Increasing the Ca²⁺ content of the sarcoplasmic reticulum (SR) in mammalian skeletal muscle fibres enhances sensitivity to caffeine-induced and Ca²⁺-induced Ca²⁺ release (Lamb et al. 2001). Here we examine what effect SR Ca²⁺ content has on the physiological release mechanism which involves an action-potential in the transverse-tubular (T-) system activating the voltage-sensors and triggering Ca²⁺ release.

Hooded rats were painlessly killed by halothane overdose and the extensor digitorum longus (EDL) muscle dissected. Single fibres were mechanically skinned under paraffin oil, attached to a force tranducer and bathed in a solution that mimicked the normal intracellular milieu (mM: K⁺, 125; Na⁺, 37; hexamethylene-diamine-tetraacetate, 50; total ATP, 8; total Mg²⁺, 8.6; free Mg²⁺, 1; creatine phosphate, 10; total EGTA, 0.05; Hepes, 90; NaN₃, 1; pH 7.10 ± 0.01 and free [Ca²⁺], 0.0001) at 24 ± 1°C. Electric field stimulation was used to elicit an action-potential in the sealed T-system, which induced a twitch response in the skinned fibre, as previously described (Posterino et al. 2000). The amount of Ca²⁺ released by an action potential could be quantified from the force response when Ca²⁺ reaccumulation by the SR was blocked (with 50 µM 2-5-di-tert-butyl-1,4-hydroquinone; TBQ) and Ca²⁺ release was rapidly buffered with a known quantity of the fast Ca²⁺ buffer, BAPTA (250 µM). The SR of the skinned EDL fibres initially contained only the amount of Ca²⁺ present endogenously, and the first twitch response elicited in TBQ/BAPTA reached ~40 % of the maximum Ca²⁺-activated force, indicating ~300 µM Ca²⁺ was released. Thereafter, two to four more twitch responses could be elicited before the SR was almost fully depleted. The SR of fibres was then near-maximally loaded with Ca²⁺ (~3-4 times endogenous level; Fryer & Stephenson, 1996), and it was found that the first response in TBQ/BAPTA was very similar to that with the endogenous SR Ca²⁺ content (~40 % of maximum force) and the response to successive stimuli remained at approximately the same level for up to 12 repetitions before declining over the last three to four twitches as the SR became depleted as above.

These data indicate that the amount of Ca²⁺ released by an action potential is virtually unchanged when the SR Ca²⁺ content is increased from the endogenous level to near maximum capacity.