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.