Differential Assembly of Native Epithelial Sodium Channel Complexes Across Mouse Epithelial Tissues
Arpita Bharadwaj1, Joshua Curry2, Xiao-Tong Su2
1Vollum Institute, Oregon Health and Science University, Portland, Oregon.
Key Points:
The new mouse line, epithelial sodium channel γ - mVenus FLAG (ENaC γ -VF), expresses a γ subunit tagged with mVenus and 3xFLAG that retains wild-type physiological function at baseline. The tags allow fluorescence-based detection and affinity purification of native γ -containing epithelial sodium channel complexes under mild, nondenaturing conditions. γ -containing complexes are heterogeneous, including αβγ channels as well as complexes associated with channel biogenesis, trafficking, and regulation.
Background:
The epithelial sodium channel (ENaC) governs sodium and fluid absorption in kidney, lung, and colon epithelia, but the molecular organization of native ENaC complexes in vivo remains poorly defined. Low abundance and biochemical instability have limited direct analysis of ENaC assembly, composition, and regulatory associations in native tissues.
Methods:
We generated a knock-in mouse in which the endogenous ENaC γ subunit was fused to a fluorescent protein and affinity tag, while preserving physiologic channel function. We validated channel activity in vivo using electrolyte measurements and pharmacologic inhibition and assessed channel pharmacology by radioligand binding. Native ENaC complexes were analyzed from lung, kidney, and colon using fluorescence-detection size-exclusion chromatography, single-molecule pull-down, fluorescent antibody fragments, and mass spectrometry.
Results:
The tagged γ subunit preserved normal ENaC function in vivo . Native ENaC complexes directly isolated from lung, kidney, and colon, revealed marked tissue-to-tissue differences in channel abundance and apparent complex size. Dual-color fluorescence analyses distinguished fully assembled channels from broader γ -containing assemblies. Proteomic analysis identified regulatory proteins associated with γ -containing complexes, indicating that ENaC exists in multiple assembly and regulatory states in vivo .
Conclusions:
Native ENaC architecture is heterogeneous across epithelial tissues. Endogenous tagging enables direct molecular interrogation of ENaC assembly and regulation in vivo .
