The epithelial sodium channel (ENaC) is believed to form a heteromeric channel composed of three homologous subunits α, β, and γ (αβγENaC). In humans an additional δ-subunit exists, but little is known about its physiological properties. In heterologous expression systems, δENaC has functional similarities with αENaC. Interestingly, in its δβγ-configuration ENaC has been reported to be activated by extracellular protons. In contrast, conflicting data exist regarding the pH-sensitivity of αβγENaC which may be due to species differences. Here we investigated the effect of extracellular pH changes on the activity of human αβγ- and δβγENaC expressed in Xenopus laevis oocytes.

Human αβγ- or δβγENaC was expressed in Xenopus laevis oocytes. The oocytes were superfused with ND96 adjusted to pH 7.4, 7.9, or 8.4 (HEPES buffer) or 6.9, 6.4, 5.9, 5.4, or 4.9 (MES buffer). Amiloride (100 µM)-sensitive whole-cell currents (δI_ami) were measured using the two-electrode voltage-clamp technique at different pH values. To estimate channel open probability (Po), ENaC was maximally stimulated by proteolytic activation or by using a βS520C mutant ENaC which increases its Po to ~1 after exposure to the sulfhydryl reagent MTSET.

In δβγENaC expressing oocytes step changes of extracellular pH from pH 7.4 to acidic values increased δI_ami. This stimulatory effect increased with decreasing pH values in a sigmoidal manner. At pH 4.9 δI_ami reached ~250% of its initial value. In contrast, in αβγENaC expressing oocytes extracellular acidification had a bimodal effect. A step change of extracellular pH from 7.4 to 4.9 reduced δI_ami to ~85%, whereas changing to pH 6.9 caused a modest stimulation to ~125%. In both, αβγ- and δβγENaC expressing oocytes, alkalinisation of the bath solution caused a decrease of δI_ami. A step change from pH 7.4 to 8.4 reduced δI_ami to ~90% and ~70% in αβγ- and δβγENaC expressing oocytes, respectively. The pH sensitivity of ENaC was reduced after increasing channel Po by proteolytic stimulation or by exposing mutant ENaC to MTSET. These findings suggest that the pH effects on ENaC activity can be attributed to changes in Po.

We conclude that extracellular acidification over a broad range of pH values has a robust stimulatory effect on δβγENaC. In contrast, in the αβγ-configuration, ENaC is stimulated by moderate but inhibited by strong extracellular acidification. Overall, the pH-sensitivity of ENaC is more pronounced in the δβγ- than in the αβγ-configuration which may have physiological implications for ENaC regulation in vivo.