Simultaneous internalization and binding of calcium during the initial phase of calcium uptake by the sarcoplasmic reticulum calcium pump

Abstract

The kinetics of Ca2+ transport mediated by the sarcoplasmic reticulum (SR) Ca-ATPase were investigated by rapid kinetic techniques that either measure the disappearance of Ca2+ from the medium [stopped-flow photometry of Ca2+ indicators or rapid filtration (method 1)] or directly detect the changes in the accessibility of Ca2+ to the exterior of the membrane, i.e., occlusion of Ca2+ within the Ca pump and Ca2+ transport into the lumen of SR vesicles [EGTA quench (method 2)]. SR vesicles were preincubated in micromolar Ca2+ to form the E.2Cacyt intermediate of the Ca-ATPase, and then Ca2+ transport was initiated by addition of ATP. It was found that Ca2+ uptake measured by method 1 began with no lag phase, in spite of the prediction of kinetic models of the Ca-ATPase. Instead, the time course of Ca2+ uptake was found to have two components: a fast and a slow phase, similar to that obtained using method 2, although the rate constant of the fast phase determined by method 1 was considerably lower than that measured by method 2. The fast phase of Ca2+ uptake measured by method 1 was not influenced by either Ca2+ ionophore or detergent treatment, whereas the slow phase was diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

“…In the case of the Ca# + -ATPase of skeletal-muscle sarcoplasmic reticulum (SR), two Ca# + ions and one ATP molecule bind to the ATPase so that full coupling results in the transport of 2 Ca# + ions for each molecule of ATP hydrolysed. Recent experiments are consistent with a model in which two pairs of sites for Ca# + exist on the Ca# + -ATPase, transport of Ca# + by the Ca# + -ATPase corresponding to transfer of Ca# + from a cytoplasmic pair of sites to a lumenal pair of sites, driven by phosphorylation of the ATPase by ATP [1][2][3], as shown in Scheme 1. Ca# + is released into the lumen of the SR from the lumenal pair of sites on the phosphorylated intermediate, and dephosphorylation of E2P then allows recycling to E1 [4].…”

Luminal dissociation of Ca2+ from the phosphorylated Ca2+-ATPase is sequential and gated by Mg2+

“…This alternating site model predicts that binding of Ca# + to the Ca# + -ATPase from the luminal side of the membrane (to E2) will be competitive with binding of Ca# + from the cytoplasmic side of the membrane (to E1). However, a variety of kinetic and equilibrium experiments failed to observe any such effect ; instead they were consistent with a model in which two pairs of sites for Ca# + exist on the Ca# + -ATPase, transport of Ca# + by the Ca# + -ATPase corresponding to transfer of Ca# + from a cytoplasmic pair of sites to a luminal pair of sites, driven by phosphorylation of the ATPase by ATP [2][3][4].…”

“…In an operational sense, the two sites are located one ' above ' the other because the binding of Ca# + to the ' outer ' site prevents the release of Ca# + from the ' inner ' site [5][6][7][8] ; this could correspond to a physical location of the outer site above the inner site, as suggested in Figure 1, but would be equally consistent with a side-by-side arrangement, with the translocation of Ca# + across the membrane then taking an angular course [25]. After phosphorylation of the ATPase, the two Ca# + ions are transferred from the pair of cytoplasmic sites to a pair of luminal sites [2][3][4]. Because the only negatively charged residues in the putative transmembrane α-helices of the Ca# + -ATPase are the four postulated to make up the pair of highaffinity cytoplasmic sites, the pair of low-affinity luminal sites must be made up of residues on the luminal side of the SR membrane.…”

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

“…Recently, Mészá ros and Bak (21,22) and Jencks and coworkers (23,24) proposed that the cytoplasmic and luminal sites could be distinct; i.e. there are four Ca 2ϩ sites on the ATPase.…”