Antagonism of the Calcium -Activated Chloride Channel TMEM16A; Beneficial Therapeutic Effects on Airway Epithelium and Smooth Muscle

项目总结/摘要 这个建议的K 08指导职业发展奖将帮助我实现我的目标，成为一个独立的 在钙激活氯离子通道在哮喘病理学中的作用方面具有专业知识的医生-科学家。 哮喘的发病机制涉及慢性气道炎症、粘液分泌增加和肥大 和气道平滑肌的高反应性。我们的实验室已经证明钙的拮抗作用- 激活的氯离子通道TMEM 16 A通过阻断钙流来松弛气道平滑肌（ASM）， 质膜和肌浆网（SR）。目前的建议将扩大这项研究， 证实了TMEM 16 A在ASM和气道中的表达、功能、机制和治疗潜力 上皮在ASM中，我们将证明TMEM 16 A通过共定位在SR膜上表达 通过免疫组织化学检测SR蛋白，研究TMEM 16 A在阻断SR钙流中的作用， 用钙离子显像法测定小鼠肺片兰尼碱和IP 3受体，并证实急性吸入 的TMEM 16 A拮抗剂在体内减弱支气管收缩。在人类气道上皮中，我们还将 证明了TMEM 16 A在杯状细胞产生粘液和炎性细胞因子分泌中的作用， 包括人类上皮和ASM。我们将证明TMEM 16 A拮抗作用降低MUC 5AC（一种免疫抑制剂）。 气道粘液的重要组分）和Th 2炎性细胞因子。因此，我们将 证明除了作为急性支气管舒张剂的有效性之外，TMEM 16 A拮抗作用还具有 作为慢性药物的治疗潜力，以减少粘液产生和Th 2炎症。这项建议会 利用诸如共聚焦显微镜、小鼠外周肺切片和同时钙离子注入的技术， 振荡测量，IP 3解开，一种新的遗传模型与诱导平滑肌特异性敲- TMEM 16 A、siRNA敲低、气道阻力的体内灵活性分析和组织学分析。一 指导委员会由成功的医生科学家组成，提供科学专业知识的各个方面， 研究计划包括离子通道、Th 2肺部炎症、上皮细胞粘液产生和临床哮喘。 一个全面的校内和校外课程和培训计划，辅之以额外的 肺组织病理学、钙成像和统计学方面的顾问专业知识将扩大研究培训， 职业发展。该研究课题是理想的途径，在上皮和 免疫细胞生物学和TMEM 16 A调节SR/ER钙释放的细胞生物学中的中心作用。这 指导，培训和新的研究方向的机会是一个理想的计划，一个独立的道路， 研究生涯。

PROJECT SUMMARY/ABSTRACT This proposed K08 Mentored Career Development Award will help me reach my goal to become an independent physician-scientist with expertise in the role of calcium-activated chloride channels in the pathology of asthma. The pathogenesis of asthma involves chronic airway inflammation, increased mucus secretion, and hypertrophy and hyperresponsiveness of airway smooth muscle. Our lab has demonstrated that antagonism of the calcium- activated chloride channel TMEM16A relaxes airway smooth muscle (ASM) through blockade of calcium flux at both the plasma membrane and sarcoplasmic reticulum (SR). The current proposal will expand this research by demonstrating TMEM16A's expression, function, mechanisms and therapeutic potential in both ASM and airway epithelium. In ASM, we will demonstrate that TMEM16A is expressed on the SR membrane by co-localization with SR proteins by immunohistochemistry, investigate TMEM16A's role in blocking SR calcium flux from both the ryanodine and IP3 receptor in murine lung slices with calcium imaging, and demonstrate that acute inhalation of TMEM16A antagonists attenuates bronchoconstriction in vivo. In human airway epithelium, we will also demonstrate TMEM16A's role in mucus production by goblet cells and secretion of inflammatory cytokines by both human epithelium and ASM. We will demonstrate TMEM16A antagonism decreases MUC5AC (an important component of airway mucus) and Th2 inflammatory cytokines by qPCR and ELISA. Thus, we will demonstrate that in addition to its effectiveness as an acute bronchorelaxant, TMEM16A antagonism also has therapeutic potential as a chronic drug to decrease mucus production and Th2 inflammation. This proposal will utilize techniques such as confocal microscopy, mouse peripheral lung slices with simultaneous calcium oscillation measurements, IP3 uncaging, a novel genetic model with an inducible smooth muscle specific knock- out of TMEM16A, siRNA knockdown, in vivo flexivent analysis of airway resistance and histologic analysis. A mentoring committee composed of successful physician-scientists offer scientific expertise in each aspect of the research plan including ion channels, Th2 lung inflammation, epithelial cell mucus production and clinical asthma. A comprehensive plan of intramural and extramural coursework and training complemented by additional consultant expertise in lung histopathology, calcium imaging and statistics will expand research training and career development. The research topic is ideal for a path to independent research in areas of epithelial and immune cell biology and the central role in cell biology of TMEM16A modulation of SR/ER calcium release. This mentoring, training and opportunities for novel research directions are an ideal plan for a path to an independent research career.