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Confocal imaging of lumenal and cytosolic [Ca²⁺] during Ca²⁺ release in skeletal muscle

Rapid Ca²⁺ release from the sarcoplasmic reticulum (SR) is essential for normal contraction of muscle. To study Ca²⁺ release in muscle we simultaneously imaged mag-indo-1 trapped in SR with cytosolic rhod-2 in skinned skeletal muscle fibres of frog using confocal microscopy. [Ca²⁺] depletion inside the SR measured during spontaneous Ca²⁺ sparks, termed “skraps”, did not simply follow the Ca²⁺ release time course observed in the cytosolic Ca²⁺ image, but showed a ∼20 ms delay between the peak of the spark and the nadir of the skrap. A similar result was observed when Ca²⁺ release was induced by an action potential. This suggests that depletion continues even after Ca²⁺ release channels have closed. A further intriguing observation was an intra-SR Ca²⁺ transient during prolonged Ca²⁺ release induced by lowering [Mg²⁺]_cyto. Such an increase in [Ca²⁺]_SR during a decrease in total SR [Ca] would violate mass conservation laws. However, these observations can be explained in the framework of the properties of calsequestrin (CSQ), a Ca²⁺-buffering protein attached to the lumenal side of Ca²⁺ release channels (Dulhunty et al., 2006): (i) CaCSQ represents a third compartment, a proximate source of Ca²⁺ for release and is invisible to the monitoring dye, thus explaining the apparent delay between cytosolic release and lumenal Ca²⁺ depletion; and (ii) CSQ depolymerizes as the total SR [Ca] falls. Thus as CSQ breaks into dimers and monomers, the capacity of CSQ for Ca²⁺ drops, resulting in a Ca²⁺ transient within the SR during prolonged release. The strategic location and reduction in dimensionality of Ca²⁺-adsorbed linear polymers of CSQ could deliver Ca²⁺ more efficiently to the release channels than lumenal Ca²⁺.

Dulhunty, A.F., Varsanyi, M., Wei, L. & Beard, N.A. (2006) Proceedings of the Australian Physiological Society, 37: 26P