Loss of the alpha subunit distal furin cleavage site blunts ENaC activation following Na(+) restriction.

Epithelial Na(+) channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (α(F2M) mice). On a normal Na(+) control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and α(F2M) mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male α(F2M) mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (I(SC)). Following dietary Na(+) restriction, WT and α(F2M) mice had similar natriuretic and colonic I(SC) responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na(+)-restricted α(F2M) mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na(+)-restricted α(F2M) vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na(+) restriction. KEY POINTS: The epithelial Na(+) channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (α(F2M) mice). We found that α(F2M) mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted α(F2M) mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.