Contents

Imaging tubular system, sarcoplasmic reticulum and myoplasmic calcium with novel fluorescence methods

The precise control of contraction in skeletal muscle critically depends on the rapid and precise delivery of Ca²⁺ from the specialized internal store, the sarcoplasmic reticulum (SR), which is under voltage control from the tightly apposed tubular (t-) system. Regulation of Ca²⁺ in the SR and cytoplasm during periods of muscle work is complex and involves influxes of Ca²⁺ from the t-system. A major problem is that measurements of voltage and store-dependent Ca²⁺ fluxes from the t-system of skeletal muscle are not possible with standard electrophysiological techniques. Methods to simultaneously image Ca²⁺ in two subcellular compartments of single muscle fibres using laser scanning confocal microscopy have recently been developed. This has largely involved trapping one Ca²⁺ sensitive dye in the t-system of a mechanically skinned fibre and introducing a second spectrally separate Ca²⁺ indicator to the cytoplasm. By stimulating release of Ca²⁺ directly from SR or via t-system depolarization, voltage dependent and independent fluxes across the t-system can be spatiotemporally resolved against the release flux of Ca²⁺ from SR. These measurements have identified an action potential-induced Ca²⁺ flux across the t-system and an ultra-rapid store-operated Ca²⁺ entry (SOCE) mechanism in skeletal muscle. This has led to the development of a working model for SOCE that is relevant within the large Ca²⁺ release fluxes initiated by excitation-contraction coupling. Potential roles of the action potential-induced Ca²⁺ flux in skeletal muscle remain speculative.