Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus Oocytes -: Role of furin-mediated endogenous proteolysis

The epithelial sodium channel ( ENaC) is preferentially assembled into heteromeric alpha beta gamma complexes. The alpha and gamma ( not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alpha beta, alpha gamma, beta gamma, and alpha beta gamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I-Na). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/ or gamma subunit furin consensus cleavage sites (alpha(mu)t and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/ or gamma subunits. ( ii) The alpha subunit alone ( or in combination with beta and/ or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)beta gamma, and I-Na is decreased by 60%. ( iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alpha beta gamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal INa of alpha beta gamma(mut) was similar to alpha beta gamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alpha beta gamma heteromers. Basal I-Na is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.