Mechanism of thermal uncoupling of Ca 2+-ATPase of sarcoplasmic reticulum as revealed by thapsigargin stabilization

Abstract

Thermal uncoupling of the Ca 2+ pump of skeletal muscle sarcoplasmic reticulum is specifically blocked by binding of Ca 2+ to the high affinity sites, having identical characteristics to the Ca 2+ transport sites (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994–1001). The present study has investigated the nature of the decreased nett Ca 2+ transport in the uncoupling process. Ca 2+ uptake in the presence and absence of oxalate, Ca 2+ retention following passive Ca 2+ loading and Ca 2+-dependent ATPase activity were inactivated at pH 7.0 and 37°C, with rate constants of 0.12, 0.047, 0.053 and 0.001 min −1, respectively. Activation energies were in the range 72–76 kcal/mol, indicating a common irreversible protein conformational transition. A thermodynamic analysis of parallel or consecutive inactivation pathways revealed that loss of Ca 2+ transfer and ATPase activity occurred on the same pump unit, making the existence of a predominant uncoupled intermediate unlikely. Decreased passive Ca + loading, an index of the number of intact vesicles, correlated with decreased active uptake in the absence of oxalate, indicating increased vesicle permeability. Thapsigargin, at a 1:1 stoichiometry, stabilised the Ca-ATPase against thermal inactivation, while previously inactivated Ca-ATPases appeared not to bind TG. Protection by TG suggests that the origin of inactivation is in the transmembrane and stalk regions of the ATPase. We propose that protein unfolding results in inefficient gating of a small percentage of ATPases with subsequent uncoupling of the entire vesicle.