2-DIMENSIONAL CONFOCAL IMAGES OF ORGANIZATION, DENSITY, AND GATING OFFOCAL CA2+ RELEASE SITES IN RAT CARDIAC MYOCYTES

In cardiac myocytes Ca2+ cross-signaling between Ca2+ channels and rya nodine receptors takes place by exchange of Ca2+ signals in microdomai ns surrounding dyadic junctions, allowing first the activation and the n the inactivation of the two Ca2+-transporting proteins. To explore t he details of Ca2+ signaling between the two sets of receptors we meas ured the two-dimensional cellular distribution of Ca2+ at 240 Hz by us ing a novel confocal imaging technique, Ca2+ channel-triggered Ca2+ tr ansients could be resolved into dynamic ''Ca2+ stripes'' composed of h undreds of discrete focal Ca2+ releases, appearing as bright fluoresce nce spots (radius congruent to 0.5 mu m) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca2+ releases triggered stochastically by Ca2+ current (I-Ca) changed little in duration (congruent to 7 ms) an d size (congruent to 100,000 Ca ions) between -40 and +60 mV, but thei r frequency of activation and first latency mirrored the kinetics and voltage dependence of I-Ca. The resolution of 0.95 +/- 0.13 reproducib le focal Ca2+ release sites per mu m(3) in highly Ca2+-buffered cells, where diffusion of Ca2+ is limited to 50 nm, suggests the presence of about one independent, functional Ca2+ release site per half sarcomer e, The density and distribution of Ca2+ release sites suggest they cor respond to dyadic junctions. The abrupt onset and termination of focal Ca2+ releases indicate that the cluster of ryanodine receptors in ind ividual dyadic junctions may operate in a coordinated fashion.