Expression and functional role of the gamma subunit of the Na,K-ATPase in mammalian cells

The functional role of the gamma subunit of the Na,K-ATPase was studied usi ng rat gamma cDNA-transfected HEK-293 cells and an antiserum (gamma C33) sp ecific for gamma, Although the sequence for gamma was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initi ator methionine despite the expression of a gamma C33-reactive doublet on i mmunoblots. Kinetic analysis of the enzyme of transfected compared with con trol cells and of gamma C33-treated kidney pumps shows that gamma regulates the apparent affinity for ATP, Thus, gamma-transfected cells have a decrea sed K-ATP' as shown in measurements of (i) K-ATP' of Na,K-ATPase activity a nd (ii) K+ inhibition of Na-ATPase at 1 mu M ATP, Consistent with the behav ior of gamma-transfected cells, gamma C33 pretreatment increases K-ATP' of the kidney enzyme and K+ inhibition (1 mu M ATP) of both kidney and gamma-t ransfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma subunit stabilizes the E-2(K) for m of the enzyme (Therien, A. G., Goldshleger, R,, Karlish, S, J., and Blost ein, R, (1997) J, Biol, Chem, 272, 32628-32634), Overall, our data demonstr ate that gamma is a tissue (kidney)-specific regulator of the Na,K-ATPase t hat can increase the apparent affinity of the enzyme for ATP in a manner th at is reversible by anti-gamma antiserum.