The γ-subunit of ENaC is more important for channel surface expression than the β-subunit

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and is composed of three homologous subunits: α, β, and γ. Only heteromultimeric channels made of αβγENaC are efficiently expressed at the cell surface, resulting in maximally amiloride-sensitive currents. To study the relative importance of various regions of the β- and γ-subunits for the expression of functional ENaC channels at the cell surface, we constructed hemagglutinin (HA)-tagged β-γ-chimeric subunits composed of β- and γ-subunit regions and coexpressed them with HA-tagged αβ- and αγ-subunits in Xenopus laevis oocytes. The whole cell amiloride-sensitive sodium current (Δ I ami) and surface expression of channels were assessed in parallel using the two-electrode voltage-clamp technique and a chemiluminescence assay. Because coexpression of αγENaC resulted in larger Δ I ami and surface expression compared with coexpression of αβENaC, we hypothesized that the γ-subunit is more important for ENaC trafficking than the β-subunit. Using chimeras, we demonstrated that channel activity is largely preserved when the highly conserved second cysteine rich domains (CRD2) of the β- and γ-subunits are exchanged. In contrast, exchanging the whole extracellular loops of the β- and the γ-subunits largely reduced ENaC currents and ENaC expression in the membrane. This indicates that there is limited interchangeability between molecular regions of the two subunits. Interestingly, our chimera studies demonstrated that the intracellular termini and the two transmembrane domains of γENaC are more important for the expression of functional channels at the cell surface than the corresponding regions of βENaC.