Lipid Regulation of Transient Receptor Potential Channels

描述（由申请人提供）：本授权更新提案的目的是了解磷酸肌醇，特别是磷脂酰肌醇4，5-二磷酸[PI（4，5）P2]或PIP 2调节感觉TRP通道的分子机制。TRPV 1通道被热、辣椒素、低pH值和大量其他疼痛产生刺激物激活。它们通过促炎剂如缓激肽激活G蛋白偶联受体（GPCR）对低级刺激敏感。热痛觉过敏是神经病理性疼痛综合征中的一种令人不安的症状，敏化在其中起着重要的作用。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TRPV 1的致敏有关。另一方面，辣椒素对TRPV 1的强烈药理学激活导致TRPV 1的脱敏，这一现象被认为是辣椒素作为局部镇痛剂的基础。有趣的是，PLC激活在TRPV 1的脱敏中也起着至关重要的作用。这个提议的主要目标是了解两种现象，敏化和脱敏，对通道活性的相反结果，可以由相同的信号通路介导。与许多其他TRP通道类似，TRPV 1的活性取决于PI（4，5）P2的存在。此外，PI（4，5）P2对完整细胞的通道也有部分抑制作用。W发现感觉神经元中缓激肽激活GPCR导致PI（4，5）P2的选择性降低，但不导致其前体PI（4）P的选择性降低。由于PI（4）P和PI（4，5）P2都可以支持TRPV 1，因此这不会显著限制通道活性。另一方面，PI（4，5）P2的选择性降低与PKC协同增强TRPV 1活性。这种效应通过破坏微管蛋白细胞骨架来模拟。另一方面，在脱敏过程中，通过饱和辣椒素浓度的TRPV 1刺激导致PI（4，5）P2和PI（4）P两者的强烈消耗，从而导致通道活性降低。冷和薄荷醇敏感的TRPM 8在GPCR激活时被缓激肽抑制，大概是因为该通道的活性特异性地依赖于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醇敏感性TRP通道调节的特异性，这取决于它们的脂质特异性谱。在这个建议中，我们将系统地检查这一假设使用广泛的方法，包括研究重组和天然通道的感觉神经元，使用各种荧光为基础的脂质传感器，以监测水平的磷酸肌醇在活细胞，单细胞真实的时间PCR，膜片钳和研究辣椒素诱发的神经冲动在离体皮肤神经制备。我们提出了以下具体目标：目的1评价PLC激活的作用，在致敏TRPV 1的促炎剂。目的2探讨PLC激活在辣椒素电流脱敏中的作用。目的3探讨PLC激活在GPCR和Ca ~（2+）内流调控TRPM 8中的作用。

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.