Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy

Intra-sarcomeric gradients of [Ca2+] during activation of action potential stimulated frog single fibres were investigated with the Ca2+ indicator fluo-3 and confocal and two-photon microscopy. The object of these experiments was to look for evidence of extra-junctional Ca2+ release and examine the microscopic diffusion of Ca2+ within the sarcomere.By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line-scanning rate (∼8000 lines s−1), although the laser scanning microscope was limited to < 1000 lines s−1. At this high time resolution, the time course of fluorescence changes was very different at the z- and m-lines, with a significant delay (∼1 ms; 22 °C) between the rise of fluorescence at the z-line and the m-line.To calculate the expected fluorescence changes, we used a multi-compartment model of Ca2+ movements in the half-sarcomere in which Ca2+ release was restricted to triadic junctions (located at z-lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca2+ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results.We conclude that there is no experimental evidence for Ca2+ release at locations other than at z-lines. In addition, our calculations support the conclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important in shaping the Ca2+ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre

Ca2+ sparks activate K+ and Cl− channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes

Local changes in cytosolic [Ca2+] were imaged with a wide-field, high-speed, digital imaging system while membrane currents were simultaneously recorded using whole-cell, perforated patch recording in freshly dissociated guinea-pig tracheal myocytes.Depending on membrane potential, Ca2+ sparks triggered ‘spontaneous’ transient inward currents (STICs), ‘spontaneous’ transient outward currents (STOCs) and biphasic currents in which the outward phase always preceded the inward (STOICs). The outward currents resulted from the opening of large-conductance Ca2+-activated K+ (BK) channels and the inward currents from Ca2+-activated Cl− (ClCa) channels.A single Ca2+ spark elicited both phases of a STOIC, and sparks originating from the same site triggered STOCs, STICs and STOICs, depending on membrane potential.STOCs had a shorter time to peak (TTP) than Ca2+ sparks and a much shorter half-time of decay. In contrast, STICs had a somewhat longer TTP than sparks but the same half-time of decay. Thus, the STIC, not the STOC, more closely reflected the time course of cytosolic Ca2+ elevation during a Ca2+ spark.These findings suggest that ClCa channels and BK channels may be organized spatially in quite different ways in relation to points of Ca2+ release from intracellular Ca2+ stores. The results also suggest that Ca2+ sparks may have functions in smooth muscle not previously suggested, such as a stabilizing effect on membrane potential and hence on the contractile state of the cell, or as activators of voltage-gated Ca2+ channels due to depolarization mediated by STICs