Characterisation of thapsigargin-releasable Ca2+ from the Ca2+-ATPase of sarcoplasmic reticulum at limiting [Ca2+]

The Ca2+ binding sites of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using Ca-45(2+), have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca2+-limited state, [Ca-2](lim), where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48-60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca2+-ATPase, released a fraction of total Ca2+ at [Ca2+](lim) that accumulated during active transport. Maximal Ca2+ release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca2+ resistant to TG. The amount of Ca2+ released depended on the incubation time at [Ca2+](lim), being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0.13 to 0.03 s(-1). The rapidly released early fraction declined with time and k = 0.13 min(-1). Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca2+ followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 muM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca2+ release mechanism. It is concluded that the Ca2+ released by TG is from the occluded Ca2+ fraction. The Ca2+ occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite 'in line' sequential binding mechanism for Ca2+ transport. (C) 2000 Elsevier Science B.V. All rights reserved.