The nervous system uses two forms of fast synaptic transmission, chemical and electrical, that both contribute to the dynamic computations that create thought, feelings, and actions. Chemical synapses are well studied, and the biochemical mechanisms by which neurotransmitter is released and received are well understood. By contrast, we know relatively little about the macromolecular complex of the electrical synapse. Electrical synapses are made from tens to thousands of gap junction channels that create direct, low-resistance routes of cytoplasmic communication between neurons. They contributed to sophisticated function in neural circuit computation, they display plasticity through a number of short- and long-term mechanisms, and their assembly is regulated during development. Together, this all suggest a complex macromolecular structure that controls their formation and function, yet a critical barrier to progress in the field remains in the identification of the proteins of the electrical synapse. The overarching goal of the proposal is to establish zebrafish electrical synapses as a model to understand their proteomic diversity.
Aim1 will demonstrate that electrical synapse proteins can be identified using genome engineered zebrafish that express electrical synapse proteins tagged with TurboID. TurboID is an evolved E.coli protein that allows for in vivo, proximity-depending labeling of proteins with biotin. Such biotinylated proteins can then be efficiently isolated from the animal and analyzed using mass spectrometry.
Aim2 will then assess the biochemical interactions and cellular localization of the identified proteins using expression systems for protein-protein interactions and in vivo immunohistochemistry in zebrafish. If successful, this grant will fundamentally shift the understanding of electrical synapses, revealing proteins involved in trafficking pathways, synaptic structure, and functional regulation. The proposed studies will provide novel insight into the molecular complexes of the electrical synapse in a model vertebrate, providing a foundation for the identification of targets for therapy of neurodevelopmental disorders.
The human brain contains billions of neurons organized into neural circuits that form the basis for perception, thought, and behavior. This study examines electrical synapses, which are critical to neural circuit function in all animals, yet we do not know the variety of proteins that create them. This proposal aims to uncover the complete set of proteins at electrical synapses and will shift our understanding of these structures to a new point of view in which they are appreciated for their molecular complexity.
