Lipid Regulation of Transient Receptor Potential Channels

瞬时受体电位通道的脂质调节

• 批准号: 7623470
• 负责人: Tibor Rohacs
• 金额: $ 34.13万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ 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 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623470
• 关键词: Afferent Neurons; Bacteria; Biological Process; Calcium Channel; Calcium Signaling; Capsaicin; Cell membrane; Cellular Membrane; Complex; Data; Dependence; Detergents; Exhibits; G-Protein-Coupled Receptors; Goals; Ion Channel; Lipid Bilayers; Lipids; Mammalian Cell; Measures; Mediating; Membrane; Menthol; Molecular Biology; Neurons; Nociception; Pain management; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Physiological; Play; Property; Protein Isoforms; Protein Kinase C; Proteins; Receptor Protein-Tyrosine Kinases; Regulation; Relative (related person); Research; Role; Sirolimus; System; TRPV1 gene; Techniques; Temperature; Testing; Vesicle; Work; base; citrate carrier; desensitization; icilin; inhibitor/antagonist; insight; novel; receptor; reconstitution; research study; tool

DESCRIPTION (provided by applicant): Phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2 or PIP2) regulate many if not all Transient Receptor Potential (TRP) channels. We will study the regulation of the cold-sensitive TRPM8 and the warmth-sensitive TRPV1 by phosphoinositides. We will focus on elucidating the mechanism and physiological importance of the regulation of these two channels by these lipids. TRPM8 and TRPV1 are expressed in sensory neurons and play important roles in thermosensation, as well as nociception. In Aim 1 we will study the regulation of TRPV1 by phosphoinositides. Our preliminary data and results from others indicate that PIP2 exerts dual control over TRPV1 channels, showing either inhibitory or stimulatory effects in certain settings. Our main hypothesis is that depletion of PIP2 (and PIP) by the activation of various PLC isoforms have differential effects on the channel, depending on the extent of depletion. To test this hypothesis we will combine novel and well established techniques in expression systems, as well as study TRPV1 in sensory neurons. In Aim 2 we will examine the physiological role of PIP2 in the regulation of TRPM8 currents. We have shown that PIP2 activates TRPM8 and that selective depletion of PIP2 is sufficient to inhibit these channels. Our hypothesis is that Ca2+ mediated activation of PLC and the ensuing depletion of PIP2 leads to desensitization of TRPM8 currents. TRPM8 is also inhibited by activators of protein kinase C (PKC), thus PKC mediated inhibition of TRPM8 may also underlie its desensitization. To elucidate the interplay between these two alternative mechanisms, and to define their relative contributions to desensitization, we will use the following approaches. We will test inhibitors and activators of PKC in conjunction with increasing or decreasing PIP2 using various tools, and measure TRPM8 desensitization. Experiments will be performed both in a mammalian expression system and native DRG neurons. In Aim 3 we will study the regulation of TRPM8 in a lipid bilayer system. The plasma membrane is a complex system consisting of a mixture of phospholipids and proteins, where we have limited tools to control the lipid composition of the membrane. Also, in a cellular membrane direct effects of regulatory molecules are hard to differentiate from indirect effects through other proteins. To overcome these limitations, we have purified the TRPM8 protein, incorporated it into planar lipid bilayers, and showed that it exhibits menthol-activated PIP2- dependent activity. We also reconstituted TRPM8 from mammalian cells expressing TRPM8, via native membrane vesicles. These systems allow full control of the phospholipid content of the membrane, therefore serves as a unique tool to study fundamental questions in lipid gating of TRPM8 channels that could not be answered with other techniques. Our data will provide mechanistic insight into the regulation of temperature sensitive TRP channel, and may also serve as a basis for better local pain control.

描述（由申请人提供）：磷酸肌醇，如磷脂酰肌醇4,5-二磷酸（Ptdlns(4,5)P2或PIP2）调节许多（如果不是全部的话）瞬时受体电位（TRP）通道。我们将研究磷酸肌苷对冷敏感的TRPM8和热敏感的TRPV1的调控作用。我们将重点阐明这些脂质调节这两个通道的机制和生理重要性。TRPM8和TRPV1在感觉神经元中表达，在热感觉和伤害感受中发挥重要作用。在Aim 1中，我们将研究磷酸肌苷对TRPV1的调控。我们的初步数据和其他人的结果表明，PIP2对TRPV1通道施加双重控制，在某些情况下表现出抑制或刺激作用。我们的主要假设是，通过激活各种PLC同工异构体来消耗PIP2（和PIP）对通道有不同的影响，这取决于消耗的程度。为了验证这一假设，我们将在表达系统中结合新的和成熟的技术，并研究感觉神经元中的TRPV1。在Aim 2中，我们将研究PIP2在调节TRPM8电流中的生理作用。我们已经证明PIP2激活TRPM8，并且选择性地耗尽PIP2足以抑制这些通道。我们的假设是Ca2+介导的PLC激活和随后的PIP2耗竭导致TRPM8电流的脱敏。TRPM8也受到蛋白激酶C （PKC）激活剂的抑制，因此PKC介导的TRPM8抑制也可能是其脱敏的基础。为了阐明这两种替代机制之间的相互作用，并定义它们对脱敏的相对贡献，我们将使用以下方法。我们将使用各种工具测试PKC抑制剂和激活剂与PIP2增加或减少的关系，并测量TRPM8脱敏。实验将在哺乳动物表达系统和原生DRG神经元中进行。在Aim 3中，我们将研究TRPM8在脂质双分子层系统中的调控作用。质膜是一个复杂的系统，由磷脂和蛋白质的混合物组成，我们有有限的工具来控制膜的脂质组成。此外，在细胞膜中，调节分子的直接作用与通过其他蛋白质产生的间接作用很难区分。为了克服这些限制，我们纯化了TRPM8蛋白，将其整合到平面脂质双分子层中，并表明它具有薄荷醇激活的PIP2依赖活性。我们也通过天然膜泡从表达TRPM8的哺乳动物细胞中重组了TRPM8。这些系统可以完全控制膜的磷脂含量，因此可以作为一种独特的工具来研究TRPM8通道脂质门控的基本问题，这些问题是其他技术无法回答的。我们的数据将提供对温度敏感TRP通道调节的机制，也可能为更好地控制局部疼痛提供基础。