Ca2+ activated TMEM16 channels and their physiological roles in the brain

描述（由申请人提供）：Ca 2+参与细胞生命的各个方面。Ca ~（2+）激活的离子通道主要是Ca ~（2+）激活的K ~+（KCa）通道、Ca ~（2+）激活的Cl ~-通道和Ca ~（2+）激活的非选择性阳离子通道（CAN），它们感知细胞内Ca ~（2+）水平的变化，并积极调节细胞膜兴奋性、离子稳态和下游细胞信号级联。这些通道在循环和神经系统的生理学中大量表达并积极参与，并且与中风和许多神经系统疾病的病理生理学有关，例如癫痫、疼痛和运动障碍，包括共济失调和震颤。与KCa通道相比，CaCCs和CAN的了解要少得多，主要是由于其分子鉴定的困难。在2008年，我们的实验室鉴定了TMEM 16家族中的TMEM 16 A和16 B形成了长期寻找的CaCC。我最近证明了TMEM 16 F不是CaCC，而是形成了一种新的CAN通道，具有小的单通道电导（SCAN）和最小的阴离子渗透性。我们的体外和体内实验进一步说明，TMEM 16 F-SCAN通道是凝血过程中血小板中脂质紊乱所必需的，TMEM 16 F敲除小鼠在与Ca 2+依赖性磷脂酰丝氨酸（PS）暴露中血小板缺乏相关的颈动脉血栓形成中表现出出血缺陷和保护作用。该建议的重点是确定钙激活的TMEM 16通道的分子特性及其在血小板和小脑浦肯野细胞中的细胞功能。在目标I中，我将指出对离子选择性和Ca 2+结合重要的关键残基，以了解TMEM 16通道功能的分子机制。在目的II中，我将使用电生理学和体外/体内成像技术的组合，研究血小板和小脑浦肯野细胞中的TMEM 16 F-SCAN通道的生理功能。对这一提议的研究将有助于在分子水平上理解TMEM 16通道，帮助定义它们的生理功能，并为开发新的治疗方法提供见解，以靶向这些通道来预防和治疗神经系统疾病和中风。

DESCRIPTION (provided by applicant): Ca2+ is involved in every aspect of cellular life. Ca2+-activated ion channels, mainly Ca2+-activated K+ (KCa) channels, Ca2+-activated Cl- channels (CaCC) and Ca2+-activated non-selective cation (CAN channels), sense changes of internal Ca2+ level and actively regulate membrane excitability, ion homeostasis and downstream cell signaling cascade. These channels are abundantly expressed and actively involved in the physiology of the circulatory and nervous systems, and have been linked to pathophysiology of stroke and many neurological disorders, such as epilepsy, pain, and movement disorders including ataxia and tremor. Compared with KCa channels, CaCCs and CANs are much less understood mainly due to the difficulties involved in their molecular identification. In 2008, our laboratory identified that TMEM16A and 16B in the TMEM16 family form the long- sought CaCCs. I recently demonstrated that TMEM16F, instead of being a CaCC, forms a novel CAN channel with small single channel conductance (SCAN) and minimal anion permeability. Our in vitro and in vivo experiments further illustrate that TMEM16F-SCAN channels are required for lipid scrambling in platelets during blood coagulation and TMEM16F knockout mice exhibit bleeding defects and protection in carotid artery thrombosis associated with platelet deficiency in Ca2+-dependent phosphatidylserine (PS) exposure. The focus of this proposal is to define the molecular properties of Ca2+-activated TMEM16 channels and their cellular functions in platelets and in cerebellar Purkinje cells. In Aim I, I will pinpoint the key residues that are important for ion selectivity and Ca2+ binding to understand the molecular mechanisms underlying the functions of TMEM16 channels. In Aim II, I will investigate the physiological functions of the TMEM16F-SCAN channels in platelets and cerebellar Purkinje cells using a combination of electrophysiology and in vitro/in vivo imaging techniques. Studies of this proposal will facilitate the understanding of TMEM16 channels at the molecular level, help define their physiological functions, and provide insights to develop new therapeutics to target these channels to prevent and treat neurological disorders and stroke.