This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The formation and maintenance of synapses are fundamental bases for the nervous system to function as an information-conveying cellular device. Our previous research showed that the interaction of voltage-gated calcium channels (VGCCs, PQ- and N-type) and synaptic laminins (laminin(S2) is required for organization of active zones (AZs) in motor nerve terminals. The role of presynaptic VGCCs in synaptic transmission is well established but a role in organization of AZs, by interacting with extracellular ligands, is a new concept. Thus, we hypothesize that VGCCs at nerve terminals are important scaffolding proteins, intracellularly and extracellularly, to organize AZs, and aim to gain insight into the mechanism of AZs organization at synapsesby these three specific aims. (1) We will systematically screen the direct interaction of N-, PQ-type VGCCs and AZ proteins to seek intracellular mediators of these AZ organizers. We will biochemically assay the interaction using recombinant proteins of known AZ proteins. In addition, we will access the functional role of such interaction for the presynaptic differentiation by inhibiting the interaction in cultured primary motor neurons. Furthermore, we will access the effect of such interaction on the channel physiology by electrophysiological recordings. (2) We will analyze the N-type VGCC deficient mice to assess its role in presynaptic differentiation during early development, because the N-type VGCC interacts with lamininfi>2. Furthermore, we will analyze mice lacking both N- and PQ-type of VGCCs because these two VGCCs switch expression pattern at the NMJ during development and may have alternative or additional roles for AZ formation as a scaffolding proteins. (3) We hypothesize that VGCCs at the brain synapse binds to novel ligand(s) to organize the nerve terminal, because Iamininfl2 is absent at the synaptic cleft of neuron-neuron synapses in the brain. This work will lead to an identification of a novel general mechanism to organize /maintain the synaptic terminal. Preliminary data support this hypothesis and suggest that extracellular interaction is important for AZ organization in neuron-neuron synapse. Through this work, we aim to gain insight into the development/maintenance of AZs at the synapse and characterize the role of VGCCs as scaffolding proteins for presynaptic organisation. These results should provide basic knowledge applicable to gain insight into the mechanism underlying the neurological disorders and aging related changes.
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