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

描述（由申请人提供）：Ca2+参与细胞生命的各个方面。Ca2+激活离子通道主要是Ca2+激活K+ (KCa)通道、Ca2+激活Cl-通道（CaCC）和Ca2+激活非选择性阳离子（CAN）通道，它们感知体内Ca2+水平的变化，积极调节膜兴奋性、离子稳态和下游细胞信号级联。这些通道大量表达并积极参与循环系统和神经系统的生理活动，并与中风和许多神经系统疾病的病理生理有关，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，人们对CaCCs和can通道的了解要少得多，这主要是由于它们的分子鉴定存在困难。2008年，我们的实验室发现TMEM16家族中的TMEM16A和16B形成了长期寻找的CaCCs。我最近证明了TMEM16F不是CaCC，而是形成一种具有小单通道电导（SCAN）和最小阴离子渗透性的新型CAN通道。我们的体外和体内实验进一步表明，TMEM16F- scan通道是凝血过程中血小板脂质混乱所必需的，TMEM16F敲除小鼠在Ca2+依赖性磷脂酰丝氨酸（PS）暴露中表现出出血缺陷和与血小板缺乏相关的颈动脉血栓形成保护。本研究的重点是确定Ca2+激活的TMEM16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Aim I中，我将指出对离子选择性和Ca2+结合很重要的关键残基，以了解TMEM16通道功能的分子机制。在Aim II中，我将使用电生理学和体外/体内成像技术的结合来研究血小板和小脑浦肯野细胞中TMEM16F-SCAN通道的生理功能。这项研究将有助于在分子水平上理解TMEM16通道，帮助确定其生理功能，并为开发针对这些通道的新疗法提供见解，以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.