Epithelial Junctions Depend on Intercellular Trans-interactions between the Na,K-ATPase β1 Subunits

N-Glycans of the Na,K-ATPase β1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the β1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous β1 subunit with various YFP-linked β1 subunits expressed in Madin-Darby canine kidney cells was used to assess β1-β1 interactions. The amount of co-precipitated endogenous dog β1 was greater with dog YFP-β1 than with rat YFP-β1, showing that amino acid-mediated interactions are important for β1-β1 binding. Co-precipitation of β1 was also less with the unglycosylated YFP-β1 than with glycosylated YFP-β1, indicating a role for N-glycans. Mixing cells expressing dog YFP-β1 with non-transfected cells increased the amount of co-precipitated β1, confirming the presence of intercellular (YFP-β1)-β1 complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated β1 subunits. The decrease in β1 co-precipitation both with rat YFP-β1 and unglycosylated YFP-β1 was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-β1)-β1 co-precipitation and strengthened intercellular junctions. Therefore, interactions between the β1 subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the β1 subunit N-glycan structure can regulate stability and tightness of intercellular junctions.