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

Abstract

The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of α and β subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the β subunit, a glycosylated protein, had less than 30% identity to the mammalian β subunit, it was suggested that the β subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm β subunit with the mammalian β subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm α and cloned rat β1 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 β subunit was replaced with the rat β1 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 β subunit did not affect the K+ affinity. These results support the involvement of the β subunit in the Na+ and K+ affinities of Na+/K+-ATPase.

Keywords

• Silkworm
• Rat
• Hybrid Na+/K+-ATPase
• Na+ affinity, K+ affinity
• BM-N cell