Differential contributions of α and γ ENaC subunit cleavage to channel activity

Abstract

Maturation of the epithelial sodium channel (ENaC) involves furin‐dependent cleavage at two extracellular sites within the α subunit and at a single site within the γ subunit. Channels carrying mutations at the furin cleavage sites within the α subunit of mouse ENaC (αR205/A, R231/Aβγ) are uncleaved and have very low activity. We recently identified a putative prostasin cleavage site (γRKRK186) that is distal to the furin cleavage site (γRKRK143) in the γ subunit. We also demonstrated that the tract αD206‐R231, between the furin cleavage sites in the α subunit, as well as the tract γE144‐K186, between the furin and prostasin cleavages sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of α and γ subunit inhibitory tracts, respectively. We used the Xenopus laevis oocyte expression system to examine, at steady state level, the functional contribution of the presence and absence of the α and/or γ inhibitory domains. Co‐expression of either wild type channels (αβγ) or channels carrying mutations in the furin cleavage sites of the α subunit (αR205/A, R231/Aβγ) with prostasin leads to an increase in currents to a similar level. Mutation of both furin and prostasin cleavage sites in the γ subunit impaired prostasin activation of wild type and mutant ENaCs. Oocytes expressing either wild type or furin‐resistant mutant α subunits with furin‐resistant γ subunits lacking the inhibitory tract (αβγR143/AΔE144‐K186 and αR205/A, R231/AβγR143/AΔE144‐K186, respectively) exhibited currents 2–3 fold larger than oocytes expressing wild type channels. Our results indicate that release of the γ inhibitory tract has a dominant effect with regard to activating ENaC.