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Effect of changing the [Mg2+] on ryanodine receptor leak activity in rat skeletal muscle

R.H. Choi and B.S. Launikonis, School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia.

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.