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

说明(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调节。长期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。作为高血压、囊性纤维化和其他疾病的潜在治疗靶点，人们对CaCCs产生了浓厚的兴趣，人们已经做出了广泛的努力来确定CaCCs的分子特性。由于几个已报道的候选分子的通道特性和表达模式与天然CaCC的通道特性和表达模式不匹配，几年前我们开始了表达克隆的工作，导致了非洲爪哇和小鼠TMEM16A以及小鼠TMEM16B为CACC亚单位。2008年，与我们的研究几乎同时发表了两项研究，这三项研究都得出了相同的结论，即哺乳动物的TMEM16A对应于CACC。到目前为止，对TMEM16A基因敲除小鼠的研究表明，TMEM16A在外分泌腺和呼吸道上皮细胞的CACC中是必需的。随着TMEM16家族作为一个新的离子通道家族的出现，即使是最基本的问题也是开放的，现在分子和遗传学研究都可以解决：钙是如何激活CACC的？CACC通道中有多少个TMEM16A亚基？TMEM16A是否对应于背根神经节(DRG)感觉神经元中的CACC？TMEM16A在失神经后是否上调，如果是，它是否影响神经再生和/或神经病理性疼痛？失神经可导致DRG神经元CACC表达上调，这是神经元性CACC的最好例子之一，因此本研究的一个具体目的是研究TMEM16A在有或不有坐骨神经病变的DRG神经元CACC中的参与，并探讨TMEM16A在神经损伤后或糖尿病、疱疹和癌症等疾病后发展的疼痛敏感性和神经病理性疼痛中的潜在作用。为了更好地了解细胞内钙离子如何控制CACC通道的流量和活性，我们将对TMEM16A进行生化和突变研究，TMEM16A可以异源表达来产生CACC。我们还将使用多种方法来确定CACC的化学计量--这是更好地了解CACC的功能和监管以及CaCC的多样性的一个重要问题。 与公共健康相关：钙激活的氯离子通道(CaCC)具有重要的生理功能，包括调节中枢和外周神经系统神经元的信号处理。最近建立了TMEM16家族的“功能未知的跨膜蛋白”，作为一个新的离子通道家族，包括TMEM16A和TMEM16B作为CACC亚基，我们建议使用异源表达系统来研究CACC通道的工作原理。我们还将描述背根神经节(DRG)内源性CACC的特征，并研究TMEM16A在疼痛敏感性和神经病理性疼痛中的作用。考虑到DRG感觉神经元的CACC在失神经后上调，我们设计了实验，为未来研究CACC在神经损伤后发生的神经再生和/或神经病理性疼痛中的潜在作用奠定基础，这些疼痛发生在糖尿病、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.