The gamma-subunit of ENaC is more important for channel surface expression than the beta-subunit

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and is composed of three homologous subunits: alpha, beta, and gamma. Only heteromultimeric channels made of alphabetagammaENaC are efficiently expressed at the cell surface, resulting in maximally amiloride-sensitive currents. To study the relative importance of various regions of the beta- and gamma-subunits for the expression of functional ENaC channels at the cell surface, we constructed hemagglutinin (HA)-tagged beta-gamma-chimeric subunits composed of beta- and gamma-subunit regions and coexpressed them with HA-tagged alphabeta- and alphagamma-subunits in Xenopus laevis oocytes. The whole cell amiloride-sensitive sodium current (DeltaI(ami)) and surface expression of channels were assessed in parallel using the two-electrode voltage-clamp technique and a chemiluminescence assay. Because coexpression of alphagammaENaC resulted in larger DeltaI(ami) and surface expression compared with coexpression of alphabetaENaC, we hypothesized that the gamma-subunit is more important for ENaC trafficking than the beta-subunit. Using chimeras, we demonstrated that channel activity is largely preserved when the highly conserved second cysteine rich domains (CRD2) of the beta- and gamma-subunits are exchanged. In contrast, exchanging the whole extracellular loops of the beta- and the gamma-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 gammaENaC are more important for the expression of functional channels at the cell surface than the corresponding regions of betaENaC.