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

描述（由申请人提供）：瞬时受体电位香草样蛋白1通道（TRPV1）是伤害感受器的关键组成部分，也是膀胱中的机械传感器和神经元中的渗透传感器。水通道蛋白-4 （AQP-4）存在于中枢神经系统的神经胶质细胞中，对大脑的水稳态至关重要。尽管TRPV1和AQP-4在正常机体功能和几种病理条件中都很重要，但目前对这些通道的功能如何被双分子层组成或它们的膜微结构域位置所改变知之甚少。这些都是基本的问题，因为许多离子通道的功能已经被特定的膜脂修饰，如磷脂酰肌醇-4,5二磷酸（PIP2），以及由胆固醇存在控制的膜材料特性。此外，一些通道被隔离在富含胆固醇的质膜微域（称为筏）中。本应用分别采用膜片钳电生理和微管抽吸测量双分子层胆固醇浓度对TRPV1和AQP-4通道特性的影响。此外，将采用各种技术，包括共聚焦显微镜和冷冻断裂电子显微镜，来确定TRPV1和AQP-4的微结构域位置，并测试通道隔离到筏中的可能机制。我们的假设是：(1)特定的蛋白-脂质和蛋白-蛋白相互作用，包括通道的同质寡聚化，是微结构域之间通道分选的关键因素；(2)双层材料特性（受磷脂烃链组成和胆固醇含量控制）调节TRPV1和AQP-4的功能；(3)酰基化的多碱性细胞质蛋白可以将PIP2等关键调节脂质隔离到膜双层细胞质小叶的筏结构域。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条件中至关重要。AQP-4在脑胶质细胞中表达，与脑水肿和多种脑疾病有关。TRPV1存在于包括疼痛感受器在内的身体许多区域，与肠道疾病、接触性皮炎以及一般疼痛有关。

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.