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1 Department of Medicine, Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
* To whom correspondence should be addressed. E-mail: hughey{at}dom.pitt.edu.
Epithelial Na+ channels (ENaC) are inhibited by extracellular Na+, a process referred to as Na+ self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in 
or one site in 
blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus oocytes. Cleavage of subunits was also blocked by these mutations when expressed in MDCK cells, and both subunit cleavage and channel activity was blocked when wild type subunits were expressed in furin-deficient CHO cells. We now report that channels with mutant alpha subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+ self-inhibition response, while channels with a mutant subunit showed a modestly enhanced Na+ self-inhibition response. Analysis of Na+ self-inhibition at varying [Na+] indicates that channels containing mutant alpha subunits exhibit an increased Na+ affinity. At the single channel level, channels with a mutant alpha subunit had a low open probability (Po) in the presence of a high external [Na+] in the patch pipette. Po dramatically increased when trypsin was also present, or when a low external [Na+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na+ self-inhibition, and that activation requires that the alpha subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC open probability and extracellular [Na+], supporting the notion that Na+ self-inhibition reflects an open probability reduction due to high extracellular [Na+].
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