Programme Contents

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

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 K_m 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.