Chimeric Ca 2+-ATPase/Na +,K +-ATPase molecules : Their phosphoenzyme intermediates and sensitivity to Ca 2+ and thapsigargin

Abstract

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 Ca 2+ and to be phosphorylated from ATP with normal apparent affinity for Ca 2+ 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 Ca 2+. The other chimera, which consisted of the NH 2-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 Ca 2+, 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 NH 2-terminal one-third of the Ca 2+-ATPase molecule.