Project Summary: Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment, while the mechanisms that drive and/or maintain chronic pain remain unclear. We have shown that chronic nociceptor hyperexcitability after severe injury is maintained by cAMP signaling through multiple cAMP effectors, including PKA, EPAC and HCN channels. These pathways are enhanced by AKAP-mediated complex formation with AC and show significant cross-talk with Ras/MAPK signaling. Activation of cAMP- and Ras-mediated pathways initiate at the plasma membrane (PM) and are uniquely sensitive to clustering of lipids within the PM. We have also shown that spinal cord injury reduces AC inhibition by Gαi, resulting in reduced potency of opioids in DRG neurons. This reduced sensitivity can be mimicked in DRG neurons from naïve animals by overnight exposure to neurotrophic factors or by a 5 min, modest depolarization that approaches the firing threshold of DRG neurons after severe injury. Importantly, nociceptor hyperexcitability and reductions in opioid potency, induced by either injury, neurotrophic factors or acute depolarization, can be reversed by inhibition of Ras-dependent signaling or reorganization of lipids in the plasma membrane. We hypothesize that the sustained depolarization that occurs in many injury models drives alterations in PM lipid organization, leading to increased ERK signaling and decreased opioid responses. Release of neurotrophic factors reinforce these pathways and, in conjunction with cAMP signaling, drives nociceptor hyperexcitability and a chronic pain state. To address these hypotheses, we propose three Aims. 1) Determine the mechanism for reduced MOR-Gαi inhibition of AC by C-Raf, 2) Define the mechanism of Ras activation and nociceptor hyperexcitability by depolarization and SCI, and 3) Define functional consequences of interactions among depolarization and cell signaling by cAMP, C-Raf, and ERK. Importantly, our model identifies multiple FDA-approved drugs that could simultaneously enhance endogenous opioid responses and block nociceptor hyperexcitability after severe injury.