Pannexins are ATP release membrane channels implicated in the formation and maintenance of inflammatory 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. We have recently demonstrated that the activity of pannexin 1 (Panx1) in synovial fluids collected from dog patients correlates with the crude pain scale assessed by a clinician, making this channel an excellent candidate for potential pain biosensors. However, our current pain scale relies on subjective judgment, leaving the possibility that the trend we observed may be underestimated or overestimated. Furthermore, our current cell-based assay system requires substantial preparation time, making it impractical for use in clinical settings. 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 unequivocally assess the potential of Panx1 and related membrane receptors as pain markers using objective gait analysis in dogs undergoing tibial plateau-leveling osteotomy (TPLO). Based on the preliminary studies, the central hypothesis is that pannexins are activated by signaling molecules enriched in the extracellular fluids of dogs suffering from inflammatory joint pain. To attain the overall objectives, the following two specific aims will be performed:1) Correlate Panx1 activity with pain as assessed by objective gait analysis using body fluid collected from dogs before and after joint surgery and 2) Expand the list of potential pain biosensors beyond Panx1. These research aims will be executed by using cell biological, biochemical, and biophysical in vitro experiments. These results are expected to have a profound positive impact because they will serve as strong foundation to investigate the pathological 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 joint pain in canine patients.