THE AMINO-TERMINAL 200 AMINO-ACIDS OF THE PLASMA-MEMBRANE NA+,K+-ATPASE ALPHA-SUBUNIT CONFER OUABAIN SENSITIVITY ON THE SARCOPLASMIC-RETICULUM CA2+-ATPASE

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K+-ATPase but not the sarcoplasmic reticulum (SR) Ca2+-ATPase, whereas thapsigargin specifically blocks the SR Ca2+-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca2+-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K+-ATPase alpha1 subunit (Met1 to Asp200), retained thapsigargin- and Ca2+-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca2+-ATPase. Moreover, this Ca2+-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca2+-ATPase were replaced with the corresponding portion of the Na+,K+-ATPase, lost the thapsigargin-sensitive Ca2+-ATPase activity seen in CCC and [n/c]CC. [H-3]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K+-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K+-ATPase alpha1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca2+-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K+-ATPase alpha1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca2+-ATPase, and the segment Ile163 to Gly354 of the SR Ca2+-ATPase is critical for thapsigargin- and Ca2+-sensitive ATPase activity.