In skeletal muscle, the release of Ca2+ from the sarcoplasmic reticulum (SR) is mediated by the ryanodine receptors (RyR) which are activated by the voltage-sensors of the transverse tubule (t-sys). Cytoplasmic Mg2+ provides inhibition of the RyR at rest to reduce Ca2+ leakage by binding to a low affinity inhibitory site. Ca2+ leaked from the RyR is resequestered by the SR Ca2+ATPase (SERCA) pump. In the present study we aimed to assess the effect of [Mg2+]cyto on SR Ca2+ handling and specifically RyR Ca2+ leak. To do this, mechanically skinned fibres were loaded with Ca2+ dependent indicators inside the SR or t-sys. Measurements of Ca2+ dependent fluorescence from inside the t-sys allowed determination of t-sys Ca2+ handling activity that was directly dependent on the [Ca2+] in the junctional space, set by RyR Ca2+ leak (Cully et al., 2015).
The use of animals in this study was approved by the Animal Ethics Committee at the University of Queensland. 2 month old Wistar rats were culled by C02 asphyxiation and the extensor digitorum longus (EDL) muscles were removed. Bundles of fibres were isolated and exposed to a Ringer solution containing Rhod-5N and then mechanically skinned, trapping the dye in the t-sys. For SR dye loading, mechanically skinned fibres were mounted to an experimental chamber, and bathed in an internal solution with 10μM Fluo-5N AM, 10μM carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and 0.05% Pluronic detergent for 1 hour at 30°C. An additional 1 hour incubation at room temperature in the absence of Fluo-5N AM was given to allow for complete hydrolysis. The release of SR Ca2+ was induced by exposing the cell to a 0.01mM Mg2+ solution containing 30 mM caffeine. The SR and t-sys were then loaded in solutions with varying amounts of free [Ca2+] and with the free [Mg2+] set at 0.13, 1, or 10mM. Rhod-5N t-sys signals were calibrated with [Ca2+] as described (Cully et al., 2013). Fluorescence signals were imaged on an Olympus FV1000 confocal microscope.
Steady state [Ca2+]t–sys over a range of [Ca2+]cyto, were reduced in the presence of 10mM [Mg2+]cyto, presumably due to reduced [Ca2+] in the junctional space. This indicates blocked RyR Ca2+ leak by 10mM Mg2+. An in-situ calibration of intra-SR Fluo-5N determined a Kd value of 418±36μM, allowing direct comparison of [Ca2+]SR between fibres. At 1 and 10 mM [Mg2+]cyto the free [Ca2+]SR was similar. This suggests that the block of RyR Ca2+ leak do not significantly change [Ca2+]SR. Lowering the [Mg2+]cyto to 0.13mM significantly reduced the steady state [Ca2+]t-sys, and this was partially restored in the presence of 1mM tetracaine (0.13mM Mg2+), an RyR inhibitor. This suggests that the potentially increased uptake of Ca2+ in the presence of greater junctional space [Ca2+] (due to increased RyR Ca2+ leak) was offset by Ca2+ influx from the t-sys via a store dependent entry pathway (Cully et al., 2015). Lowering [Mg2+]cyto from 1 to 0.13mM minimally altered the free [Ca2+]SR, demonstrating that SOCE is active despite the presence of high free [Ca2+]SR.
Cully TR, Edwards JN, Shannon TR, Launikonis BS. (2013) Ca2+ uptake by the tubular (t-) system membrane of rat fast-twitch muscle. Proc Aust Physiol Soc 44, 61P.
Cully TR, Roberts L, Fassett R, Raastad T, Sax J, Coombes JS, Launikonis BS, (2015) The tubular (t-) system is a dynamic Ca2+-buffer in human skeletal muscle fibres. Proc Aust Physiol Soc 46, 104P.