Excitation-contraction (EC) coupling in skeletal muscle is the process by which an action potential (AP) activates a global increase in [Ca2+]i which then induces contraction. The global increase in [Ca2+]i is mediated by the release of Ca2+ from the lumen of the sarcoplasmic reticulum (SR) through the opening of ryanodine receptors (RyR1). We examined the structure and function of the SR Ca2+ store dynamically, using the low affinity Ca2+ indicator, fluo-5N, which when loaded as an -AM derivative concentrates in the lumen of the SR. Fluo-5N has been used extensively to measure SR Ca2+ and to characterize the Ca2+ stores in cardiac myocytes, with high spatial and temporal resolution (Brochet et al 2005; Wu and Bers 2006). Using dissociated flexor digitorum brevis (FDB) fibers in culture, we studied the dynamics of fluo-5N in the SR and connecting compartments. Fluo-5N accumulates in transverse structures that align with Z-disks, consistent with the location of a major compartment of the SR in adult skeletal muscle. That these structures are primarily SR in nature is indicated by the fact that fluo-5N fluorescence decreases when fibers are exposed to caffeine. FRAP experiments demonstrated similar recovery constants for SR-trapped fluo-5N to those we have previously recorded in rat cardiac myocytes. We will be using fluo-5N to study the organization of the SR, the changes in Ca2+ dynamics in the SR during EC coupling, and the role of different proteins of the SR in regulating SR [Ca2+].
