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

描述(申请人提供)：钙离子参与细胞生活的方方面面。钙激活的离子通道，主要是钙激活的钾通道(Kca)、钙激活的氯离子通道(CACC)和钙激活的非选择性阳离子通道(CaN通道)，可以感知细胞内钙离子水平的变化，并主动调节膜兴奋性、离子动态平衡和下游细胞信号级联反应。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学，并与中风和许多神经疾病的病理生理学有关，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，对CaCCs和CaN的了解较少，主要是因为它们的分子鉴定存在困难。2008年，我们的实验室鉴定了TMEM16家族中的TMEM16A和16B形成了长期寻找的CaCC。我最近证明，TMEM16F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表明，在血液凝固过程中，TMEM16F扫描通道对于血小板中的脂质扰乱是必需的，并且TMEM16F基因敲除小鼠在钙依赖的磷脂酰丝氨酸(PS)暴露中表现出出血缺陷和对与血小板缺乏相关的颈动脉血栓形成的保护作用。这项建议的重点是确定钙激活的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。在目标I中，我将精确定位对离子选择性和钙结合至关重要的关键残基，以了解TMEM16通道功能的分子机制。在AIM II中，我将结合电生理学和体外/体内成像技术，研究TMEM16F扫描通道在血小板和小脑浦肯野细胞中的生理功能。对这一提议的研究将有助于在分子水平上了解TMEM16通道，有助于确定它们的生理功能，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.