Epithelial junctions depend on intercellular trans-interactions between the Na,K-ATPase β 1 subunits

Abstract

N-Glycans of the Na,K-ATPase β 1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the β 1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous β 1 subunit with various YFPlinked β 1 subunits expressed in Madin-Darby canine kidney cells was used to assess β 1-β 1 interactions. The amount of coprecipitated endogenous dog β 1 was greater with dog YFP-β 1 than with rat YFP-β 1, showing that amino acid-mediated interactions are important for β 1-β 1 binding. Co-precipitation of β 1 was also less with the unglycosylated YFP-β 1 than with glycosylated YFP-β 1, indicating a role for N-glycans. Mixing cells expressing dog YFP-β 1 with non-transfected cells increased the amount of co-precipitated β 1, confirming the presence of intercellular (YFP-β 1)-β 1 complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated β 1 subunits. The decrease in β 1 co-precipitation both with rat YFP-β 1 and unglycosylated YFP-β 1 was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. ReducingN-glycan branching by specific inhibitors increased (YFP-β 1)-β 1 co-precipitation and strengthened intercellular junctions. Therefore, interactions between the β 1 subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the β 1 subunit N-glycan structure can regulate stability and tightness of intercellular junctions.