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

项目摘要/摘要 这个K08导师职业发展奖将帮助我实现成为一名独立人士的目标 内科医生-科学家，擅长研究钙激活的氯离子通道在哮喘病理中的作用。 哮喘的发病机制包括慢性呼吸道炎症、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了钙离子的拮抗作用- 激活的氯通道TMEM16A通过阻断钙离子通道而松弛气道平滑肌(ASM 质膜和肌浆网(SR)。目前的提案将通过以下方式扩大这项研究 TMEM16A在ASM和呼吸道的表达、功能、机制及治疗潜力的研究 上皮组织。在ASM中，我们将通过共定位的方式证明TMEM16A在SR膜上表达 用免疫组织化学方法研究TMEM16A在阻断两侧SR钙离子流动中的作用 用钙显像法测定小鼠肺切片中兰尼定和IP3受体的含量，并证实急性吸入 TMEM16A拮抗剂可在体内减轻支气管收缩。在人类呼吸道上皮细胞中，我们还将 证明TMEM16A在杯状细胞产生粘液和分泌炎性细胞因子中的作用 包括人类上皮细胞和ASM。我们将演示TMEM16A拮抗剂降低MUC5AC(和 呼吸道粘液的重要成分)和Th2炎性细胞因子。因此，我们将 证明TMEM16A拮抗剂除了作为一种急性支气管松弛剂的有效性外，还具有 作为一种慢性药物的治疗潜力，以减少粘液产生和Th2炎症。这项提议将 利用共聚焦显微镜、小鼠外周肺切片同时含钙等技术 振荡测量，IP3去化，一种新的具有可诱导的平滑肌特异性敲击的遗传模型- 取出TMEM16A，siRNA敲除，进行体内气道屈曲阻力分析和组织学分析。一个 由成功的医生和科学家组成的指导委员会在每一个方面提供科学专业知识 研究计划包括离子通道、Th2肺炎症、上皮细胞黏液产生和临床哮喘。 一项全面的校内外课程和培训计划，并辅之以额外的 在肺组织病理学、钙成像和统计学方面的顾问专业知识将扩大研究培训和 职业发展。该研究主题是一条通往上皮和上皮组织领域独立研究的理想之路 免疫细胞生物学及TMEM16A调节SR/ER钙释放的细胞生物学中心作用这 对于新的研究方向的指导、培训和机会是通往独立道路的理想计划 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.