Ca2+Translocation across Sarcoplasmic Reticulum ATPase Randomizes the Two Transported Ions

Denis Canet, Vincent Forge, Florent Guillain, Elisabeth Mintz
1996 Journal of Biological Chemistry  
Cytoplasmic Ca 2؉ dissociation is sequential, and the Ca 2؉ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca 2؉ is freely exchangeable from the cytoplasm, whereas the deeper Ca 2؉ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5°C, pH 8, 300 mM K ؉ ), luminal Ca 2؉ dissociation is sequential as well, so that the representation of two superimposed Ca 2؉ ions still holds on the
more » ... ill holds on the phosphoenzyme, with the superficial Ca 2؉ facing the lumen freely exchangeable and the deeper Ca 2؉ blocked by the superficial Ca 2؉ . Under the same conditions, we have investigated whether a prebuilt Ca 2؉ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca 2؉ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca 2؉ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation. Sarcoplasmic reticulum ATPase is a membranous enzyme that pumps Ca 2ϩ from the cytoplasm of muscle cells into the reticulum lumen, requiring ATP hydrolysis. During its cycle, each ATPase monomer transports two Ca 2ϩ ions (Scheme 1). During transport, the Ca 2ϩ sites change their orientation and affinity, depending on whether the ATPase is phosphorylated. The high affinity transport sites of the nonphosphorylated ATPase are accessible from the cytoplasm, whereas once the ATPase has been phosphorylated the transport sites have lower affinity and are accessible from the lumen. This allows Ca 2ϩ release into the SR 1 lumen and is followed by dephosphorylation. Ca 2ϩ binding to E, the Ca 2ϩ -deprived nonphosphorylated ATPase, has been well characterized. Two Ca 2ϩ ions bind sequentially with high affinity and positive cooperativity (1, 2). In 1982, Dupont (3) showed that the dissociation of one-half of the 45 Ca 2ϩ bound to Ca 2 E was impaired by the presence of excess 40 Ca 2ϩ in the medium. This was interpreted in 1987 by Inesi (4)
doi:10.1074/jbc.271.34.20566 pmid:8702801 fatcat:e3rbcyar7bfnznmklum3jcip24