Question: 

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (a, b, g). The recently published crystal structure of the related channel ASIC1a suggests that ENaC is likely to be a trimer; however, the subunit stoichiometry is still controversial. In this study we used atomic force microscopy (AFM) imaging of channels decorated by antibodies against subunit-specific epitope tags on the a-, b- and g-ENaC subunits to determine the subunit stoichiometry and arrangement of ENaC.

Methods: 

In addition to wild-type (WT) ENaC subunits, five epitope-tagged subunits were used: aHA/V5, bHA/V5, gHA/V5, aHA/FLAG, and bHA/His₆. To investigate a possible effect of the tags on the functional properties of ENaC, different combinations of tagged and untagged ENaC subunits were heterologously expressed in Xenopus laevis oocytes. Immunoblot analysis confirmed expression of all tagged ENaC subunits with the expected cleavage products. Amiloride-sensitive whole-cell currents (DI_ami) were determined by the two-electrode voltage-clamp technique, and patch-clamp experiments were performed to determine single-channel conductances. tsA 201 cells expressing HA/V5-tagged a-, b- or g-ENaC or the triply-tagged channel (aHA/FLAG-, bHA/His₆-, and gHA/V5-ENaC) were solubilized in 1% TX-100. Proteins were isolated by affinity chromatography, using the epitope tags. The proteins were incubated with antibodies to the tags, and the resulting protein-antibody complexes were adsorbed on mica support and imaged by AFM.

Results: 

Robust DI_ami was detectable with all combinations of tagged or untagged ENaC subunits expressed in oocytes, demonstrating that the tags do not abolish ENaC function. The triply-tagged channel showed the same single-channel conductance (5.2 pS) and ion selectivity as WT ENaC. Using AFM experiments we showed that for a-, b- and g-ENaC alone pairs of antibodies decorate the channel at an angle of 120° indicating that the individual subunits assemble as homotrimers. A similar approach demonstrated that abg-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers.

Conclusions: 

We have shown that abg-ENaC assembles as a heterotrimer, that a-, b- and g-ENaC can each form homotrimers, and that ENaC trimers interact to form dimers- and trimers-of-trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains 8-9 subunits.