Ion channels are intimately involved in all aspects of nervous system function because they give rise to electrical signals that transmit information throughout the nervous system. Information transfer is optimized by dynamically tuning ion channels. Aberrations in ion channel modulation can profoundly disrupt nervous system performance. Identifying the mechanisms that modulate ion channels will provide important insights into the wide array of diseases stemming from their dysregulation, including chronic pain. Kv4 proteins are the pore- forming subunits of the ion channels that mediate a transient potassium current called ISA. Characterizing Kv4 modulation will be broadly impactful because ISA critically shapes electrical signaling throughout the nervous system and in the heart. Kv4 proteins exist in macromolecular complexes wherein they interact with several ancillary subunits (Kv4 interactome). These interactions determine the functional expression of ISA and are likely to be key targets for modulation. Protein-protein interactions can be regulated by the process of SUMOylation, whereby Small Ubiquitin-like MOdifier (SUMO) peptides are post-translationally conjugated to a target protein to promote or prevent its interaction with another protein. Aim I tests the hypothesis that Kv4 SUMOylation alters ISA in HEK cells by adjusting Kv4 stability, surface expression and/or the Kv4 interactome. Kv4 SUMOylation sites will be mutated. Wild type or mutated Kv4 will be co-expressed with ancillary subunits in HEK cells, and SUMOylation will be experimentally manipulated. Four experiments will then be conducted: ISA will be measured with whole cell patch clamp. Kv4 stability will be measured with pulse-chase experiments. Kv4 surface expression will be measured with biotinylation experiments. Changes in the Kv4 interactome will be quantified with stable isotope labeling of amino acids in HEK cell cultures combined with mass spectrometry. Integrating the results of these four experiments will illuminate if/how Kv4 SUMOylation modulates ISA. Aim II tests the hypothesis that SUMO regulates ISA in nociceptors. Nociceptors are sensory neurons that initiate pain signaling. A reduced ISA contributes to nociceptor hyper-excitability in chronic pain. Nociceptor ion channel hyper-SUMOylation contributes to chronic pain. Nociceptors can be subdivided into five distinct cell types based on their transcriptomes. Previous work has ignored this heterogeneity, which has hampered data interpretation and efforts to understand the mechanisms underpinning nociceptor hyper- excitability during chronic pain. This proposal uses a set of genetically modified mouse lines where each line has a specific nociceptor cell type labeled in vivo. Whole cell patch clamp will be used to examine SUMO modulation of ISA in each cell type. The results of this proposal will provide fundamental information on if/how SUMOylation modulates Kv4 to sculpt ISA. These mechanistic insights are a prereq...