ABSTRACT Chloride channels contribute to airway health and disease by modulating airway secretion and mucociliary function. We discovered that the channel regulator CLCA1 is a potent and specific potentiator of the TMEM16A calcium-activated chloride channel, but there remains a major gap in understanding how this pair influences airway biology. In order to fill this gap and develop therapies for mucus-obstructive diseases that target CLCA1-mediated TMEM16A potentiation, the following are required: 1) a comprehensive understanding of TMEM16A isoform expression patterns and biophysical properties in diseased airway; 2) an understanding of how CLCA1-TMEM16A impact mucus properties in disease; and 3) the structural basis for CLCA1 potentiation of TMEM16A. These points are addressed by each aim of this project. Since it is challenging to fully investigate the potential mucus-modifying abilities of other channels in normal human airways due to the dominant activity of cystic fibrosis transmembrane regulator (CFTR), we will utilize human cystic fibrosis (CF) models to study CLCA1-TMEM16A biology in more depth. Investigating this system in CF also provides an opportunity to evaluate other channels that may compensate for CFTR as therapeutic targets for patients unresponsive to CFTR modulators. We have made a series of technical advances to facilitate our proposed studies investigating the role this pair plays in human airway biology. First, we discovered a minimal domain of CLCA1 (CLCA1 VWA) that potentiates TMEM16A in cells, is stable and can be purified in large quantities for functional studies. We have also devised ex vivo airway and human cellular models, using cystic fibrosis (CF) airway tissue, primary CF cells and cell lines for detailed studies of TMEM16A activation in regulating mucociliary function. Last, we have developed the tools required to determine the molecular structure of the CLCA1- TMEM16A complex. To address the function of the CLCA1-TMEM16A system in human airway health and disease, we propose a multidisciplinary investigation that incorporates banked human specimens, single cell and spatial ‘omics, multicellular human models and state-of-the-art structural approaches. In Aim 1, we will use single-nucleus RNAseq and merFISH to investigate expression dynamics of TMEM16A isoforms in CF disease. In Aim 2, we will potentiate TMEM16A with CLCA1 VWA in CF airway tissue and cellular models to characterize the impact of TMEM16A activation on beneficial mucus properties. In Aim 3, we will define the molecular mechanism for CLCA1 activation of TMEM16A using cryo-electron microscopy (cryo-EM). These studies will examine the impact of CLCA1 potentiation of TMEM16A in human airway disease, which will provide critical insight into broader regulation of TMEM16 family members relevant to lung biology and produce the molecular framework to develop much needed novel therapeutics for muco-obstructive diseases.

PROJECT NARRATIVE (RELEVANCE): We identified calcium-activated chloride channel regulator 1 (CLCA1) protein as a specific potentiator of the calcium-activated chloride channel TMEM16A, however, this channel and modulator play an underappreciated role in human airway biology, in part due to the challenges of studying the system in the setting of broader airway channel activity. Cystic fibrosis represents an ideal context to study the CLCA1-TMEM16A system in human airway disease, allowing for both the unmasked investigation of TMEM16A channel activity (in the absence of CFTR) and the opportunity to evaluate a unique potential therapeutic angle for patients unresponsive to CFTR modulator therapy. In this proposal we investigate the cellular and tissue expression patterns of TMEM16A in human disease, the potential beneficial impact of CLCA1 on TMEM16A-mediated airway function, and determine the molecular features of the CLCA1-TMEM16A interaction in order to produce the molecular framework needed to develop new therapeutics for diseases hallmarked by mucus obstruction, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).