Synaptic communication in the nervous system is accomplished by two major classes of synapses, chemical and electrical, which operate in different ways. Electrical synapses are formed by gap junctions between neurons and allow passage of electrical current directly between cells, providing fast and often bi-directional communication. Cellular control of the magnitude of this communication refines local and long-range neural network functions, and is an important component of network plasticity. Electrical synapse plasticity plays a particularly important role in sensory adaptation in the vertebrate retina, where changes in coupling of some networks exceed an order of magnitude. The long-term goals of this established research project are to elucidate the mechanisms that control plasticity of electrical synapses. In recent work, we have discovered an intimate relationship between the actin cytoskeleton and functional control of coupling in Connexin 36 (Cx36) gap junctions. This appears to integrate the main components of excitatory and inhibitory signaling and switching between those modes. This project will investigate those links, using a combination of cell culture and mouse retina model systems. We will test three specific hypotheses about regulation of Cx36 functional coupling in the following ways: (1) Signaling protein complexes that regulate Cx36 coupling are associated with the actin cytoskeleton. Using proximity labeling and quantitative proteomic techniques, we will identify signaling components involved in the regulation of Cx36 coupling. We will further investigate the dynamic changes in proximity of these components to Cx36 during regulatory signaling. (2) Phosphorylation of Cx36 alters its association with signaling components. We will investigate how phosphorylation of certain residues of Cx36 regulates the association of some signaling components. (3) RhoA and Cdc42 signaling pathways modulate functional plasticity. We will investigate how pathways that control cytoskeletal remodeling influence coupling of Cx36. The proposed studies will elucidate mechanisms central to control of electrical synapse functional plasticity. Knowledge of these mechanisms will provide a great deal of insight not only into the control of visual adaptation processes in the retina, but also electrical synapse plasticity throughout the brain. This will allow the development of targeted therapies for disorders in which gap junctions play a role.