Differential Assembly of Native ENaC Complexes Across Mouse Epithelial Tissues

The epithelial sodium channel (ENaC) governs sodium and fluid absorption in the lung, kidney, and colon, but the organization of native ENaC complexes has remained difficult to define because of their low abundance and biochemical instability. To enable direct analysis of native assemblies, we generated a knock-in mouse in which the endogenous γ subunit is fused at its C terminus to mVenus, a 3C protease cleavage site, and a 3xFLAG epitope (ENaCγ-VF). The tag preserves physiological ENaC function, as ENaCγ-VF mice display normal electrolyte handling, benzamil affinity, and amiloride-sensitive Na⁺:K⁺ responses indistinguishable from wild-type animals. Using fluorescence-detection size-exclusion chromatography and single-molecule pull-down, we directly monitor intact native ENaC complexes from lung, kidney, and colon and uncover marked tissue-to-tissue differences in channel abundance and apparent complex size. Dual-color analysis with a fluorescent Fab against ENaCα marks fully assembled αβγ channels, while γ-based fluorescence reports the broader population of γ-containing assemblies. In combination, the ENaCγ-VF line provides a biochemical anchor for identifying regulatory and trafficking proteins that co-purify with native ENaC complexes. These data show that ENaC architecture <i>in vivo</i> is heterogeneous, and establish ENaCγ-VF mice as a platform for dissecting how epithelial environments shape ENaC assembly, composition, and regulation.