Analysis of osmotic stress induced Ca²⁺ spark termination in mammalian skeletal muscle

Ca²⁺ sparks represent synchronous opening of the ryanodine receptor (RyR) Ca²⁺ release channels located at the sarcoplasmic reticulum (SR) membrane. Whereas a quantal nature of Ca²⁺ sparks has been defined in cardiac muscle, the regulation of Ca²⁺ sparks in skeletal muscle has not been well-studied. Osmotic-stress applied to an intact skeletal muscle fiber can produce brief Ca²⁺ sparks and prolonged Ca²⁺ burst events. Here, we show that termination of Ca²⁺ bursts occurs in a step wise and quantal manner. Ca²⁺ burst events display kinetic features that are consistent with the involvement of both stochastic attrition and coordinated closure of RyR channels in the termination of SR Ca²⁺ release. Elemental unitary transition steps could be defined with a mean ΔF/F₀ of ~0.28, corresponding to the gating of 1-2 RyR channels. Moreover, the amplitude of the elemental transition steps declines at the later stage of the burst event. In tandem Ca²⁺ burst events where two Ca²⁺ bursts occur at the same position within a fiber in rapid succession, the trailing event is consistently of lower amplitude than the initial event. These two complementary results suggest that SR Ca²⁺ release may be associated with local depletion of SR Ca²⁺ stores in mammalian skeletal muscle.