The role of the AP2 adaptor complex in inflammatory pain

项目摘要 炎症状态的一个主要特征是通常无害的刺激会产生疼痛。电流 止痛药包括非甾体抗炎药，其目的是阻断 前列腺素产生、皮质类固醇和阿片类药物。然而，副作用和某些情况下成瘾 与这些药物相关的潜在风险限制了它们的长期使用，特别是在慢性炎性疼痛期间。到 开发新的，非成瘾性镇痛药，仍然迫切需要了解炎症如何 产生伤害感受器放电的变化，而这种变化是疼痛感知的基础。在这份提案中，我们的目标是提供 在背根神经节（DRG）神经元中，adaptin 2网格蛋白介导的内吞作用（AP 2- CME）是炎症诱导的伤害感受器敏化的主要促进剂。我们先前已经 表明，在对蛋白激酶A（PKA）刺激的反应中，Slack KNa通道通过以下途径内化： AP 2-CME来自DRG神经元膜，这引起过度兴奋。此外，我们还表明， 抑制AP 2-CME防止PKA诱导的神经元过度兴奋。初步研究表明， 在DRG神经元内特异性地体内敲低AP 2 α亚基AP 2A 2， 炎性疼痛行为。在这里，我们将结合蛋白质化学，免疫组织化学， 电生理学、脊髓生理学、疼痛行为测定和一种新的体内基因敲除方法 以检验AP 2-CME是伤害感受器敏化的关键调节剂的假设。具体目标是：（1） 确定AP 2-CME是否控制基础兴奋性和神经传递2）为了证明 减少AP 2-CME减轻炎性疼痛。该研究项目将揭示AP 2-CME的核心作用 在疼痛信号中发挥作用。

Project Summary One of the cardinal features of inflammatory states is that normally innocuous stimuli produce pain. Current pain-relieving drugs include nonsteroidal anti-inflammatory drugs, which are aimed at the interdiction of prostaglandin production, corticosteroids and opioids. However, the side effects and some cases addiction potential associated with these drugs limit their long-term use especially during chronic inflammatory pain. To develop novel, non-addictive analgesics, there remains an urgent need to understand how inflammation produces the change in nociceptor firing that underlies pain perception. In this proposal, we aim to provide proof of principal that in dorsal root ganglion (DRG) neurons, adaptin 2 clathrin-mediated endocytosis (AP2- CME) is a principal facilitator of inflammatory-induced nociceptor sensitization. We have previously demonstrated that in response to protein kinase A (PKA) stimulation, Slack KNa channels are internalized via AP2-CME from DRG neuronal membranes and this caused hyperexcitability. Furthermore we showed that inhibiting AP2-CME prevented PKA-induced neuronal hyperexcitability. Preliminary studies now indicate that in vivo knockdown of the AP2 alpha subunit AP2A2 specifically within DRG neurons, substantially reduces inflammatory pain behavior. Here, we will apply a combination of protein chemistry, immunohistochemistry, electrophysiology, spinal cord physiology, pain behavior assays and a novel in vivo gene knockdown approach to test the hypothesis that AP2-CME is a key regulator of nociceptor sensitization. The specific aims are 1) to determine whether AP2-CME controls basal excitability and neurotransmission 2) To demonstrate that reducing AP2-CME mitigates inflammatory pain. This research project will reveal the central role AP2-CME plays in pain signaling.