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

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Abstract
The cDNAs encoding α3-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 α3 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 Ki 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 α3 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 α3 isoform of Na+,K+-ATPase.