Effect of sarcoplasmic reticulum Ca2+ content on action potential-induced Ca2+ release in rat skeletal muscle fibres

This study examined the relationship between the level of Ca 2+ loading in the sarcoplasmic reticulum (SR) and the amount of Ca 2+ released by an action potential (AP) in fast-twitch skeletal muscle fibres of the rat. Single muscle fibres were mechanically skinned and electric field stimulation was used to induce an AP in the transverse-tubular system and a resulting twitch response. Responses were elicited in the presence of known amounts (0–0.38 mM) of BAPTA, a fast Ca 2+ buffer, with the SR Ca 2+ pump either functional or blocked by 50 μM 2,5-di-tert-butyl-1,4-hydroquinone (TBQ). When Ca 2+ reuptake was blocked, an estimate of the amount of Ca 2+ released by an AP could be derived from the size of the force response. In a fibre with the SR loaded with Ca 2+ at the endogenous level (≈1.2 mM, expressed as total Ca 2+ per litre fibre volume; approximately one-third of maximal loading), a single AP triggered the release of ≈230 μM Ca 2+ . If a second AP was elicited 10 ms after the first, only a further ≈60 μM Ca 2+ was released, the reduction probably being due to Ca 2+ inactivation of Ca 2+ release. When Ca 2+ reuptake was blocked, APs applied 15 s apart elicited similar amounts of Ca 2+ release (≈230 μM) on the first two or three occasions and then progressively less Ca 2+ was released until the SR was fully depleted after a total of approximately eight APs. When the SR was loaded to near-maximal capacity (≈3–4 mM), each AP (or pair of APs 10 ms apart) still only released approximately the same amount of Ca 2+ as that released when the fibre was endogenously loaded. Consistent with this, successive APs (15 s apart) elicited similar amounts of Ca 2+ release ≈10–16 times before the amount released declined, and the SR was fully depleted of Ca 2+ after a total release calculated to be ≈3–4 mM. When the SR was loaded maximally, increasing the [BAPTA] above 280 μM resulted in an increase in the amount of Ca 2+ released per AP, probably because the greater level of cytoplasmic Ca 2+ buffering prevented Ca 2+ inactivation from adequately limiting Ca 2+ release. These results show that the amount of Ca 2+ released by AP stimulation in rat fast-twitch fibres normally stays virtually constant over a wide range of SR Ca 2+ content, in spite of the likely large change in the electrochemical gradient for Ca 2+ . This was also found to be the case in toad twitch fibres. This constancy in Ca 2+ release should help ensure precise regulation of