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Ca2+ uptake by the tubular (t-) system membrane of rat fast-twitch muscle

T.R. Cully,1 J.N. Edwards,2 T.R. Shannon2 and B.S. Launikonis,1 1School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia and 2Department of Molecular Physiology and Biophysics, Chicago, IL 60612, USA.

The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane. The t-system forms a junction with the terminal cisternae of the sarcoplasmic reticulum (SR) at every sarcomere of skeletal muscle. At any given moment the [Ca2+] within the small volume bound by the junctional membranes will be critically determined by the leak of Ca2+ through ryanodine receptors and the net Ca2+ handling ability of the t-system. The Ca2+ in this microdomain can change rapidly on a very large scale during excitation-contraction coupling and also under situations where ryanodine receptor leak alters or the Ca2+ handling ability of the t-system may change, as during metabolic fatigue. In this study we aimed to assess the basic Ca2+ handling ability of the t-system of rat fast-twitch muscle fibres.

Wistar rats were euthanized under CO2 asphyxiation and the extensor digitorum longus (EDL) muscles were rapidly dissected under protocols approved by the Animal Ethics Committee of The University of Queensland. Isolated EDL muscles were pinned in a Petri dish above a layer of Sylgard under a layer of paraffin oil. A bundle of fibres were then isolated and exposed to a physiological solution containing (mM): fluo-5N or rhod-5N, 1-10; NaCl, 145; KCl, 3; MgCl2, 2 and HEPES, 10 (pH adjusted to 7.4 in NaOH). In some solutions all Na+ was replaced with K+. After waiting 10 – 15min, isolated fibres were mechanically skinned and transferred to an experimental chamber and bathed in an internal solution containing (mM): EGTA, 50; Na+, 36; K+, 126; Mg2+, 1; total ATP, 8; creatine phosphate, 10 and HEPES, 90 (pH adjusted to 7.1 in KOH). Free Ca2+ was adjusted in the range 0-800 nM. Ca2+-dependent fluorescence was continuously imaged on an Olympus FV1000 confocal microscope in xyt mode, with the dyes excited by laser lines 488 or 543 nm.

In situ calibration determined the half signal of fluo-5N and rhod-5N in the t-system to be close to 335 and 872 μM, respectively. Further experiments were conducted with rhod-5N as mM levels of Ca2+ were expected to be achieved in the t-system ([Ca2+]t-sys). Chronic depletion of [Ca2+]SR with caffeine reduced [Ca2+]t-sys to 0.1 mM via chronic activation of store-operated Ca2+ entry. We then exposed Ca2+-depleted preparations to 0, 50, 100 and 800 nM (n = 4, 17, 10, 16 respectively) [Ca2+]cyto in 50 mM EGTA. At [Ca2+]cyto > 100 nM the [Ca2+]t-sys reached a plateau at 1.8-1.9 mM after 3-5 s. At [Ca2+]cyto < 100 nM the [Ca2+]t-sys did not always reach this plateau and showed a biphasic uptake of Ca2+. At the plateau [Ca2+]t-sys lowering [Ca2+]cyto to < 1 nM did not cause a significant loss of [Ca2+]t-sys. There was an apparent absence of effect of removing [Na+]cyto on these results. Mathematical modeling of these results suggests that the plasma membrane CaATPase (PMCA) with its low Km for Ca2+ is the major protein responsible for t-system Ca2+ uptake in the resting muscle, despite the higher transport capacity of the Na-Ca exchanger. Furthermore, these results show that the t-system membrane is able to establish the “physiological” Ca2+ gradient from within the cytoplasm without a requirement for Ca2+ to enter the t-system from the extracellular fluid surrounding the fibre.