Lipid Regulation of Transient Receptor Potential Channels

瞬时受体电位通道的脂质调节

• 批准号: 8581450
• 负责人: Tibor Rohacs
• 金额: $ 34.78万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581450
• 关键词: Afferent Neurons; Analgesics; Bradykinin; Capsaicin; Cells; Cytoskeleton; Data; Fluorescence; G-Protein-Coupled Receptors; Goals; Grant; Heating; Hypersensitivity; Inflammatory; Ion Channel; Lead; Life; Lipids; Mediating; Medical; Menthol; Molecular; Monitor; Mus; Nerve; Neurons; Nociceptors; Pain; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Physiological; Play; Preparation; Protein Isoforms; Protein Kinase C; Receptor Activation; Recombinants; Regulation; Role; Sensory; Signal Pathway; Signal Transduction; Skin; Specificity; Spinal Ganglia; Stimulus; Symptoms; Syndrome; System; TRP channel; TRPV1 gene; Testing; Therapeutic; Thermal Hyperalgesias; Time; Tubulin; United States; Work; base; chemotherapy; chronic pain; cost; desensitization; painful neuropathy; patch clamp; public health relevance; receptor; relating to nervous system; research study; sensor

DESCRIPTION (provided by applicant): The objective of this grant renewal proposal is to understand the molecular mechanism of regulation of sensory TRP channels by phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] or PIP2. TRPV1 channels are activated by heat, capsaicin, low pH and a plethora of other pain producing stimuli. They are sensitized to low-grade stimuli through activation of G-protein coupled receptors (GPCRs) by pro-inflammatory agents such as bradykinin. Sensitization plays an important role in thermal hyperalgesia, a disturbing symptom in neuropathic pain syndromes. GPCRs involved in sensitization usually activate phospholipase C (PLC). Both protein kinase C (PKC) and the reduction in phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] have been implicated in sensitization of TRPV1. Strong pharmacological activation of TRPV1 with capsaicin on the other hand leads to desensitization of TRPV1, a phenomenon thought to underlie the use of capsaicin as a local analgesic. Intriguingly PLC activation also plays a crucial role in desensitization of TRPV1. The major goal of this proposal is to understand how two phenomena, sensitization and desensitization, with opposite results on channel activity, can be mediated by the same signaling pathway. The activity of TRPV1, similar to many other TRP channels, depends on the presence of PI(4,5)P2. In addition, PI(4,5)P2 also partially inhibits the channel in intact cell. W found that GPCR activation by bradykinin in sensory neurons leads to the selective decrease of PI(4,5)P2, but not its precursor PI(4)P. Since both PI(4)P and PI(4,5)P2 can support TRPV1, this would not significantly limit channel activity. The selective decrease in PI(4,5)P2 on the other hand synergizes with PKC to potentiate TRPV1 activity. This effect was mimicked by disruption of the tubulin cytoskeleton. During desensitization on the other hand, TRPV1 stimulation by saturating capsaicin concentrations leads to a strong depletion of both PI(4,5)P2 and PI(4)P leading to diminished channel activity. The cold and menthol sensitive TRPM8 is inhibited upon GPCR activation by bradykinin, presumably since the activity of this channel specifically depends on PI(4,5)P2. Our overall hypothesis is that differential regulation of PI(4,5)P2 and PI(4)P levels by different PLC isoforms determines specificity of regulation of phosphoinositide sensitive TRP channels, depending on their lipid specificity profile. In this proposal we will systematically examine this hypothesis using a wide range of approaches including studying recombinant and native channels in sensory neurons, using various fluorescence-based lipid sensors to monitor levels of phosphoinositides in live cells, single cell real time PCR, patch clamp and studying the nerve impulses evoked by capsaicin in the ex-vivo skin-nerve preparation. We propose the following specific aims: Aim 1 Evaluate the role of PLC activation in sensitization of TRPV1 by pro-inflammatory agents. Aim 2 Evaluate the role of PLC activation in desensitization of capsaicin-induced currents. Aim 3 Evaluate the role of PLC activation in regulation of TRPM8 by GPCRs and Ca2+ influx.

描述（由申请人提供）：本资助续期提案的目的是了解磷酸肌苷，特别是磷脂酰肌醇4,5-二磷酸[PI(4,5)P2]或PIP2调节感觉TRP通道的分子机制。TRPV1通道被热、辣椒素、低pH值和大量其他产生疼痛的刺激激活。通过促炎剂（如缓激肽）激活g蛋白偶联受体（gpcr），使它们对低强度刺激敏感。致敏在痛觉热过敏中起着重要作用，痛觉热过敏是神经性疼痛综合征中的一种令人不安的症状。参与致敏的gpcr通常激活磷脂酶C （PLC）。蛋白激酶C （PKC）和磷脂酰肌醇4,5-二磷酸[PI(4,5)P2]的减少都与TRPV1的增敏有关。另一方面，辣椒素对TRPV1的强烈药理激活会导致TRPV1的脱敏，这一现象被认为是使用辣椒素作为局部止痛药的基础。有趣的是，PLC激活在TRPV1的脱敏中也起着至关重要的作用。本提案的主要目标是了解敏化和脱敏这两种对通道活性具有相反结果的现象如何通过相同的信号通路介导。与许多其他TRP通道类似，TRPV1的活性取决于PI(4,5)P2的存在。此外，PI(4,5)P2也部分抑制完整细胞中的通道。W发现GPCR在感觉神经元中被缓激肽激活导致PI(4,5)P2选择性降低，而不是其前体PI(4)P。由于PI(4)P和PI(4,5)P2都可以支持TRPV1，因此这不会显着限制通道活动。另一方面，PI(4,5)P2的选择性减少与PKC协同作用，增强TRPV1的活性。这种效应被微管蛋白细胞骨架的破坏所模仿。另一方面，在脱敏过程中，饱和辣椒素浓度刺激TRPV1会导致PI(4,5)P2和PI(4)P的强烈耗竭，导致通道活性降低。低温和薄荷醇敏感的TRPM8在GPCR激活时被缓激肽抑制，可能是因为该通道的活性特别依赖于PI(4,5)P2。我们的总体假设是，不同PLC亚型对PI(4,5)P2和PI(4)P水平的差异调节决定了磷酸肌肽敏感TRP通道的调节特异性，这取决于它们的脂质特异性。在本研究中，我们将采用多种方法系统地检验这一假设，包括研究感觉神经元中的重组通道和天然通道，使用各种基于荧光的脂质传感器监测活细胞中磷酸肌苷的水平，单细胞实时PCR，膜片钳和研究辣椒素在离体皮肤神经制备中引起的神经冲动。我们提出以下具体目标：目的1评估PLC激活在促炎剂致敏TRPV1中的作用。目的2评估PLC激活在辣椒素诱导电流脱敏中的作用。目的3评估PLC激活在GPCRs和Ca2+内流调控TRPM8中的作用。