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

G 蛋白偶联受体和内吞作用对 Piezo2 通道的调节

基本信息

批准号：
10198568
负责人：
John Smith Del Rosario
金额：
$ 1.93万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10198568
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 G-Protein-Coupled Receptors Goals Health Human In Vitro Individual Injury Ion Channel Ligation Light Mechanics Microscopy Mitogen-Activated Protein Kinases Molecular Mus Nerve Neuraxis Neurons 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/antagonist mechanical allodynia novel optogenetics persistent symptom public health relevance receptor response transcriptome sequencing voltage clamp

项目摘要

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.

摘要机械性异常性疼痛是慢性疼痛的标志性症状，其特征在于对无害物质的疼痛反应。刺激。然而，人们对这一过程的细胞和分子调节知之甚少。近日机械激活的Piezo 2通道被鉴定为小鼠机械异常性疼痛的关键参与者，但是在损伤时负责Piezo 2通道致敏的分子和蛋白质是仍然知之甚少。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一种新的免疫应答。背根神经节（DRG）神经元和HEK 293细胞中Piezo 2电流的持久增强。的通过抑制Gβγ的活性，Piezo 2电流的增强被消除。令人惊讶的是， Gβγ-下游激酶，磷酸肌醇3-激酶（PI 3 K）和丝裂原活化蛋白激酶（MAPK）， Gβ γ对Piezo 2通道的增强作用也消失，提示Gβγ对Piezo 2通道有间接作用。因此，对于目标1（博士学位），进展），我们描述了一种新的机制，调节Piezo 2电流的Gi- 蛋白偶联受体另一方面，我们的实验室也表明，瞬时受体的激活辣椒素诱导的潜在香草素1（TRPV 1）通道对背根神经节Piezo 2电流的抑制作用神经元和异源系统。通过从细胞外去除Ca 2+来消除这种抑制作用。解决方案，证实了Ca 2+对Piezo 2通道的关键作用。我们实验室的初步数据使用总内部反射荧光（TIRF）显示，Piezo 2通道在TRPV 1通道激活后被内化在HEK 293细胞中，Piezo 2通道是通过内吞作用内化的，但Piezo 2通道是否通过内吞作用内化尚不清楚。对于目标2（F99 阶段），我们假设TRPV 1的激活诱导了Ca 2+触发的内吞作用，抑制Piezo 2电流。我们的目标是确定调节细胞活性的分子和蛋白质，机械激活的Piezo 2通道。我们希望这些新的发现可以帮助我们理解这个过程探讨触觉异常性疼痛的外周和中枢神经系统的变化系统（目标3-K 00阶段），最终目标是为机械性疼痛的治疗提供新的途径综合征