Programme Contents

The effects of membrane potential and cytoplasmic calcium concentration on calcium extrusion across the tubular system in mammalian skeletal muscle

The regulation of cytoplasmic Ca²⁺ is essential in the maintenance of skeletal muscle function and survival. This is achieved by the combined activities of ion transporters such as SERCA, NCX, PMCA, found embedded within the sarcoplasmic reticulum (SR) and plasma membranes. Upon Ca²⁺ release from the SR, [Ca²⁺]_cyto can globally reach ∼2μM and it is during such an event that highlights Ca²⁺ extrusion is an essential regulatory mechanism, as this is ∼20 times the resting [Ca²⁺]_cyto. We have shown that during cell wide Ca²⁺ release events, there is an initial increase in t-system (extracellular) Ca²⁺, followed by a net decline due to the activation of a store-operated calcium entry (SOCE) current. Although the SOCE current has recently generated significant levels of interest, information on the rates of t-system Ca²⁺ uptake (cytoplasmic Ca²⁺ extrusion) remains limited. Therefore, we aimed to characterise the effects of membrane potential and [Ca²⁺]_cyto on the rate of cytoplasmic Ca²⁺ extrusion in mammalian skeletal muscle.

Wistar rats were killed by asphyxiation in accordance to the guidelines set by the Animal Ethics Committee of the University of Queensland. Extensor digitorum longus and soleus muscles were rapidly excised, pinned out and fully immersed in paraffin oil. Small bundles of intact fibres were isolated and exposed to a Na⁺-based physiological solution containing the fluorescent dye, fluo-5N salt. Single fibres were then isolated and mechanically skinned (resulting in the trapping the dye in the t-system) and transferred to a chamber containing a K⁺ or Na⁺ -based internal solution to set the membrane potential. A ‘release solution’ with low Mg²⁺, 5mM BAPTA and 5mM caffeine was used to chronically deplete sarcoplasmic reticulum Ca²⁺ stores and activate SOCE. T-system fluo-5N fluorescence was imaged on an Olympus FV1000 confocal microscope in either xy or xyt mode in polarized and depolarized fibres with known cytoplasmic Ca²⁺ concentrations, at rest or during SR Ca²⁺ release. The net change in t-system fluo-5N signal was used as an indicator of Ca²⁺ movements across the t-system.

Fluo 5N trapped in the sealed t-system was calibrated (in situ) and in the presence of different concentrations of bovine serum albumin. Following low [Ca²⁺]_t-system (achieved by chronic activation of SOCE with ‘release solution’) the t-system could be reloaded with internal solutions containing 1 mM EGTA (either 100, 200 or 800 nM free Ca²⁺). This rate of uptake was markedly greater in depolarized cells (Na+ based solutions) compared to polarized cells (K⁺ based solutions), most likely due to an increased driving force for Ca²⁺ to exit the cell. Interestingly, vacuoles were seen in some fibres. Vacuoles retained fluo 5N for > 20mins in the presence of a ‘release solution’ whereas a fluorescence signal from transverse tubules was rapidly lost. We have also measured for the first time, a SOCE flux and t-system uptake in slow-twitch fibres from the soleus muscle which are currently being explored.