β subunit affects Na+ and K+ affinities of Na+/K+-ATPase: Na+ and K+ affinities of a hybrid Na+/K+-ATPase composed of insect α and mammalian β subunits

The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of alpha and beta subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the a subunit had more than 70% identity to the mammalian alpha subunit in the amino acid sequence, whereas the beta subunit, a glycosylated protein, had less than 30% identity to the mammalian beta subunit, it was suggested that the beta subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm beta subunit with the mammalian beta subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm alpha and cloned rat beta 1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na+/K+-ATPase, to construct a hybrid Na+/K+-ATPase, in which the silkworm beta subunit was replaced with the rat beta 1 subunit. The hybrid Na+/K+-ATPase increased the affinity for K+ by 4.1-fold and for Na+ by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm beta subunit did not affect the K+ affinity. These results support the involvement of the beta subunit in the Na+ and K+ affinities of Na+/K+-ATPase.