Oligomerization of the Na,K-ATPase in Cell Membranes

Abstract

The higher order oligomeric state of the Na,K-ATPase ␣␤ heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with ␣-subunits bearing either His 6 or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase ␣␤ heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding ␣-His/␤ and ␣-FLAG/␤ Na,K-ATPases. Co-immunoprecipitation of the Histagged ␣-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,KATPase is present in cell membranes in an oligomeric state of at least (␣␤) 2 composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the ␣-subunit results in the deletion ␣-4,5-loop-less (␣-4,5LL), which associates with ␤ but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive ␣-4,5LL/␤ heterodimer is co-expressed with wild-type ␣␤, oligomers of wild-type ␣␤ and ␣-4,5LL/␤ form in the ER, but the ␣-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric ␣␤ heterodimer in native cell membranes.

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

“…Plasmid construction, cell culture, and transfection Sheep Na + /K + -ATPase a and b open reading frames were PCR amplified from pFASTBAC dual-Na + /K + -ATPase plasmid, which was cloned and characterized as described previously (Gatto et al, 2001;Laughery et al, 2003;Laughery et al, 2004). For heterologous expression of both a-and b-subunits concomitantly, Na pump a-mcherry and b-fragments were cloned into pBud v4 (Invitrogen, Carlsbad, CA) into NotI/KpnI and SalI/XbaI sites, respectively.…”

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

“…5), even when diluted 10-fold, or when run in DDM at 15 mg/ml, the monomeric species in DDM is stable, and does not appear to be in a mobile equilibrium with other molecular species. Our findings are not inconsistent with recent work, showing by immunoprecipitation that Na ϩ ,K ϩ -ATPase expressed in baculovirus-infected insect cell membranes, and dissolved in DDM, can exist as oligomers of the ␣/␤ heterodimer, if we consider the oligomers detected in the void volume by HPLC, the presence of minor heavier species in addition to monomer in the sedimentation profiles, and the tendency of the protein to slowly auto-associ- ate with time (data not shown) (47). Of course, the characterization of Na ϩ ,K ϩ -ATPase as a monomeric species in DDM does not preclude that other oligomeric species exist in the membrane.…”

Stimulation of Na+transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells

“…While early studies concluded that the minimal functional unit is an ␣-␤ protomer (24), the monomeric or oligomeric nature of the functional Na-K-ATPase unit is not yet fully established. Recent evidence either suggests that plasma membrane ␣-␤ protomers of Na-K-ATPase are mostly monomeric (29) or oligomeric (27). It should be mentioned that in transfected cells, c-myc-tagged ␣ 1 -subunits are overexpressed and compete with endogenous ␣-subunits for limiting ␤-subunits.…”