Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

Abstract

Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca 2+ ] in the SR-measured with a calsequestrin-fused biosensor-was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca 2+ ] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR.skeletal muscle | excitation/contraction coupling | cardiac muscle | muscle diseases | catecholaminergic polymorphic ventricular tachycardia

Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum

“…Since DaNa appears to facilitate CASQ2 polymerization and enhanced Ca 2+ binding to CASQ2 at lower [Ca 2+ ] it was important to verify that DaNa increases the SR Ca 2+ storage and makes more Ca 2+ available for subsequent SR Ca 2+ release. When the free [Ca 2+ ] decreases, CASQ2 undergoes depolymerization and Ca 2+ dissociates at a faster rate than the Ca 2+ release from the SR, with CASQ2 thus providing localized intraSR Ca 2+ storage for easy release …”

“…Once inside the SR DaNa could alter Ca 2+ interactions with CASQ and therefore, ultimately alter the availability of Ca 2+ for release from the SR store. CASQ is the chief Ca 2+ buffer located in the region of the terminal cisternae in both skeletal and cardiac muscle and undergoes major Ca 2+ ‐dependent changes in its state of polymerization and Ca 2+ ‐binding abilities . Little is clearly understood regarding Ca 2+ binding and how polymerization is stimulated in cardiac CASQ (CASQ2), but Ca‐binding curves for CASQ2 using atomic absorption spectroscopy have been produced and correlated with their Ca‐dependent oligomerization…”

Mechanisms of calcium handling during a malignant hyperthermia event.

“…The SR forms an extensive continuous network that surrounds every myofibril and is the main site for Ca 2+ storage in the muscle fiber (Franzini- Armstrong & Peachey, 1981;Pinali et al, 2013;Manno et al, 2017). Structurally, the SR is organized into two functional regions which include the terminal cisternae and longitudinal SR (Franzini- Armstrong & Peachey, 1981).…”

“…The portion of the SR that comes into close contact with the t-system at is known as the terminal cisternae of the SR and functions as the main site of Ca 2+ release. The majority of Ca 2+ in the SR is bound to calsequestrin (CSQ), a high capacity Ca 2+ binding protein (Park et al, 2004;Murphy et al, 2009;Royer & Ríos, 2009;Manno et al, 2017). In the resting skeletal muscle, CSQ polymerizes into a multimeric structure and can be found in high concentrations near the terminal cisternae which is facilitated by CSQ binding to the SR membrane bound proteins triadin and junctin (Györke et al, 2004;Park et al, 2004;Dulhunty et al, 2009;Manno et al, 2017).…”

“…on CSQ causes the polymeric structure of CSQ to destabilize; thus, resulting in CSQ depolymerization and a subsequent reduction in its buffering capacity (Park et al, 2004;Manno et al, 2013b;Manno et al, 2017). The total [Ca 2+ ]SR decreases moderately during normal muscle function.…”