Imaging spatial distribution of release in single muscle fibres from Balanus nubilus using image intensification

Abstract

The physical location of the sites for Ca2+ release, triggering the contraction of a muscle fibre, is a key issue in understanding tbe physiological control mechanisms of excitation-contraction (EC) coupling [l]. The isolation of the ryanodine receptor from rabbit SR fractions [2] shows the terminal cistemal SR to contain ryanodine-sensitive Ca channels with a 1OOpS conductance. A second type of Ca channel, which has a 1OpS conductance and is IP3 sensitive at polarised potentials, has also been identified in the T system [3]. Electron microscopy has shown the ryanodine receptor to be located at tire triad, accounting for at least part of the foot structures [4]. Studies of the 1ocaliWion of Ca2+ release sites have been carried out in a number of ways. Somlyo et al. [5, 61 used an electron microprobe to compare the localisation of calcium at rest and during a tetanus. More recently, Sommer and Nassar [7] using a quick-freezing technique, which reportedly showed the appearance of ‘pits’ over the entire area of the SR examined, within the first millisecond after stimulation. Both these techniques have the drawback that they require fmation of the fibre. The aim of this study was to determine the spatial distribution of the calcium release sites in a single, intact_ electrical1 I+ excited muscle fibre, using the fluorescent Ca indicators Quin-2 and Fura[8] by employing an image intensifier. Single barnacle muscle cell sarcomere length is some g-10 pm [9] and this long sarcomere length provides the advantage that, using conventional light microscope optics, intrasarcomere detail could be better-resolved than for the shorter sarcomeres present in amphibian fibres. Prehminary accounts of the methods and results have already appeared [lo, 111.