Assembly of the Epithelial Na+Channel Evaluated Using Sucrose Gradient Sedimentation Analysis*

Three subunits, α, β, and γ, contribute to the formation of the epithelial Na+channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the α subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual β and γ subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells andin vitro. When we co-expressed β with α or with α plus γ, the β subunit assembled into a 25 S complex. Glycosylation of the α subunit was not required for assembly into a 25 S complex. We found that the α subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the α subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+channel subunits form high order oligomeric complexes and that the α subunit contains the information that facilitates such formation. Interestingly, the ability of the α, but not the β or γ, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.