Monika Sztretye

Indo-1 derivatives for local calcium sensing.

The role of calcium in signal transduction relies on the precise spatial and temporal control of its concentration. The existing means to detect fluctuations in Ca2+ concentrations with adequate temporal and spatial resolution are limited. We introduce here a method to measure Ca2+ concentrations in defined locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1 to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in single cells dissociated from muscle of adult mice expressing a nucleus-localized SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by shifted excitation and emission ratioing of confocal microscopic images of fluorescence. After permeabilizing the plasma membrane, changes in the bathing solution induced corresponding changes in nuclear [Ca2+] that were readily detected and used for a preliminary calibration of the technique. This work thus demonstrates the synthesis and application of SNAP-tag-based Ca2+ indicators that combine the spatial specificity of genetically encoded calcium indicators with the advantageous spectroscopic properties of synthetic indicators.

Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca 2+ release in skeletal muscle

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.

Paradoxical buffering of calcium by calsequestrin demonstrated for the calcium store of skeletal muscle.

Contractile activation in striated muscles requires a Ca2+ reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca2+] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca2+ signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca2+ release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca2+ permeability and inversely to its Ca2+ buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca2+ depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca2+ depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca2+], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca2+, but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca2+ as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca2+ storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.

D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle.

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.

Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor.

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala(8)]MCa, [Ala(19)]MCa, [Ala(20)]MCa, [Ala(22)]MCa, [Ala(23)]MCa, and [Ala(24)]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical (24)Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys(20), Lys(22), Arg(23), Arg(24), and Lys(8). Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.

Altered expression of triadin 95 causes parallel changes in localized Ca2+ release events and global Ca2+ signals in skeletal muscle cells in culture.

The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome‐ or adenovirus‐mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization‐evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin‐specific immunoreactivity. Functional experiments on these cells revealed that while caffeine‐evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.

Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling

Hypermuscularity associated with naturally occurring mutations in the myostatin gene as found in Compact mice results in increased muscle mass but reduced specific force. The calcium sensitivity of the contractile apparatus as assessed on chemically skinned skeletal muscle fibres under isometric conditions is not altered in these animals. While the resting calcium concentration remains unaffected, depolarization‐evoked increases in intracellular calcium concentration are suppressed. Spontaneous calcium release events from sarcoplasmic reticulum are also decreased in frequency, amplitude and spatial spread. Our results suggest that mutations in the myostatin gene are accompanied by alterations in excitation contraction coupling, which manifest as a reduction in sarcoplasmic calcium release.

Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle

•  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 [Ca2+]SR, in muscle cells of wild type or calsequestrin‐lacking mice, using two different methods to monitor [Ca2+]SR and deriving changes in total SR calcium content from simultaneous measurements of cytosolic [Ca2+]. •  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 Ca2+ 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 Ca2+ load and on the rate of its changes, a dependence that could be, at least in part, explained by the unique Ca2+ binding properties of calsequestrin. •  This study reveals Ca2+ buffering as a highly dynamic process, marking it as both a vulnerable link in diseases that involve loss of control of Ca2+ release, and a candidate for further study and intervention.

Restricting calcium currents is required for correct fiber type specification in skeletal muscle

ABSTRACT Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease. Summary: Developmental splicing of CaV1.1 restricts calcium currents in skeletal muscle, which are critical for activity-dependent regulation of fiber type specification and the prevention of mitochondrial damage.

SOCE Is Important for Maintaining Sarcoplasmic Calcium Content and Release in Skeletal Muscle Fibers

Store-operated Ca2+ entry (SOCE) is a Ca2+-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca2+ release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.