Chimeric Ca2+‐ATPase/Na+,K+‐ATPase molecules

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Abstract
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.