Abstract
The tubular (t-) system of skeletalmuscle is an internalization of the plasma membrane that maintains a large Ca2+ gradient and exchanges Ca2+ between the extracellular and intracellular environments. Little is known of the Ca2+-handling properties of the t-systemas the small Ca2+ fluxes conducted are difficult to resolvewith conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca2+](t-sys), allowing confocal measurements of Ca2+-dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca2+] transients in the t-system ([Ca2+](t-sys) (t)). Furthermore, t-system Ca2+-buffering power was determined so that t-system Ca2+ fluxes could be derived from [Ca2+](t-sys) (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca2+ induced a robust store-operated Ca2+ entry (SOCE) in fast-and slow-twitch fibres, reducing [Ca2+](t-sys) to < 0.1 mM. The rapid activation of SOCE upon Ca2+ releasewas consistentwiththe presence of STIM1L inbothfibre types. Abruptly introducing internal solutions with 1mM Mg2+ and [Ca2+](cyto) (28 nM-1.3 mu M) to Ca2+-depleted fibres generated t-system Ca2+ uptake rates dependent on [Ca2+](cyto) with [Ca2+](t-sys) reaching final plateaus in themillimolar range. For the same [Ca2+](cyto), t-system Ca2+ fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca2+ signals indicated that both membrane compartments accumulated Ca2+ at similar rates and that SOCE was activated early during SR Ca2+ depletion.