Furin cleavage activates the epithelial [Na.sup.+] channel by relieving [Na.sup.+] self-inhibition

Epithelial [Na.sup.+] channels (ENaC) are inhibited by extracellular [Na.sup.+], a process referred to as [Na.sup.+] self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in [alpha], or one site in [gamma] blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary 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.sup.+] self-inhibition response, while channels with a mutant [gamma]/-subunit showed a modestly enhanced [Na.sup.+] self-inhibition response. Analysis of [Na.sup.+] self-inhibition at varying [Na.sup.+] indicates that channels containing mutant [alpha]-subunits exhibit an increased [Na.sup.+] affinity. At the single-channel level, channels with a mutant [alpha]-subunit had a low open probability ([P.sub.o]) in the presence of a high external [Na.sup.+] in the patch pipette. [P.sub.o] dramatically increased when trypsin was also present, or when a low external [Na.sup.+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving [Na.sup.+] 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 [P.sub.o] and extracellular [Na.sup.+], supporting the notion that [Na.sup.+] self-inhibition reflects a [P.sub.o] reduction due to high extracellular [Na.sup.+]. amiloride; open probability; voltage clamp; Xenopus laevis oocyte; mutagenesis