The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.
Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]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 alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.
Ishii
T
T
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Takeyasu
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D017931:DNA Primers; D007700:Kinetics; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D010042:Ouabain; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase