TRP and AQP Channels: Modulation of Function, Raft Location by Membrane Lipids

描述(申请人提供)：瞬时受体电位香草酸1通道(TRPV1)是伤害性感受器的重要组成部分，也是膀胱的机械感受器和神经元的渗透感受器。水通道蛋白AQP-4(AQP-4)存在于中枢神经系统的神经胶质细胞中，对脑内水的稳态起着至关重要的作用。尽管TRPV1和AQP-4在正常的身体功能和一些病理条件下都是重要的，但目前关于这些通道的功能是如何被双层组成或其膜微域位置改变的知之甚少。这些都是基本问题，因为许多离子通道的功能已经被证明被特定的膜脂改变，如磷脂酰肌醇-4，5-二磷酸(PIP2)，以及受胆固醇存在控制的膜材料性质。此外，一些通道被隔离在富含胆固醇的质膜微域中，称为RAFT。这项应用使用膜片钳电生理学和微管吸入法分别测量双层胆固醇浓度对TRPV1和AQP-4通道特性的影响。此外，包括共聚焦显微镜和冷冻断裂电子显微镜在内的各种技术将被用来确定TRPV1和AQP-4的微域位置，并测试将通道隔离到筏中的可能机制。我们的假设是：(1)特定的蛋白质-脂质和蛋白质-蛋白质相互作用，包括通道的同源齐聚，是在微域之间分类通道的关键因素，(2)双层材料的性质(由磷脂碳氢链组成和胆固醇含量控制)调节TRPV1和AQP-4的功能，(3)酰化的多碱性细胞质蛋白可以将关键调节脂如PIP2隔离到双层细胞膜的细胞质小叶中的RAFT结构域中。公共卫生相关性：水通道AQP-4和瞬时受体电位通道TRPV1在正常身体功能和一些病理情况下至关重要。AQP-4在大脑的神经胶质细胞中表达，与脑水肿和各种脑部疾病有关。TRPV1存在于身体的许多区域，包括疼痛受体，与肠道疾病、接触性皮炎以及一般的疼痛有关。

DESCRIPTION (provided by applicant): The Transient Receptor Potential Vanilloid 1 channel (TRPV1) is a key component of nociceptors and is also a mechanosensor in the urinary bladder and osmosensor in neurons. The water channel Aquaporin-4 (AQP-4) is found in glial cells in the CNS and is critical for water homeostasis in the brain. Although both TRPV1 and AQP-4 are important in normal body function and several pathological conditions, little is currently known about how the functions of these channels are modified by bilayer composition or their membrane microdomain locations. These are fundamental issues because the functions of many ion channels have been shown to be modified by specific membrane lipids such as phosphatidylinositol-4,5 bisphosphate (PIP2), as well as by membrane material properties controlled by the presence of cholesterol. Moreover, some channels are sequestered into cholesterol-rich plasma membrane microdomains called rafts. This application uses patch clamp electrophysiology and micropipette aspiration to measure the role of bilayer cholesterol concentration on the channel properties of TRPV1 and AQP-4, respectively. In addition, a variety of techniques, including confocal microscopy and freeze-fracture electron microscopy, will be employed to determine the microdomain locations of TRPV1 and AQP-4 and test possible mechanisms for the sequestering of channels into rafts. Our hypotheses are that: (1) specific protein-lipid and protein- protein interactions, including homo-oligomerization of the channels, are key factors in sorting channels between microdomains, (2) bilayer material properties (as controlled by phospholipid hydrocarbon chain composition and cholesterol content) modulate the function of both TRPV1 and AQP-4, and (3) acylated polybasic cytoplasmic proteins can sequester key regulatory lipids such as PIP2 into raft domains in the cytoplasmic leaflet of membrane bilayers. PUBLIC HEALTH RELEVANCE: The water channel AQP-4 and the Transient Receptor Potential channel TRPV1 are critically important in normal body function and several pathological conditions. AQP-4, expressed in glial cells in the brain, is implicated in brain edema and a variety of cerebral disorders. TRPV1, found in many regions of the body including pain receptors, is implicated in bowel disease, contact dermatitis, as well as pain in general.