Thermal Uncoupling of the Ca²⁺‐transporting ATPase in Sarcoplasmic Reticulum

Abstract

It is known that the light fraction of rabbit skeletal muscle sarcoplasmic reticulum vesicles can release Ca2+ from the intravesicular space, although the Ca2+-conductive channels are present only in the heavy fraction of sarcoplasmic reticulum vesicles. To study the possible pathways of the Ca2+ leakage from light vesicles we have used a short-term treatment for 4.5 min at 45°C which quickly decreases the efficiency of Ca2+-transporting ATPase operation without any visible effects on the hydrolytic activity of the Ca2+-ATPase in the membranes. The treatment of the vesicles decreased the negative membrane surface potential created by the Ca2+-ATPase. Comparative titration of control and heat-treated preparations of light sarcoplasmic reticulum vesicles by K+, Na+, Mg2+, and Ca2+ revealed clear differences in their surface properties. The short-term heating resulted in release of Ca2+ from the vesicles previously loaded with 45Ca2+, which indicates an increase in passive membrane permeability to Ca2+. Study of Ca2+-ATPase protein arrangement in the membrane indicated that the heat treatment induced protein oligomerization and some of the Ca2+-ATPase molecules acquired intermolecular and intramolecular covalent bonds. From these data, we have concluded that the changes in the surface and structure properties of the vesicle membranes after the short-term heat treatment were the result of clustering of the CaZ+-ATPase molecules. This protein rearrangement may create channels for calcium leakage from light sarcoplasmic reticulum vesicles.The sarcoplasmic reticulum (SR) plays a key role in excitation-contraction coupling between nerve impulses and muscle contraction. The skeletal muscle SR consists of two morphologically and functionally distinct regions, namely the lateral sacs (heavy fraction of vesicles after centrifugation in a sucrose density gradient) and the longitudinal tubules (light fraction of vesicles) which differ in protein composition [l]. The lateral sacs contain Ca2+ channels and provide fast release of stored Ca2+ [l, 21. The role of the longitudinal tubules, which are rich in CaZ+-ATPase protein, is calcium accumulation. The latter process involves active transport in which Ca" translocation is coupled with ATP hydrolysis [3]. It is known that short-term heating of SR vesicles at 42-45 "C leads to a quick decrease of Ca2+-transporting ATPase (Ca'+ pump) efficiency without an effect on the Ca2+-ATPase hydrolytic activity [4, 53. Studying the mechanism of the Ca2+ pump thermal uncoupling may be useful for understanding the possible pathways of Ca" release from SR. New pathways for Caz+ release from SR could be found which may be relevant to the regulation of the Caz+ exchange in muscle tissue under normal and pathological conditions. It has been shown that the decrease in the Ca2+ transport efficiency upon short-term heating of SR vesicles was not connected with true intramolecular uncoupling of Ca2+ pump [6]. To evaluate conformational changes of the Caz+-ATPase after heat treatment, kinetic paramete...

No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+‐ATPase from short‐term stimulated rat muscle

“…3b) indicated no changes in the SERCA content. Under certain conditions, it has been shown that SERCA forms high molecular mass complexes (Geimonen et al. 1994).…”

“…It has been shown that although SERCA is able to function as a monomer, protein–protein interactions between monomers may play an important role in enzyme stabilization and cooperative kinetics (Martonosi 1995). Oligomerization of SERCA has been observed after exposure to high temperature (45 °C) (Geimonen et al. 1994).…”

Paradoxical effects of prior activity on human sarcoplasmic reticulum Ca2+-ATPase response to exercise

“…Moreover, this type of exercise results in a pronounced thermal stress with muscle temperatures, which, in the case of horses, reach 43°C (4). Incubation of SR vesicles (7,14) or homogenates (43) at this temperature is known to induce large perturbations in Ca 2ϩ handling with loss of coupling efficiency. It is known that cells preexposed to thermal stress quickly acquire a resistance, an effect possibly mediated by increased expression of heat shock proteins (HSPs) (26,39).…”

Melittin-Induced Inhibition and Aggregation of Ca-ATPase in Skeletal Muscle Sarcoplasmic Reticulum: A Comparative Study

“…Incubation of SR vesicles with cupric phenanthroline for 2-30 min results in a slow and gradual decrease of the peak area of the Ca-ATPase monomer and in the appearance of new protein bands with high molecular masses (cross-linked enzyme oligomers) on the electrophoretograms. We have also demonstrated earlier that short-term heat treatment of SR vesicles at 45°C induces the formation of stable aggregates consisting of Ca-ATPase molecules in SR membranes (28,29,32). After such treatment, even 1-2 min incubation of SR preparations with cupric phenanthroline is sufficient for cross-linking of all the Ca-ATPase protein aggregates.…”