Cathepsin B cleaves and activates the alpha subunit of the epithelial sodium channel

Abstract

Several proteases have been found to cleave ENaC subunits to produce the active form of the heterotrimeric channel. ENaC cleavage is thought to occur primarily in the trans‐Golgi network (TGN) by serine proteases. ENaC may undergo additional cleavage after it is inserted into the apical membrane or while it is being recycled in recycling endosomes. Therefore, we investigated if secreted proteases could cleave and activate ENaC subunits. We performed two‐dimensional gel electrophoresis after concentrating condition medium taken from the apical and basolateral sides of 2F3 cells that formed tight junctions and demonstrated a tight resistance. We identified a protein secreted onto the apical side of 2F3 cells by mass spectrometry, as the cysteine protease cathepsin B. We expressed and purified full length recombinant alpha, beta, and gamma subunits of ENaC as GST fusion proteins. We identified a putative site for cathepsin B within the alpha subunit of ENaC that is somewhat conserved between species. We demonstrate that active cathepsin B cleaves the alpha subunit, but not the beta or gamma subunits of ENaC in vitro. We use single channel patch clamp studies and short circuit current experiments to show ENaC activity decreases in response to a specific cathepsin B inhibitor after being directly applied onto the apical side of 2F3 cells. These results suggest a novel mechanism for the proteolytic regulation of ENaC.