The mechanisms that terminate Ca2+ release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca2+ concentration inside the sarcoplasmic reticulum (SR), [Ca2+]SR, simultaneously with that in the cytosol, [Ca2+]c, during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca2+ release flux (derived from [Ca2+]c(t)) over the gradient that drives it (essentially equal to [Ca2+]SR) provided directly, for the first time, a dynamic measure of the permeability to Ca2+ of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca2+]SR stabilized at ∼35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ∼10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca2+ release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca2+]SR, sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca2+ release.

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

Current fluorescent monitors of free [Ca2+] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca2+. “D4cpv-Casq1,” expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective KD of 222 µM and a dynamic range [(Rmax − Rmin)/Rmin] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, Rmax, and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca2+]SR in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca2+]SR was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca2+ concentration.

Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca2+]cytosol. 42% of MHS patients have hyperglycemia. We show that phosphorylated glycogen phosphorylase (GPa), glycogen synthase (GSa) – respectively activated and inactivated by phosphorylation – and their Ca2+-dependent kinase (PhK), are elevated in microsomal extracts from MHS patients’ muscle. Glycogen and glucose transporter GLUT4 are decreased. [Ca2+]cytosol, increased to MHS levels, promoted GP phosphorylation. Imaging at ~100 nm resolution located GPa at sarcoplasmic reticulum (SR) junctional cisternae, and apo-GP at Z disk. MHS muscle therefore has a wide-ranging alteration in glucose metabolism: high [Ca2+]cytosol activates PhK, which inhibits GS, activates GP and moves it toward the SR, favoring glycogenolysis. The alterations probably cause these patients’ hyperglycemia. For basic studies, MHS emerges as a variable stressor, which forces glucose pathways from the normal to the diseased range, thereby exposing novel metabolic links.

Key points• The signal for skeletal muscle contraction is a rapid increase in cytosolic Ca 2+ concentration, which requires the coordinated opening of ryanodine receptor (RyR) channels in the sarcoplasmic reticulum.• Channel opening is controlled by voltage-sensing dihydropyridine receptors (DHPRs) of plasma membrane and T tubules. Whether or not their signal is amplified by Ca 2+ -induced Ca 2+ release (CICR) is controversial.• We used two-photon lysis of an advanced Ca 2+ cage to produce local Ca 2+ concentration transients that were large, fast, reproducible and quantifiable, while monitoring the cellular response with a dual confocal laser scanner.• Single frog muscle cells in physiological solutions responded to transients greater than 0.28 μM with propagated CICR waves.• Mouse cells did not respond to stimuli up to 8 μM, unless channel opening drugs were present.• We conclude that CICR contributes to physiological Ca 2+ release in frog but not mouse muscle.• Mice and presumably other mammals do have a capability for CICR that is normally inhibited.It could be manifested under special circumstances, including diseases.Abstract The contribution of Ca 2+ -induced Ca 2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca 2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca 2+ ] rapidly and reversibly, up to 8 μM, levels similar to those reached during physiological activity. They improve over previous paradigms in rate of rise, locality and reproducibility. Use of NDBF-EGTA allowed for the separate modification of resting [Ca 2+ ], trigger [Ca 2+ ] and resting [Mg 2+ ]. In frog muscle, SLICs elicited propagated responses that had the characteristics of CICR. The threshold [Ca 2+ ] for triggering a response was 0.5 μM or less. As this value is much lower than concentrations prevailing near channels during normal activity, the result supports participation of CICR in the physiological control of contraction in amphibian muscle. As SLICs were applied outside cells, the primary stimulus was Ca 2+ , rather than the radiation or subproducts of photorelease. Therefore the responses qualify as 'classic' CICR. By contrast, mouse muscle fibres did not respond unless channel-opening drugs were present at substantial concentrations, an observation contrary to the physiological involvement of CICR in mammalian excitation-contraction coupling. In mouse muscle, the propagating wave had a substantially lower release flux, which together with a much higher threshold justified the absence of response when drugs were not present. The differences in flux and threshold may be ascribed to the absence of ryanodine receptor 3 (RyR3) isoforms in adult mammalian muscle.

Key points• The rise in cytosolic calcium ion concentration that triggers muscle contraction requires release of a large amount of calcium from the cellular store, sarcoplasmic reticulum (SR), where it is stored bound, largely to the protein calsequestrin.• Binding of calcium by calsequestrin is a complex process, believed to involve changes in protein conformation and aggregation. We want to know to what extent these properties, observed in vitro, apply inside cells.• We measured the calcium buffering power of the SR, defined as the ratio of change in total SR calcium by change in free [Ca 2+ ] SR , in muscle cells of wild type or calsequestrin-lacking mice, using two different methods to monitor [Ca 2+ ] SR and deriving changes in total SR calcium content from simultaneous measurements of cytosolic [Ca 2+ ].• The average buffering power during a large, depleting calcium release event was 157 in the wild type and 40 in the calsequestrin-null mice, suggesting that three-quarters of the calcium released normally comes from the calsequestrin-bound pool.• The Ca 2+ buffering ability of the SR is different from that of the equivalent concentration of calsequestrin in aqueous solution, the SR exhibiting greater affinity and cooperativity. We conclude that calsequestrin adopts different properties inside cells.• SR buffering power depends on the SR Ca 2+ load and on the rate of its changes, a dependence that could be, at least in part, explained by the unique Ca 2+ binding properties of calsequestrin.• This study reveals Ca 2+ buffering as a highly dynamic process, marking it as both a vulnerable link in diseases that involve loss of control of Ca 2+ release, and a candidate for further study and intervention.Abstract The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca 2+ ], was determined in fast-twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca 2+ ] were measured integrating Ca 2+ release flux, determined with a cytosolic [Ca 2+ ] monitor. Free [Ca 2+ ] SR was measured using the cameleon D4cpv-Casq1. In 34 wild-type (WT) cells average B during the depolarization (ON phase) was 157 (SEM 26), implying that of 157 ions released, 156 were bound inside the SR. B was significantly greater when BAPTA, which increases release flux, was present in the cytosol. B was greater early in the pulse -when flux was greatest -than at its end, and greater in the ON than in the OFF. In 29 Casq1-null cells, B was 40 (3.6). The difference suggests that 75% of the releasable calcium is normally bound to calsequestrin. In the nulls the difference in B between ON and OFF was less than in the WT but still significant. This difference and the associated decay in B during the ON were not artifacts of a slow SR monitor, as they were also found in the WT when [Ca 2+ ] SR was tracked with the fast dye fluo-5N. The calcium buffering power, binding capacity and non-linear binding properties of the SR measured here could be accounted for ...

Key points• Malignant Hyperthermia (MH) affects the Ca 2+ movements that control muscle contraction. We measured Ca 2+ movements in skeletal muscle of "Y522S" mice, with a tyrosine-to-serine mutation in the RyR channel that causes MH in mice and humans.• In YS cells, [Ca 2+ ] inside the Ca 2+ store (sarcoplasmic reticulum, SR) was 45% of that in the wild type (WT), but the SR membrane permeability increased 2-fold, resulting in Ca 2+ release of initially normal value.• During Ca 2+ release, cytosolic [Ca 2+ ] and SR Ca 2+ buffering power evolved differently in YS and WT. These variables became similar in WT exposed to BAPTA, an inhibitor of Ca 2+ -dependent inactivation (CDI) of the RyR, suggesting that tyrosine 522 is involved in CDI.• Similar paradoxical observations in YS and WT cells with reduced content of the SR protein calsequestrin, revealed the importance of balance between SR Ca permeability (increased in YS) and storage capability (decreased when calsequestrin is low).Abstract Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca 2+ channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell 'cores' . Imaging cytosolic and intra-SR [Ca 2+ ] in muscle cells of heterozygous YS mice we determined Ca 2+ release flux activated by clamp depolarization, permeability (P) of the SR membrane (ratio of flux and [Ca 2+ ] gradient) and SR Ca 2+ buffering power (B). In YS cells resting [Ca 2+ ] SR was 45% of the value in normal littermates (WT). P was more than doubled, so that initial flux was normal. Measuring [Ca 2+ ] SR (t) revealed dynamic changes in B(t). The alterations were similar to those caused by cytosolic BAPTA, which promotes release by hampering Ca 2+ -dependent inactivation (CDI). The [Ca 2+ ] transients showed abnormal 'breaks' , decaying phases after an initial rise, traced to a collapse in flux and P. Similar breaks occurred in WT myofibres with calsequestrin reduced by siRNA; calsequestrin content, however, was normal in YS muscle. Thus, the Y522S mutation causes greater openness of the RyR1, lowers resting [Ca 2+ ] SR and alters SR Ca 2+ buffering in a way that copies the functional instability observed upon reduction of calsequestrin content. The similarities with the effects of BAPTA suggest that the mutation, occurring near the cytosolic vestibule of the channel, reduces CDI as one of its primary effects. The unstable SR buffering, mimicked by silencing of calsequestrin, may help precipitate the loss of Ca 2+ control that defines a fulminant MH event.

A novel category of diseases of striated muscle is proposed, the couplonopathies, as those that affect components of the couplon and thereby alter its operation. Couplons are the functional units of intracellular calcium release in excitation–contraction coupling. They comprise dihydropyridine receptors, ryanodine receptors (Ca2+ release channels), and a growing list of ancillary proteins whose alteration may lead to disease. Within a generally similar plan, the couplons of skeletal and cardiac muscle show, in a few places, marked structural divergence associated with critical differences in the mechanisms whereby they fulfill their signaling role. Most important among these are the presence of a mechanical or allosteric communication between voltage sensors and Ca2+ release channels, exclusive to the skeletal couplon, and the smaller capacity of the Ca stores in cardiac muscle, which results in greater swings of store concentration during physiological function. Consideration of these structural and functional differences affords insights into the pathogenesis of several couplonopathies. The exclusive mechanical connection of the skeletal couplon explains differences in pathogenesis between malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT), conditions most commonly caused by mutations in homologous regions of the skeletal and cardiac Ca2+ release channels. Based on mechanistic considerations applicable to both couplons, we identify the plasmalemma as a site of secondary modifications, typically an increase in store-operated calcium entry, that are relevant in MH pathogenesis. Similar considerations help explain the different consequences that mutations in triadin and calsequestrin have in these two tissues. As more information is gathered on the composition of cardiac and skeletal couplons, this comparative and mechanistic approach to couplonopathies should be useful to understand pathogenesis, clarify diagnosis, and propose tissue-specific drug development.