Synaptic Organization and Modulation of Kainate Receptors: Investigating the Structure, Dynamics, and Function in the Context of Trans-Synaptic Junctions

摘要 海人藻酸受体是离子型谷氨酸受体家族的成员，与癫痫和脑出血有关。 多发性神经认知缺陷，如自闭症、精神分裂症和智力低下。这些受体可以是 在突触前膜上表达，调节神经递质释放和突触后 参与兴奋性信号的细胞膜。这些通道是钙渗透的，因此， 谷氨酸的激活导致细胞内钙信号和突触反应。然而，他们目前 活性可以通过蛋白质的相互作用来调节。这种相互作用会导致稳态电流增加， 导致钙通透性增加和差异性突触反应。最近，突触前神经新素3β和 细胞外支架类C1q蛋白已被发现与突触后红藻氨酸建立连接 苔藓纤维上的受体-CA3突触。这些相互作用已被发现调节红藻氨酸受体 然而，在膜上的募集，它们对红藻氨酸受体门控特性的调节作用仍然存在 未知。 在我的论文工作中，我打算研究红藻氨酸胞外区的蛋白质相互作用。 受体调节它们的门控特性。作为F99训练的一部分，我将使用电生理学来调查 与Neuresin 3和类C1q蛋白相互作用引起的电流调制 使用smFRET的构象变化(目标1)。这项工作将使我们对门控机制有更深入的了解 并有助于解释这些连接在突触后兴奋中的作用 发信号。 为了我博士后的训练，我打算继续探索分子之间的联系 用电子显微镜观察这些连接。因此，对于本提案的K00阶段，我计划在 进行低温电子显微镜和低温电子显微镜实验。使用这些技术将使我能够调查 在这些突触连接的背景下，这些蛋白质之间的连接(目标2)。这些信息 在这些实验中获得的结果将使我们深入了解 突触分裂和进一步了解这些连接在突触形态中所起的作用。 了解这一点将使我们能够将这一连接的生物学破坏与行为缺陷联系起来。 总而言之，这项提议的目的是给我必要的培训，以调查职能和 这些连接的结构功能，以了解它们在生理条件下的作用。建议的培训 根据这项提议，加上我之前的培训，我将获得成为一名 独立调查员。

Abstract Kainate receptors are members of the ionotropic glutamate receptor family and are implicated in epilepsy and in multiple neurocognitive deficits such as autism, schizophrenia, and mental retardation. These receptors can be found to express in the presynaptic membrane where they regulate neurotransmitter release and postsynaptic membrane where it is involved in excitatory signaling. These channels are calcium-permeable and, therefore, activation by glutamate lead to intracellular calcium signaling and synaptic response. However, their current activity can be modulated by protein interactions. Such interactions can lead to increase steady-state currents, causing increase calcium permeability and differential synaptic response. Recently, presynaptic Neurexin3β and extracellular scaffolding C1q-like protein have been revealed to create a junction with postsynaptic kainate receptors at mossy fibers-CA3 synapses. These interactions have been found to regulate kainate receptor recruitment at the membrane, however, their modulatory effect on kainate receptor gating properties still remains unknown. For my dissertation work, I propose to study how protein interaction at the extracellular domain of kainate receptor modulate their gating properties. As part of the F99 training, I will use electrophysiology to investigate the current modulation caused by interactions with Neurexin 3 and C1q-like protein and investigate the conformational changes using smFRET (Aim 1). This work will give us more insight into the gating mechanisms caused by the modulation of these proteins and help explain the role of these junctions in postsynaptic excitatory signaling. For my training as a post-doctoral student, I intend to continue exploring the macromolecule contacts between these junctions using electron microscopy. Therefore, for the K00 phase of this proposal I plan to train in performing CryoEM and CryoET experiments. Using these technologies will allow me to investigate the connections made between these proteins in the context of these synaptic junctions (Aim 2). The information obtained in these experiments will give us insight into the structure and organization of this junctions within the synaptic cleft and further our understanding of the role these junctions play in synapse morphology. Understanding this will allow us to correlate biological disruption of this junction to behavior deficits. In summary, the objective of this proposal is to give me the training necessary to investigate the functional and structural function of these junctions to understand their role in physiological conditions. The training proposed under this proposal alongside my previous training will give me the framework necessary to become an independent investigator.