APPS November 2002 Meeting Abstract 1358

It is not clear if increasing the sarcoplasmic reticulum (SR) Ca²⁺ content will increase, decrease or have no effect on the skeletal muscle ryanodine receptor Ca²⁺ efflux rates^{1, 2}. We examined this using single mechanically-skinned fast-twitch muscle fibres from the rat that retain functional E-C coupling. Skinned fibres were mounted on a force transducer and then transferred to a bath containing a solution that mimicked the normal intracellular milieu (ie. (mM): K⁺, 125; Na⁺, 37; hexamethylene-diamine-tetraacetate (HDTA^2-), 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). Twitch responses were elicited by electric field stimulation as previously described³.

To quantify the amount of Ca²⁺ released with each twitch, Ca²⁺ reaccumulation by the SR was prevented by inhibiting the SR Ca²⁺-ATPases with 50 µM TBQ and Ca²⁺ release was rapidly buffered with a known quantity of the fast Ca²⁺ buffer, BAPTA (250 µM), such that the twitch size was submaximal and a sensitive indicator of Ca²⁺ release. In fibres that initially contained the endogenous level of SR Ca²⁺, the first twitch elicited in TBQ/BAPTA was ~40% of the maximum Ca²⁺-activated force (and was indicative of ~300 µM Ca²⁺ released). Thereafter, 2-3 more twitches were elicited (each progressively smaller) before the SR was almost fully depleted. When the SR of fibres was then near-maximally loaded with Ca²⁺ (~3-4 times endogenous level), the first and as many as 12 subsequent twitches (total of 15) were virtually unchanged (~40% of maximum force) with the last 3-4 twitches declining as the SR became depleted as above. This data shows that raising the SR Ca²⁺ content does not affect the amount of Ca²⁺ released with each twitch.