Lipid Regulation of Transient Receptor Potential Channels

说明（由申请人提供）：磷酸肌醇，如磷脂酰肌醇4，5-二磷酸（PtdIns（4，5）P2或PIP 2）调节许多（如果不是全部）瞬时受体电位（TRP）通道。我们将研究磷酸肌醇对冷敏感性TRPM 8和热敏感性TRPV 1的调节。我们将着重阐明这两个通道的调节机制和生理重要性，这些脂质。TRPM 8和TRPV 1在感觉神经元中表达，并且在温度感觉和伤害感受中起重要作用。在目的1中，我们将研究磷酸肌醇对TRPV 1的调节。我们的初步数据和其他人的结果表明，PIP 2对TRPV 1通道施加双重控制，在某些情况下显示出抑制或刺激作用。我们的主要假设是，通过激活各种PLC亚型消耗PIP 2（和PIP）对通道具有不同的影响，这取决于消耗的程度。为了验证这一假设，我们将联合收割机结合表达系统中新颖且成熟的技术，并研究感觉神经元中的TRPV 1。在目的2中，我们将研究PIP 2在TRPM 8电流调节中的生理作用。我们已经表明，PIP 2激活TRPM 8，并且选择性耗尽PIP 2足以抑制这些通道。我们的假设是，Ca 2+介导的PLC的激活和随后的PIP 2的耗尽导致TRPM 8电流的脱敏。TRPM 8也被蛋白激酶C（PKC）的激活剂抑制，因此PKC介导的TRPM 8的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，并确定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的抑制剂和激活剂与增加或减少PIP 2的结合，并测量TRPM 8脱敏。实验将在哺乳动物表达系统和天然DRG神经元中进行。在目标3中，我们将研究TRPM 8在脂质双层系统中的调节。质膜是由磷脂和蛋白质的混合物组成的复杂系统，其中我们具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很难与通过其他蛋白质的间接作用区分开来。为了克服这些局限性，我们已经纯化了TRPM 8蛋白，将其纳入平面脂质双层，并表明它表现出薄荷醇激活的PIP 2依赖性活性。我们还通过天然膜囊泡从表达TRPM 8的哺乳动物细胞重建TRPM 8。这些系统允许完全控制膜的磷脂含量，因此作为一个独特的工具，研究TRPM 8通道的脂质门控的基本问题，不能用其他技术回答。我们的研究结果将为温度敏感性TRP通道的调控机制提供理论依据，也可能为更好地控制局部疼痛提供依据。

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.