Pannexins comprise a unique family of heptameric large-pore channels that are emerging as novel targets for treating common, yet hard to cure diseases such as hypertension and chronic pain. Previous studies indicate that Panx1 is activated through stimulation of structurally unrelated receptors such as G proteincoupled receptors, ligand-gated ion channels, and tumor necrosis factor receptors. However, it remains unclear what cellular mechanism(s) actually open and close the Panx1 channel downstream of such seemingly unrelated stimuli. Furthermore, Panx2 and 3 are severely understudied and essentially nothing is known about the activation mechanisms of these subtypes. The long-term goal is to elucidate the mechanisms underlying pannexin gating, regulation, and physiological signaling pathways. The specific objectives for this proposal are to identify the physiological pannexin activators and elucidate the subtype-specific activation mechanisms. The central hypothesis is that both Panx1 and 2 are directly activated by naturally occurring signaling molecules in living cells and that Panx1 specifically requires posttranslational modifications to be "primed" for its activation. The rationale for the proposed research is that once the direct activation-stimuli and the subtypespecific mechanisms are identified, it will enable us to fill the critical gap in the pannexin-dependent signaling pathway by connecting the upstream cell-stimulation and the downstream ATP-permeable membrane pore formation. To attain the overall objectives, the following three specific aims will be performed:1) Identify the direct pannexin activators for living cells; 2) Elucidate the role of the N-terminal domain (NTD) in pannexin activation; and 3) Uncover the subtype-specific structural features of pannexins. These research aims will be executed by using a combination of a cell-based pannexin activity assay, electrophysiology, functional reconstitution, and cryo-EM. The research proposed in this application is innovative because it introduces a novel concept that pannexins—including the understudied Panx2—are directly activated by signaling molecules produced downstream of various stimuli in living cells. It is also innovative because it will provide important insights into the structure of the open channel and why Panx2 and 3 behave differently from Panx1. The proposed research is significant because it will provide concrete molecular mechanisms for the missing link in the pannexin signaling function. The proposed research is also expected to provide a strong structural foundation for subtype specific mechanisms of pannexin channels. These results are expected to have profound positive impact not only because they provide detailed basic mechanisms, but also because they will open a new door for screening/designing pannexin-specific inhibitors- much-needed molecular tools that have great potentials to serve as novel therapeutics for a variety of currently uncurable diseases.