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

分子、遗传

• 批准号: 8039058
• 负责人: LILY Y JAN
• 金额: $ 30.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8039058
• 关键词: Action Potentials; Affect; Afferent Neurons; Ambystoma; Axotomy; Binding; Biochemical; Bioinformatics; Calcium; Calmodulin; Cell physiology; Chloride Channels; Cohort Studies; Collaborations; Complement; Complex; Cystic Fibrosis; Denervation; Diabetes Mellitus; Disease; Dominant-Negative Mutation; Epithelial Cells; Exocrine Glands; Family; Feedback; Future; Generations; Green Algae; Herpes zoster disease; Hypertension; Injection of therapeutic agent; Integral Membrane Protein; Interleukin-4; Ion Channel; Knock-out; Knockout Mice; Knowledge; Malignant Neoplasms; Mammals; Mechanics; Messenger RNA; Microarray Analysis; Microscopy; Mind; Molecular; Molecular Genetics; Mus; Mutagenesis; Mutation; Nerve Regeneration; Neurons; Nociception; Oocytes; Organism; Pain; Pattern; Peptides; Peripheral; Peripheral Nervous System; Physiological; Potassium Channel; Property; Protons; Publishing; Regulation; Reporting; Role; Sensory; Signal Transduction; Spinal Ganglia; System; Testing; Time; Transcript; Up-Regulation; Work; Xenopus; Xenopus oocyte; airway epithelium; allodynia; chemotherapy; computerized data processing; design; expression cloning; interest; mutant; nerve injury; novel; painful neuropathy; postnatal; prevent; research study; response; sciatic nerve lesion; single molecule; stoichiometry; therapeutic target; trafficking; voltage

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.

描述（由申请人提供）：钙活化的氯化物通道（CACC）具有重要的生理功能，包括调节各种中枢和周围神经元的信号处理。例如，CACC有助于感官输入的信号扩增以及感觉和中央神经元的兴奋性调节。长期目标是了解这些渠道的工作原理以及它们如何调节神经元活动。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标的强烈兴趣，因此已经做出了广泛的努力来确定CACC的分子认同。由于几种报告的分子候选者的通道特性和表达模式与天然CACC的频道和表达模式不匹配，因此几年前，我们开始进行表达克隆的承诺，从而鉴定了Xenopus和小鼠TMEM16A，以及小鼠TMEM16B作为CACC亚基。 2008年，与我们的同时发表了两项并发研究，这三个研究得出的结论与哺乳动物TMEM16A相同的结论与CACC相对应。到现在为止，对TMEM16A敲除小鼠的几项研究表明，在外分泌腺和气道上皮中，CACC需要TMEM16A。随着TMEM16的“具有未知功能的跨膜蛋白”的家族，即使是一个新的离子通道家族，即使是最基本的问题也是开放的，现在可以适合分子和遗传研究：钙如何激活CACC？ CACC通道中存在多少个TMEM16A亚基？ TMEM16A是否对应于背根神经节（DRG）的感觉神经元中的CACC？ TMEM16A是否在神经支配后调节，如果是的，它是否会影响神经再生和/或神经性疼痛？ 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糖尿病，疱疹和癌症。为了更好地了解CACC通道的流量和活动如何通过胞质钙控制，我们将对TMEM16A进行生化和诱变研究，可以异构表达以生成CACC。我们还将结合使用多种方法来确定CACC化学计量学 - 以更好地欣赏CACC功能和调节以及CACC的多样性。 公共卫生相关性：钙活化的氯化物通道（CACC）具有重要的生理功能，包括调节中枢和周围神经系统中神经元的信号处理。最近将“未知功能的跨膜蛋白”的TMEM16家族作为一种新型离子通道家族，其中包括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。我们还将表征CACC内源性的背根神经节（DRG），并检查TMEM16A在疼痛敏感性和神经性疼痛中的作用。请记住，DRG感觉神经元的CACC在进行改造后受到调节，我们设计了实验，为未来研究CACC在神经再生和/或神经性疼痛中的潜在作用的研究为神经损伤后发生的潜在作用而言，在神经损伤后发展，在糖尿病之类的疾病中，伴随着糖尿病，疱疹卵血样注射和癌症和癌症，也可以通过化学症状。