Programme Contents

The effect of intracellular [Mg²⁺] on Ca²⁺ handling by the sarcoplasmic reticulum

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 K_d 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.