Injury-Induced Plasticity of P2X Receptor Function

描述(申请人提供)：我们最近对背根神经节(DRG)神经元中嘌呤能受体(P2XR)的调节研究表明，在炎症或神经损伤后，P2X3R的活性大大增强，这种增加源于P2X3R表达上调或P2X3R转运到细胞膜的增加。这种受体敏化有助于大鼠疼痛模型中异常的疼痛反应。我们进一步表明，炎症改变了P2X3Rs的调制信号。前列腺素E_2(PGE_2)通过激活cAMP调节的鸟嘌呤核苷酸交换蛋白(EPAC)，使P2X3Rs的信号从单纯依赖PKA转变为依赖PKA和PKC。我们在这里的重点是了解EPAC在改变P2X3R表达和贩运方面可能发挥的作用。我们假设损伤促进了EPAC的激活。EPAC、细胞骨架蛋白和支架锚定蛋白复合体之间的相互作用在调节P2X3R活性中起着关键作用。体外和体内方法将被用来检验这一假说。我们将确定EPAC是否与DRGs中的P2X3Rs、肌动蛋白和AKAP相互作用，以及损伤是否改变这种相互作用。我们将进一步确定这些蛋白质是否参与了P2X3R介导的电流反应的调制。此外，我们还将确定阻断EPAC活性是否可以减少大鼠的异常疼痛行为。这些研究应该能更好地理解伤害诱导的P2XR敏化的机制。

DESCRIPTION (provided by applicant): Our recent studies of modulation of purinergic receptors (P2XR) in dorsal root ganglion (DRG) neurons show that, after inflammation or nerve injury, the activity of P2X3Rs becomes greatly enhanced The increase derives either from an upregulation of P2X3R expression or an increase in the trafficking of P2X3Rs to the cell membrane. This receptor sensitization contributes to abnormal pain responses in rat pain models. We further show that inflammation changes the modulatory signaling of P2X3Rs. Prostaglandin E2 (PGE2) modulation of P2X3Rs switches from a solely PKA-dependent to both PKA and PKC-dependent signaling as a result of the activation of cAMP-regulated guanine nucleotide exchange protein (Epac). Our focus here is to understand the role that Epac may have in changing P2X3R expression and trafficking. We hypothesize that injury facilitates Epac activation. Interactions among Epac, cytoskeletal proteins and scaffold anchoring protein complexes play critical roles in the modulation of P2X3R activity. In vitro and in vivo approaches will be used to test this hypothesis. We will determine if Epac interacts with P2X3Rs, actin and AKAP in DRGs and if injury changes the interactions. We will further determine if these proteins participate in the modulation of P2X3R-mediated current responses. In addition, we will determine if blocking Epac activity can reduce abnormal pain behaviors in rats. These studies should provide a better understanding of the mechanisms underlying injury-induced P2XR sensitization.