Sodium self‐inhibition and proton sensitivity do not correlate with proteolytic processing in epithelial sodium channels containing the δ‐subunit

Abstract

The epithelial sodium channel (ENaC) is the only constitutively‐active ion channel of the DEG/ENaC protein family and evolved as a mechanism for sodium conservation during tetrapod evolution. We recently characterised a Xenopus laevis ENaC isoform which consists of the δ‐, β‐and γ‐ENaC subunits (Wichmann et al. J Biol Chem 4;293(18):6647–6658). Currents generated by these δβγ‐ENaCs have a slow transient nature due to a pronounced sodium self‐inhibition (SSI). In contrast to canonical ENaCs composed of the α‐, β‐ and γ‐subunits, δβγ‐ENaCs are not stimulated by proteases. The δ‐subunit is not proteolytically processed during intracellular maturation and the presence of the δ‐subunit prevents cleavage of the γ‐subunit at the cell surface. We therefore hypothesised that a lack of proteolytic processing might explain the transient nature of currents generated by Xenopus δβγ‐ENaCs and might indicate an evolutionary prerequisite towards constitutive ENaC activity.We expressed αβγ‐ or δβγ‐ENaC from guinea pigs (Cavia porcellus) as well as Xenopus laevis in Xenopus oocytes and measured currents generated by those isoforms as well as their degree of SSI, response to extracellular chymotrypsin (2 μg/ml) and extracellular acidification (pH 6.0), by two‐electrode voltage‐clamp electrophysiology.In contrast to Xenopus laevis δβγ‐ENaC, currents generated by Cavia porcellus δβγ‐ENaC did not have a transient form and had a profoundly reduced SSI compared to the Cavia porcellus αβγ‐ENaC isoform. Like our previous observations using Xenopus laevis ENaCs, Cavia porcellus αβγ‐ENaC was robustly activated by extracellular chymotrypsin, whereas Cavia porcellus δβγ‐ENaC was insensitive to extracellular protease. Xenopus laevis δβγ‐ENaC was strongly activated by extracellular acidification due to inhibition of SSI, whereas Cavia porcellus δβγ‐ENaC was insensitive to extracellular acidification.We conclude that the absence of proteolytic ENaC activation is not directly linked to a transient ion channel activity. By contrast, a strong SSI and proton sensitivity correlate with a more transient ENaC activity, whereas a lack of SSI and proton insensitivity renders ENaC constitutively‐open. These findings might suggest that changes in the kinetics of SSI and proton sensitivity rather than proteolytic processing determined ENaC evolution towards a constitutively‐active ion channel.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.