们在不同功能状态下的差异。辅助亚基在离子调谐中的作用 预计通道选通动力学将对电路动力学产生重大影响。总而言之，结果 这项研究的成果将有助于我们从机制上理解AMPAR的功能，并有助于开发 新的治疗化合物可以缓解神经科和精神科的AMPAR调节失调 疾病，如阿尔茨海默病、中风、自闭症、拉斯穆森和边缘脑炎，以及癫痫。

The AMPA type ionotropic glutamate receptors (AMPARs), a ligand gated ion channel activated by the neuro- transmitter glutamate, mediate the majority of excitatory neurotransmission in the brain. The signals trans- duced by these complexes are critical for synaptic plasticity, learning and memory. AMPAR auxiliary subunits regulate trafficking and gating modulation of AMPARs. In this proposal we will investigate the mechanism of AMPAR regulation by their auxiliary subunits. The two major AMPAR auxiliary subunits, in the hippocampus, cortex, and striatum, are TARPs and cornichons (CNIHs). The TARPs are extensively studied and therapeutic compounds to alleviate seizure are already available to target γ-8 TARP, a hippocampus enriched TARP. On the other hand, our understanding on CNIHs is limited. Within the CNIH family, CNIH2/3 is known to function as AMPAR auxiliary subunits. In humans, the N-terminus of CNIH2 that forms the interaction interface with AMPAR is intolerant to missense mutations, indicating an essential role of CNIH2-AMPAR interaction in hu- mans. Our hypothesis is that CNIHs play fundamental roles in regulating AMPAR gating during synaptic transmission and plasticity. To further establish this hypothesis, we will study the functional mechanism of complexes made of GluA2 subunit of AMPAR and CNIH3 as a model. Our lab has recently solved the cryo-EM structure of GluA2/CNIH3 complex in GluA2:CNIH3=4:4 stoichiometry at high resolution. In Aim 1 we hypothe- size that the GluA2/CNIH3 complex could exists in other stoichiometry, and propose to reveal the architecture of complex in GluA2:CNIH3=4:2 stoichiometry using cryo-EM. CNIH1 is currently not categorized as AMPAR auxiliary subunit. However the cryo-EM structure of the GluA2/CNIH3 complex tells us that CNIH1 possess AMPAR binding motif that is present in CNIH2/3. The cryo-EM structure also revealed the presence of lipids surrounding the complex. We hypothesize that these lipids may play important functional roles in AMPAR gat- ing modulation. In Aim2 we will test roles of CNIH1 and lipids in gating modulation of AMPAR. Finally, we hy- pothesize that revealing the allosteric gating modulation mechanism of CNIH3 would require obtaining snap- shots of lipid embedded GluA2/CNIH3 complex in channel closed, open, and desensitized states. In Aim 3, we propose to solve high resolution cryo-EM structures of GluA2/CNIH3 complex embedded in a lipid bilayer mi- metic environment, and compare them in different functional states. The role of auxiliary subunits in tuning ion channel gating kinetics is predicted to have significant impact on circuit dynamics. In summary, the outcomes of this study are expected to advance our mechanistic understanding of AMPAR function and assist developing new therapeutic compounds that can alleviate dysregulation of AMPARs seen in neurological and psychiatric disorders, such as Alzheimer's disease, stroke, autism, Rasmussen's and limbic encephalitis, and seizure.