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

The functional role of the gamma subunit of the Na,K-ATPase was studied using rat gamma cDNA-transfected HEK-293 cells and an antiserum (gamma C33) specific for gamma, Although the sequence for gamma was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a gamma C33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of gamma C33-treated kidney pumps shows that gamma regulates the apparent affinity for ATP, Thus, gamma-transfected cells have a decreased K-ATP' as shown in measurements of (i) K-ATP' of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 mu M ATP, Consistent with the behavior 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-transfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma subunit stabilizes the E-2(K) form of the enzyme (Therien, A. G., Goldshleger, R,, Karlish, S, J., and Blostein, R, (1997) J, Biol, Chem, 272, 32628-32634), Overall, our data demonstrate that gamma is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum.