Molecular, Genetic & Physiological Studies of Calcium-activated Chloride Channels

描述（由申请人提供）：钙激活氯离子通道（CaCC）具有重要的生理功能，包括调节各种中枢和外周神经元的信号处理。例如，CaCC有助于感觉输入的信号放大和感觉神经元和中枢神经元的兴奋性的调节。长期的目标是了解这些通道是如何工作的，以及它们是如何调节神经元活动的。反映了对CaCC作为高血压、囊性纤维化和其他疾病的潜在治疗靶点的强烈兴趣，已经进行了广泛的努力来确定CaCC的分子身份。由于几种报道的分子候选物的通道特性和表达模式与天然CaCC的不匹配，几年前我们开始进行表达克隆，导致非洲爪蟾和小鼠TMEM 16 A以及小鼠TMEM 16 B被鉴定为CaCC亚基。2008年，与我们的研究同时发表了两项并行研究，所有三项研究都得出了相同的结论，即哺乳动物TMEM 16 A对应于CaCC。到目前为止，TMEM 16 A敲除小鼠的几项研究已经表明，TMEM 16 A是外分泌腺和气道上皮中CaCC所必需的。随着TMEM 16家族的“功能未知的跨膜蛋白”作为一种新的离子通道家族出现，即使是最基本的问题也是开放的，现在可以进行分子和遗传学研究：钙如何激活CaCC？CaCC通道中存在多少个TMEM 16 A亚基？TMEM 16 A是否对应于背根神经节（DRG）感觉神经元中的CaCC？TMEM 16 A在去神经支配后是否上调，如果是，它是否影响神经再生和/或神经性疼痛？去神经支配引起DRG神经元的CaCC上调-神经元CaCC的最佳实例之一，因此本提议的一个具体目的是检查TMEM 16 A在有或没有坐骨神经损伤的DRG神经元的CaCC中的参与，并探索TMEM 16 A在疼痛敏感性和神经性疼痛中的潜在作用，其在神经损伤后或在如糖尿病、疱疹和癌症的疾病中发展。为了更好地理解CaCC通道的交通和活性如何受到胞质钙的控制，我们将对TMEM 16 A进行生物化学和诱变研究，TMEM 16 A可以异源表达以产生CaCC。我们还将使用多种方法的组合来确定CaCC的化学计量-这是一个重要的问题，可以更好地了解CaCC的功能和调节以及CaCC的多样性。 公共卫生相关性：钙激活氯离子通道（CaCCs）具有重要的生理功能，包括调节中枢和外周神经系统中神经元的信号处理。最近建立了TMEM 16家族的“未知功能的跨膜蛋白”作为一种新的离子通道家族，其中包括TMEM 16 A和TMEM 16 B作为CaCC亚基，我们建议使用异源表达系统来研究CaCC通道如何工作。我们还将表征背根神经节（DRG）内源性CaCC，并检查TMEM 16 A在疼痛敏感性和神经性疼痛中的作用。考虑到DRG感觉神经元的CaCC在去神经支配后上调，我们设计了我们的实验，为将来研究CaCC在神经再生和/或神经病理性疼痛中的潜在作用奠定基础，神经损伤后，糖尿病，带状疱疹注射和癌症等疾病中，也可能由化疗诱导。

DESCRIPTION (provided by applicant): Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of a variety of central and peripheral neurons. For example, CaCC contributes to signal amplification of sensory inputs and regulation of excitability of both sensory and central neurons. The long- term objectives are to understand how these channels work, and how they regulate neuronal activity. Reflecting an intense interest in CaCCs as potential therapeutic targets for hypertension, cystic fibrosis and other diseases, there have been extensive efforts to determine the molecular identity of CaCCs. Because the channel properties and expression patterns of several reported molecular candidates do not match those for native CaCCs, several years ago we began the undertaking for expression cloning, leading to the identification of Xenopus and mouse TMEM16A, as well as mouse TMEM16B as CaCC subunits. In 2008, two concurrent studies were published around the same time as ours, and all three reached the same conclusion that mammalian TMEM16A corresponds to CaCC. By now, several studies of TMEM16A knockout mice have shown that TMEM16A is required for CaCC in exocrine glands and airway epithelia. With the TMEM16 family of "transmembrane proteins with unknown function" emerging as a novel family of ion channels, even the most basic questions are open and now amenable to molecular and genetic studies: How does calcium activate CaCC? How many TMEM16A subunits are present in a CaCC channel? Does TMEM16A correspond to the CaCC in sensory neurons of the dorsal root ganglion (DRG)? Is TMEM16A up regulated following denervation and, if so, does it influence nerve regeneration and/or neuropathic pain? Denervation causes up regulation of CaCC of DRG neurons - one of the best examples of neuronal CaCC, hence one specific aim of this proposal is to examine the involvement of TMEM16A in CaCC of DRG neurons with or without sciatic nerve lesion, and to explore potential roles of TMEM16A in pain sensitivity and neuropathic pain, which develops after nerve injury or in diseases like diabetes, herpes, and cancer. To better understand how CaCC channel traffic and activity may be controlled by cytosolic calcium, we will carry out biochemical and mutagenesis studies of TMEM16A, which can be heterogeneously expressed to generate CaCC. We will also use a combination of approaches to determine the CaCC stoichiometry - an important question for better appreciation of CaCC function and regulation, and the diversity of CaCCs. PUBLIC HEALTH RELEVANCE: Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of neurons in the central and peripheral nervous system. Having recently established the TMEM16 family of "transmembrane proteins of unknown function" as a novel ion channel family that includes TMEM16A and TMEM16B as CaCC subunits, we propose to use heterologous expression systems to study how CaCC channels work. We will also characterize CaCC endogenous to the dorsal root ganglion (DRG) and examine the role of TMEM16A in pain sensitivity and neuropathic pain. Bearing in mind that CaCC of DRG sensory neurons is up regulated after denervation, we have designed our experiments to lay the groundwork for future studies of the potential roles of CaCC in nerve regeneration and/or neuropathic pain, which develops after nerve injury, in diseases like diabetes, herpes zoster injection and cancer, and may also be induced by chemotherapy.