The Polarized Distribution of Na⁺,K⁺-ATPase: Role of the Interaction between β Subunits

Abstract

Na+,K+-ATPase polarity depends on the interaction between the β subunits of Na+,K+-ATPases located on neighboring cells. In the present work, we use energy transfer methods (FRET), in vivo to demonstrate that these β subunits interact directly at the intercellular space of epithelial cells.

Identification of the amino-acid region involved in the intercellular interaction between the Na,K-ATPase β1 subunits

“…Western blot analysis of the culture media of transfected cells using the 464.8 antibody against b 1 detected a secreted protein of molecular mass ,45 kDa (Fig. 7A), similar to the results reported for CHO cells (Padilla-Benavides et al, 2010). Deglycosylation of this protein by peptide:N-glycosidase F (PNGase F) yielded a core protein of molecular mass ,28 kDa (Fig.…”

“…Stable MDCK cell lines expressing wild-type and mutated YFP-b 1 were obtained as described previously (Vagin et al, 2005). The vector encoding a secreted extracellular domain of the dog b 1 subunit (Secdog b 1 ) (Padilla-Benavides et al, 2010) constructed in the Fambrough laboratory was a generous gift from Liora Shoshani. This vector encodes a chimera containing 29 N-terminal amino acid residues of DPPIV and amino acid residues 64-303 of the dog Na + /K + -ATPase b 1 subunit.…”

“…The cytoplasmic domain of the Na + /K + -ATPase is linked to the cytoplasmic domain of E-cadherin via an ankyrin and spectrin cytoskeleton (Kizhatil et al, 2007;Nelson et al, 1990;Nelson and Veshnock, 1987), whereas the extracellular domain of the Na + /K + -ATPase b 1 subunit interacts with the b 1 subunit of the apposed cell (Padilla-Benavides et al, 2010;Tokhtaeva et al, 2011). The removal of N-glycans from the Na + /K + -ATPase b 1 subunit or alteration of the amino acid sequence of one of the interacting b 1 subunits by 'swapping species' weakens b 1 -b 1 binding (Tokhtaeva et al, 2011), indicating that both N-glycans and particular amino acids are important for binding.…”

“…To express Sec-dog b 1 , human embryonic kidney (HEK-293) cells were transiently transfected with the construct, which was generated in the Fambrough laboratory and was used previously to express the secreted extracellular domain of the dog b 1 subunit in CHO cells (Padilla-Benavides et al, 2010). This construct encodes for the chimera containing a cytoplasmic and a transmembrane domain of dipeptidyl peptidase 4 (DPPIV) and an extracellular domain of the dog Na + /K + -ATPase b 1 subunit.…”

Ouabain modulates ciliogenesis in epithelial cells

“…Ouabain and Na + ,K with the fact that the Na + ,K + -ATPase located at the intercellular cell borders is only accessible from the basal side (20,(22)(23)(24). This finding suggests that this enzyme may act as receptor for low concentrations of ouabain acting on cilliogenesis.…”