CHIMERIC CA2+-ATPASE/NA+,K+-ATPASE MOLECULES - THEIR PHOSPHOENZYME INTERMEDIATES AND SENSITIVITY TO CA2+ AND THAPSIGARGIN

Chimeric molecules consisting of parts from the sarcoplasmic reticulum Ca2+-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 Ca2+-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 Ca2+-ATPase, was unable to phosphorylate from ATP, but phosphorylated from inorganic phosphate in a Ca2+-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 Ca2+-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 Ca2+-binding sites. The data moreover show that thapsigargin sensitivity is dependent on residues in the NH2-terminal one-third of the Ca2+-ATPase molecule.