Deconstructing calsequestrin. Complex buffering in the calcium store of skeletal muscle

Abstract

Since its discovery in 1971, calsequestrin has been recognized as the main Ca(2+) binding protein inside the sarcoplasmic reticulum (SR), the organelle that stores and upon demand mobilizes Ca(2+) for contractile activation of muscle. This article reviews the potential roles of calsequestrin in excitation-contraction coupling of skeletal muscle. It first considers the quantitative demands for a structure that binds Ca(2+) inside the SR in view of the amounts of the ion that must be mobilized to elicit muscle contraction. It briefly discusses existing evidence, largely gathered in cardiac muscle, of two roles for calsequestrin: as Ca(2+) reservoir and as modulator of the activity of Ca(2+) release channels, and then considers the results of an incipient body of work that manipulates the cellular endowment of calsequestrin. The observations include evidence that both the Ca(2+) buffering capacity of calsequestrin in solution and that of the SR in intact cells decay as the free Ca(2+) concentration is lowered. Together with puzzling observations of increase of Ca(2+) inside the SR, in cells or vesicular fractions, upon activation of Ca(2+) release, this is interpreted as evidence that the Ca(2+) buffering in the SR is non-linear, and is optimized for support of Ca(2+) release at the physiological levels of SR Ca(2+) concentration. Such non-linearity of buffering is qualitatively explained by a speculation that puts together ideas first proposed by others. The speculation pictures calsequestrin polymers as 'wires' that both bind Ca(2+) and efficiently deliver it near the release channels. In spite of the kinetic changes, the functional studies reveal that cells devoid of calsequestrin are still capable of releasing large amounts of Ca(2+) into the myoplasm, consistent with the long term viability and apparent good health of mice engineered for calsequestrin ablation. The experiments therefore suggest that other molecules are capable of providing sites for reversible binding of large amounts of Ca(2+) inside the sarcoplasmic reticulum.

Figure 1. The calsequestrin network in skeletal muscle

A, electron microscopic image from mouse FDB. Colorization by hand, according to the following code: yellow, Casq network; blue-green, ‘tendrils’ connecting calsequestrin network to junctional SR; orange, RyRs. B, deep etch image from toadfish muscle. Arrows indicate tendrils. Unpublished images of C. Franzini-Armstrong and S. Boncompagni.

Figure 2. Ca²⁺ binding properties of calsequestrins in aqueous solution

A, fractional occupancy (Y=[bound Ca²⁺]/[total Casq]) was plotted against unbound ligand concentration. Inset: magnified view of the Ca²⁺ range 0–0.7 mm. B, scatchard-type plot of the same data, which shows that the dissociation constant (slope) varies according to the degree of ligand binding. sCSQ represents Casq1, cCSQ is Casq2 and ΔC27 a mutant with the last 27 residues deleted. Note that the mutant shows no transitions in binding capacity. Reproduced from Park et al. (2004).

Figure 3. ‘Skraps’ of depletion inside the skeletal SR

A, surface representation of an average of 6000 sparks (yellow) in line-scans of fluorescence of fluo-4 inside membrane-permeabilized frog semitendinosus muscle fibres. Above the spark, in ‘rainbow’ palette, is the average of simultaneously recorded skraps, from SEER ratio images of fluorescence of mag-indo 1 inside the SR. B, evolution of spark (blue) and skrap (red) amplitude at their spatial maxima. The time to nadir of the skrap (red bracket) outlasts by ∼60 ms the time to peak of the spark (blue bracket). This delay, confirmed with other dyes, suggests the existence of a ‘proximate source’ for Ca²⁺ release that is different from the free lumenal SR Ca²⁺ monitored by mag-indo 1. Modified from Launikonis et al. (2006).

Figure 4. Intra-SR release of Ca²⁺. Simultaneous imaging of cytosolic and SR [Ca²⁺] in a frog muscle fibre with permeabilized plasma membrane

A, xy scans of fluorescence F₃ of rhod-2 in cytosol. B, [Ca²⁺]_SR derived from ratios of simultaneous SEER images of mag-indo 1 inside SR. C, image averages of F₃/F_3,0 (red) and [Ca²⁺]_SR (blue). During acquisition of the 2nd set (arrow in A), the solution was changed to one with low Mg²⁺ (upper blue trace in C), eliciting Ca²⁺ release. An increase in [Ca²⁺]_SR followed shortly after the beginning of the cytosolic transient. Green, time course of net Ca²⁺ release flux. The second peak of release flux, accompanied by a peak of intra-SR release (blue), again implies input from an additional source, presumably calsequestrin. Republished from Launikonis et al. (2006).

Figure 5. The NFRC, an index of non-exponential decay of release flux

A, line scan of rhod-2 fluorescence in a voltage-clamped mouse FDB cell stimulated by a 400 ms pulse to 0 mV. B, cytosolic [Ca²⁺](t) derived from the averaged line scan. C, black trace, release flux derived from the record in B, showing a ‘shoulder’ or sigmoidal decay, starting at level QS following the early peak and ending at steady level S. Green trace, NFRC(t), calculated according to equation in text. Note its steady growth during the time of the shoulder of flux. If flux decayed exponentially, NFRC would be constant. Calculation of NFRC is stopped when becomes small compared with noise. Reproduced from Royer et al. (2008).

Figure 6. Aggregation-dependent buffering by calsequestrin?

A pictorial summary of ideas (MacLennan & Reithmeier, 1998; Park et al. 2003, 2004) which may help explain the observations. A, linear polymers of Casq1, with stereotyped alternation of front-to-front and back-to-back interactions, linked to the channel by triadin or junctin (green). Polymers feature a layer where Ca²⁺ ions (red) are adsorbed and may diffuse length-wise. These putative ‘calcium wires’, which copy structures visible in EM images (Fig. 1), link to a TC-wide network of Casq1 molecules that is not shown. B–D, sequential changes of the polymeric Casq1 network proposed to occur during Ca²⁺ release. Progressive depletion leads to de-aggregation. If the Ca²⁺ layer adsorbed on calcium wires is delivered to the channels more rapidly than dissolved Ca²⁺, the buffering power B of the SR will decay as the Ca²⁺ wires are emptied or their connection to the channels collapse. The evolution of B is depicted by the height of the orange bar.