The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric αβγ complexes. The α and γ (not β) 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 (α, β, γ, αβ, αγ, βγ, and αβγ). We assayed corresponding channel function as amiloride-sensitive sodium transport (INa). We varied furin-mediated proteolysis by mutating the P1 site in α and/or γ subunit furin consensus cleavage sites (αmut and γmut). Our findings were as follows. (i) The β subunit alone is not transported to the cell surface nor cleaved upon assembly with the α and/or γ subunits. (ii) The α subunit alone (or in combination with β and/or γ) is efficiently transported to the cell surface; a surface-expressed 65-kDa α ENaC fragment is undetected in αmutβγ, and INa is decreased by 60%. (iii) The γ subunit alone does not appear at the cell surface; γ co-expressed with α reaches the surface but is not detectably cleaved; and γ in αβγ complexes appears mainly as a 76-kDa species in the surface pool. Although basal INa of αβγmut was similar to αβγ, γmut was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of α and γ in αβγ heteromers. Basal INa is reduced by preventing furin-mediated cleavage of the α, but not γ, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases
