FUNDAMENTAL CALCIUM-RELEASE EVENTS REVEALED BY 2-PHOTON EXCITATION PHOTOLYSIS OF CAGED CALCIUM IN GUINEA-PIG CARDIAC MYOCYTES

1. In cardiac muscle, 'Ca2+ sparks' have been proposed to underlie Ca2 +-induced Ca2+ release (CICR), and to result from openings of clusters of Ca2+ channels (ryanodine receptors; RyRs) located in the sarcoplas mic reticulum membrane. 2. To investigate the elementary nature of the se Ca2+ signals directly, a diffraction-limited point source of Ca2+ w as created in single cardiac myocytes by two-photon excitation photoly sis of caged Ca2+. Simultaneously concentration profiles of released C a2+ were imaged at high temporal and spatial resolution with a laser-s canning confocal microscope. 3. This approach enabled us to generate a nd detect photolytic Ca2+ signals that closely resembled the Ca2+ spar ks occurring naturally, not only in amplitude and size, but also in th eir ability to trigger additional Ca2+ sparks or Ca2+ waves. 4. Surpri singly, at low photolytic power minuscule events with estimated Ca2+ r elease fluxes 20-40 times smaller than those calculated for a typical Ca2+ spark were directly resolved. These events appeared to arise from the opening of a more limited number of RyRs (possibly one) or from R yRs exhibiting a different gating mode and may correspond to the elusi ve 'Ca2+ quark'. 5. The Ca2+ quark represents the fundamental Ca2+ rel ease event of excitable cells implementing hierarchical Ca2+ signallin g systems with Ca2+ release events of various but distinct amplitude l evels (i.e. Ca2+ quarks, Ca2+ sparks and full cellular Ca2+ transients ). 6. A graded recruitment of nanoscopic Ca2+ release domains (i.e. Ca 2+ quarks) exhibiting variable degrees of spatial coherence and coupli ng may then build up intermediate Ca2+ signalling events (i.e. Ca2+ sp arks). This mechanism suggests the existence of Ca2+ sparks caused by gating of a variable fraction of RyRs from within an individual cluste r. Additional mobilization of a variable number of these Ca2+ sparks e nables cardiac cells to show graded cellular Ca2+ transients. Similar recruitment processes may underlie regulation of Ca2+ signalling on th e cellular level in general.