Homeostatic Regulatory Mechanisms in Nociceptors

伤害感受器的稳态调节机制

• 批准号: 10452662
• 负责人: John Smith Del Rosario
• 金额: $ 8.44万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10452662
• 关键词: 1-Phosphatidylinositol 4-Kinase; Address; Adverse effects; Affect; Afferent Neurons; Agonist; American; Animal Model; Behavioral; Biochemical; Biological Assay; Biotinylation; Capsaicin; Cell membrane; Cell surface; Cells; Charge; Chemosensitization; Chloroquine; Coupled; Data; Doctor of Philosophy; Drug Targeting; Electrophysiology (science); Endocytosis; Environment; Excision; Fluorescence; Goals; Health; Human; In Vitro; Individual; Injury; Ion Channel; Ligation; Light; Mechanics; Microscopy; Mitogen-Activated Protein Kinases; Molecular; Mus; Nerve; Neuraxis; Neurons; Nociceptors; Pain; Pathway interactions; Peripheral; Persistent pain; Pharmacology; Phase; Phosphatidylinositols; Phosphotransferases; Piezo 2 ion channel; Play; Postdoctoral Fellow; Preparation; Prevalence; Process; Proteins; RNA; Regulation; Reporting; Role; Second Messenger Systems; Sensory Process; Skin; Spinal Ganglia; Stimulus; Symptoms; Syndrome; System; Tactile; Techniques; Touch sensation; Vanilloid; allodynia; chronic pain; disability; experience; experimental study; extracellular; immunocytochemistry; improved; in vivo; inhibitor; mechanical allodynia; novel; optogenetics; persistent symptom; public health relevance; receptor; response; transcriptome sequencing; voltage clamp

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.

抽象的 机械性异常性症是慢性疼痛的标志性症状，其特征是对无害的痛苦反应 刺激。然而，对于此过程的细胞和分子调节知之甚少。最近， 机械激活的压电通道被确定为小鼠机械性异常尼亚的关键参与者， 人类，但是负责受伤时负责敏感性的分子和蛋白质是 仍然很了解。我们实验室的最新数据表明，Gi-protein偶联受体的激活会影响A 背根神经节（DRG）神经元和HEK293细胞中压电2电流的持久增强。这 通过抑制Gβγ的活性来消除压电2电流的增强。令人惊讶的是，抑制 Gβγ-下游激酶，磷酸肌醇3-激酶（PI3K）和促分裂原激活的蛋白激酶（MAPK）， 还废除了压电2电流的增强，这表明Gβγ对压电2通道的间接作用。 因此，对于AIM 1（Ph.D.进度），我们描述了通过Gi-i-gi对压电2电流调节的新机理 蛋白偶联受体。另一方面，我们的实验室还表明了瞬态受体的激活 辣椒素的潜在香草素1（TRPV1）通道可抑制DRG中的压电2电流 神经元和异源系统。通过从细胞外去除Ca2+来消除这种抑制作用 解决方案，确认Ca2+在压电2通道上的关键作用。我们实验室中使用总体内部数据的初步数据 反射荧光（TIRF）表明，在激活TRPV1通道时，压电2通道被内在化。 在HEK293细胞中，但是尚不清楚通过内吞作用内部化的压电2通道。对于目标2（F99） 阶段），我们假设TRPV1的激活会影响CA2+触发的内吞作用，该内吞作用 抑制压电2电流。我们旨在确定调节活性的分子和蛋白质 机械激活的压电通道。我们希望这些新颖的发现可以帮助我们理解过程 触觉异常症并调查影响周围的变化并影响中枢神经 系统（AIM 3-K00阶段）的最终目标是为机械呼吸的治疗提供新的途径 综合征。