Plasma membrane cation channels play critical roles in lung health: The epithelial Na+ channel (ENaC) is expressed in pulmonary epithelia where it regulates fluid homeostasis and mucus clearance.  The Orai1 Ca2+ channel is ubiquitously expressed and controls gene expression and inflammation, to list but two of its functions.  SPLUNC1 (gene name BPIFA1) is a 256 amino acid long protein that is secreted from airway surface epithelia and glands into the lung lining fluid.  We were initially interested in identifying secreted proteins that could regulate ENaC in airway epithelia.  Using a proteomic screen, we identified SPLUNC1 as a protein that could interact with ENaC. Subsequent studies confirmed that SPLUNC1 bound to and internalized ENaC to reduce Na+ and fluid absorption1.  Fluorescence resonance energy transfer (FRET) studies demonstrated that extracellular SPLUNC1 binding to ENaC caused intracellular changes in ENaC (i.e. allostery).  We also found that SPLUNC1 binding caused the ubiquitin ligase NEDD4-2 to ubiquitinate ENaC and send it to lysosomes for degradation.  Indeed, immobilized ENaC concatamers, or expression of a dominant negative form of NEDD4-2 were no longer internalized2.  We found that a region in human SPLUNC1’s N-terminus, historically called the S18 region, was responsible for the ENaC-mediated inhibition, and peptides that corresponded to this region fully replicated this inhibition.  Murine SPLUNC1 does not express the S18 region and cannot regulate ENaC.  However, we noticed that SPLUNC1-/- mice exhibited both spontaneous airway hyperreactivity and increased ex vivo airway smooth muscle contraction, which could be rescued with recombinant SPLUNC13. Further study indicated that SPLUNC1 inhibited Orai1-dependent, store operated Ca2+ entry in both human and murine tissues.  However, while ENaC inhibition was mediated by SPLUNC1’s N-terminus, Orai1 inhibition was mediated by SPLUNC1’s C-terminus.  Surprisingly, SPLUNC1’s C-terminus induced NEDD4-2-dependent ubiquitination, internalization and lysosomal degradation of Orai1, without affecting ENaC.  Cystic fibrosis is an inherited disease where mutations in the CFTR anion channel lead to chronic airway infection and inflammation.  We recently found that CF patients with reduced SPLUNC1 levels undergo more frequent acute exacerbations and have greater rates of hospitalization4.  Similarly, reduced SPLUNC1 levels correlate with increased inflammation and reduced lung function in asthma patients5. These data suggest that restoring SPLUNC1 function would benefit both asthma and CF patients. We have developed proteolytically-stable peptides based on SPLUNC1’s C-terminus, and found that they exhibit potent anti-inflammatory actions in asthma, acute infection and CF murine models, indicating that they are suitable for development as anti-inflammatory compounds.   Work in this area is ongoing in the Tarran lab.  Funded by the NIH and the CF Foundation. RT has equity in Eldec Pharmaceuticals. 1. Garcia-Caballero A et al. PNAS (2009). 2. Kim CS et al. FASEB J. (2018). 3. Wu T et al. Nat Commun.  (2017). 4. Khanal et al., ERJ, in press (2021).