Retinal neurons that form ribbon-style synapses operate over a wide dynamic range to continuously encode and relay visual information to their downstream targets. These remarkable signaling abilities are supported by a specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is a retinal-specific t-SNARE protein. It is also a Ca2+-dependent phosphoprotein that is phosphorylated at T14 by Ca2+/calmodulin-dependent protein kinase II (CaMKII). In the retina, phosphorylation at this site is governed by illumination, while results of in vitro binding studies raise the possibility that T14 phosphorylation alters the ability of syntaxin3B to form the SNARE complexes required for neurotransmitter release. We therefore hypothesize that syntaxin3B has both an essential and a modulatory role in vision. In this research program, we test this hypothesis and establish the roles of syntaxin3B and T14 modification in the mammalian retina. To achieve these goals we use a powerful multi-pronged approach. Using modern in vitro fusion assays, we will establish the role of syntaxin3B T14 modification in the catalysis of SNARE-mediated membrane fusion and determine whether T14 modulates a critical interaction with Munc18 known to facilitate SNARE complex formation. We will also establish the effects of T14 phosphorylation on exocytosis in a tractable model cell that utilizes STX3 and allows for direct, biophysical analysis of exocytosis and manipulation of T14 phosphorylation. In the mammalian retina, we take advantage of a cell-specific STX3KO mouse line to examine the roles of STX3B in multiple aspects of rod bipolar cell function, including from neurotransmitter release and calcium channel activity, to active zone ultrastructure, and to the ability to relay visual information to downstream neurons and across the retinal circuit. To this end, we use high-resolution techniques that include electrophysiological approaches and electron tomography, in addition to assays of simple visual behavior. Finally, we combine the cell-specific STX3KO mouse line with an elegant transfection method to directly study the role of STX3B T14 modification in the rod bipolar cell. We will examine the role of T14 modification in the modulation of synaptic vesicle fusogenicity, synaptic vesicle dynamics and short-term adaptation, synaptic transmission and the throughput of visual information across the retina. Together, this research program will provide a full picture of the essential and modulatory roles of STX3B in the retina and yield new insights into the pathophysiology of STX3B-related disorders of vision.
In this research program, we examine presynaptic mechanisms that regulate neurotransmitter release at retinal ribbon-style synapses. These specialized synapses, essential for vision, convey the fundamental signals we use to perceive the visual world. Knowledge of their regulation is important for developing new strategies to preserve or restore vision in patients experiencing retinal degeneration.
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