Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding α3-subunits of rat brain Na+,K+-ATPase

Abstract The cDNAs encoding α 3 -subunits of rat brain Na + ,K + -ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co-transfection method. Stably transformed cells were selected with 300 μg/ml of neomycin (G-418) for 6 weeks. Northern blot analysis using the 3′-non-translated region of the cDNA as a probe revealed that the α 3 mRNA appeared in transfected cells. Na + ,K + -ATPase activity of the transfected cells was twice that of wild-type cells. Regarding ouabain sensitivity, the Na + ,K + -ATPase showed two K i values for ouabain (8 × 10 −8 and 4.5 × 10 −5 M) in transfected cells while wild-type cells displayed only the higher value. Ouabain sensitivity of Rb + uptake also demonstrated two K i values in the transfected cells (8 × 10 −8 and 4.5 × 10 −5 M) and a K i in wild-type cells of 4 × 10 −5 M. It is concluded that α 3 is a highly ouabain-sensitive catalytic subunit of Na + ,K + -ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the α-subunit rather than the β-subunit. This is the first report on the catalytic charateristics of the α 3 isoform of Na + ,K + -ATPase.