Pannexins are ATP release membrane channels implicated in the formation and maintenance of neuropathic, inflammatory, and mechanical pain. Since these membrane channels constitute a unique protein family that is fundamentally different from the targets of commonly used analgesics such as NSAIDs and opioids, pannexins have a great potential to be novel targets for developing new analgesics. Furthermore, pannexin activity can be readily quantified using recently developed assays in the lab, making them excellent candidates for potential pain biosensors that enable objective assessment of pain for canine patients. However, how these membrane channels are activated and how they contribute to painful conditions remain unknown. The long-term goals are to develop a biosensor for assessing the type and degree of pain in canine patients and to discover new drug targets for treating chronic pain. The specific objectives of this proposal are to investigate this potential new pain biosensor, pannexin, using samples obtained from canine patients at CUHA and to deepen our understanding of how this membrane channel is activated. Based on the preliminary studies, the central hypothesis is that pan-nexins constitute membrane channels that are activated by signaling molecules enriched in the extracellular fluids of dogs suffering from inflammatory pain. To attain the overall objectives, the following two specific aims will be performed:1) Identify the types and conditions of canine extracellular fluids that activate pannexin chan-nels and 2) Determine whether the signaling molecules directly activate pannexins. These research aims will be executed by using cell biological, biochemical, and biophysical in vitro experiments. These results are ex-pected to have profound positive impact because they will serve as strong foundation to investigate the patho-logical roles of pannexins in pain signaling, a critical first step toward developing a potential new analgesic. Furthermore, our results will open a new door for further developing a simpler and more efficient pannexin-activity assay. Once established, such pannexin-mediated biosensors may serve as a novel "painometer" that has great potentials to unbiasedly assess pain scale of dog patients.