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 includingion 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.
Perioperative bronchospasm in the OR and ICU remains an important clinical problem for anesthesiologists, with poorly controlled asthma identified as a major risk factor. This proposal will demonstrate that the antagonism of the calcium-activated chloride channel TMEM16A has multiple beneficial effects on asthma including bronchodilation, reduced mucus production and reduced inflammation. The cellular mechanisms uncovered will have important implications in other organ systems, and will demonstrate TMEM16A's important role in smooth muscle contraction, secretory events and inflammation.