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-tubule). 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. The net leak of Ca2+ contributes minimally to the overall [Ca2+]cyto at rest, but increases metabolic rate and ATP turnover. Importantly the RyR Ca2+ leak sets a standing Ca2+gradient, with a high [Ca+2] in the junctional space between the SR and t-tubule membrane. In situations such as metabolic fatigue [Mg2+]cyto can increase following the consumption of MgATP to Mg2+, ADP and inorganic phosphate. In the present study we aimed to assess the effect of [Mg2+]cyto on RyR Ca2+ leak. To do this mechanically skinned fibers with the t-tubule loaded with a Ca2+ indicator were used. Measurements of Ca2+ dependent fluorescence from inside the t-tubules allowed determination of t-tubule Ca2+ATPase (PMCA) activity. This activity is directly dependent on the [Ca2+] in the junctional space, set by RyR Ca2+ leak. Thus the effect of changing [Mg2+]cyto on RyR Ca2+ leak can be determined from these measurements.
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 CO2 asphyxiation and the extensor digitorum longus (EDL) muscles were removed. The t-tubule were loaded with a Ringer solution containing Rhod-5N and then mechanically skinned, trapping the dye in the t-tubule. The preparation was then transferred to an experiment chamber containing a physiological internal solution. Rhod-5N fluorescence signals were imaged on an Olympus FV1000 confocal microscope. The release of SR Ca2+ was induced by treating the cell with a 30 mM caffeine solution. The SR and t-tubule were then loaded in solutions with 50-800nM [Ca2+] and 0.2-3mM [Mg2+]. In some experiments the skinned fiber were exposed to the same levels of [Ca2+] and [Mg2+] but in the presence of 1mM Tetracaine, a RyR inhibitor. Rhod-5N t-tubule signals were calibrated with [Ca2+] as described previously (Cully et al., 2013).
Exposing the preparation to a 30mM caffeine solution induced store-dependent entry and reduced [Ca2+]t–tubule to 0.1mM. The t-system Ca2+ uptake rate and steady state [Ca2+]t-tubule was not changed by increasing [Mg2+]cyto from 1mM to 3mM. In the presence of 1mM Tetracaine, both the [Ca2+]t-tubule and t-tubule peak uptake flux were reduced. Collectively, this suggests that RyR leak rate was not effected by raising [Mg2+]cyto from 1 to 3mM. Interestingly, lowering the [Mg2+]cyto to 0.2mM significantly reduced the steady state [Ca2+]t-tubule, suggesting that the potentially increased uptake of Ca2+ in the presence of greater junctional space [Ca2+] was offset by Ca2+ loss from the t-tubule via a store dependent entry pathway. Reducing RyR leak with 1mM Tetracaine in 0.2 mM Mg2+ partially restored the t-tubule [Ca+2].
Cully TR, Edwards JN, Shannon TR, Launikonis BS. (2013). Proceedings of the Australian Physiological Society 44: 61P.