Regulation of Piezo2 Channels by G-protein Coupled Receptors and Endocytosis

摘要 机械性异位痛是慢性疼痛的一个显著症状，其特征是对无害的疼痛做出疼痛反应。 刺激物。然而，对这一过程的细胞和分子调控知之甚少。最近， 机械激活的Piezo2通道被认为是小鼠机械性痛觉异常的关键因素，并 人类，但负责损伤后Piezo2通道敏化的分子和蛋白质是 人们对此仍知之甚少。我们实验室的最新数据表明，Gi-蛋白偶联受体的激活诱导了 背根神经节(DRG)神经元和HEK293细胞中Piezo2电流的持久增强。这个 抑制G-βγ的活性可消除对Piezo2电流的增强作用。令人惊讶的是，这种抑制 G-βγ下游激酶、磷脂酰肌醇3-激酶和丝裂原活化蛋白激酶； 也取消了Piezo2电流的增强作用，提示G-βγ对Piezo2通道有间接作用。 因此，对于目标1(博士进展)，我们描述了Gi-2调节Piezo2电流的新机制。 蛋白质偶联受体。另一方面，我们的实验室还表明，瞬时受体的激活 辣椒素对DRG Piezo2电流的抑制作用 神经元和异源系统。这种抑制作用可以通过从细胞外去除钙离子来消除。 解决方案，证实了钙在Piezo2通道上的关键作用。我们实验室的初步数据使用完全内部 反射荧光(TIRF)显示，当激活TRPV1通道时，Piezo2通道被内化 在HEK293细胞中，Piezo2通道是否通过内吞作用内化尚不清楚。目标2(F99) 阶段)，我们假设TRPV1的激活诱导了钙离子触发的内吞作用，从而协调了 抑制Piezo2电流。我们的目标是找出调节细胞活性的分子和蛋白质。 机械激活的Piezo2频道。我们希望这些新颖的发现能帮助我们理解这一过程。 并研究影响外周和影响中枢神经的变化 系统(目标3-K00阶段)，最终目标是为治疗机械性疼痛提供新的途径 综合症。

ABSTRACT Mechanical allodynia is a hallmark symptom of chronic pain characterized by painful responses to innocuous stimuli. However, little is known about the cellular and molecular regulation of this process. Recently, the mechanically activated Piezo2 channels were identified as key players of mechanical allodynia in mice and humans, but the molecules and proteins responsible for the sensitization of Piezo2 channels upon injury are still poorly understood. Recent data from our lab show that activation of Gi-protein coupled receptors induces a long-lasting potentiation of Piezo2 currents in Dorsal Root Ganglion (DRG) neurons and HEK293 cells. The potentiation of Piezo2 currents was abolished by inhibiting the activity of Gβγ. Surprisingly, the inhibition of Gβγ-downstream kinases, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), also abolished the potentiation of Piezo2 current suggesting an indirect effect of Gβγ on Piezo2 channels. Therefore, for aim 1 (Ph.D. progress), we described a novel mechanism of regulation of Piezo2 currents by Gi- protein coupled receptors. On the other hand, our lab has also shown that activation of Transient Receptor Potential Vanilloids 1 (TRPV1) channels by capsaicin leads to robust inhibition of Piezo2 currents in DRG neurons and heterologous systems. This inhibition is abolished by removing Ca2+ from the extracellular solution, confirming a pivotal role of Ca2+ on Piezo2 channels. Preliminary data in our lab using total internal reflection fluorescence (TIRF) show that Piezo2 channels are internalized upon activation of TRPV1 channels in HEK293 cells, but whether Piezo2 channels are internalized via endocytosis is not known. For aim 2 (F99 phase), we hypothesize that activation of TRPV1 induces a Ca2+-triggered endocytosis that orchestrate the inhibition of Piezo2 currents. We aim to identify molecules and proteins that regulate the activity of the mechanically activated Piezo2 channels. We hope these novel findings could help us understand the process of tactile allodynia and investigate the changes that affect the periphery and influence the central nervous systems (aim 3-K00 phase) with the ultimate goal of providing new avenues for treatments of mechanical-pain syndromes.