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

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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.