Phospholemman (FXYD1) is a single-transmembrane protein regulator of 
Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and 
phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. 
Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or 
Ser-63 relieves the inhibition. Despite the accumulated information on 
physiological effects, whole cell studies provide only limited information on 
molecular mechanisms. As a complementary approach, we utilized purified human 
Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and 
S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control 
α1β1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic 
mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on 
α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with 
α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by 
phosphomimetic mutants and also by increasing mole fractions of DOPS in the 
proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 
slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 
3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular 
modeling, which indicates that a cytoplasmic helix (residues 60-70) docks 
between the αN and αP domains in the E2 conformation, but docking is weaker in 
E1 (also for phosphomimetic mutants). Taken together with previous work showing 
that FXYD1 also raises binding affinity for the Na(+)-selective site III, these 
results provide a rather comprehensive picture of the regulatory mechanism of 
FXYD1 that complements the physiological studies.