Chimeric Ca(2+)-ATPase/Na+,K(+)-ATPase molecules. Their phosphoenzyme intermediates and sensitivity to Ca2+ and thapsigargin.
Chimeric molecules consisting of parts from the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase were expressed in COS-1 cells and analysed functionally. One chimera, in which most of the central cytoplasmic loop was derived from the Na+,K(+)-ATPase, while the transmembrane segments and the minor cytoplasmic loop came from the Ca(2+)-ATPase, was able to occlude Ca2+ and to be phosphorylated from ATP with normal apparent affinity for Ca2+ and ATP. This chimera also displayed normal sensitivity to thapsigargin, but was unable to undergo the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and to transport Ca2+. The other chimera, which consisted of the NH2-terminal two-thirds of Na+,K(+)-ATPase and the COOH-terminal one-third of Ca(2+)-ATPase, was unable to phosphorylate from ATP, but phosphorylated from inorganic phosphate in a Ca(2+)-inhibitable and thapsigargin-insensitive reaction. These results can be explained in terms of a structural model in which the non-conserved residues in the central cytoplasmic domain of the Ca(2+)-ATPase are without major importance for the binding and occlusion of Ca2+, but are involved in the E1P-->E2P conformational changes of the phosphoenzyme, whereas residues in transmembrane segments on both sides of the central cytoplasmic domain are involved in formation of the Ca(2+)-binding sites. The data moreover show that thapsigargin sensitivity is dependent on residues in the NH2-terminal one-third of the Ca(2+)-ATPase molecule.
Nørregaard
A
A
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
Vilsen
B
B
Andersen
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D011993:Recombinant Fusion Proteins; D013729:Terpenes; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium