Fundamental calcium release events revealed by two-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 sarcoplasmic reticulum membrane. 2. To investigate the elementary nature of these Ca2+ signals directly, a diffraction-limited point source of Ca2+ was created in single cardiac myocytes by two-photon excitation photolysis of caged Ca2+. Simultaneously, concentration profiles of released Ca2+ were imaged at high temporal and spatial resolution with a laser-scanning confocal microscope. 3. This approach enabled us to generate and detect photolytic Ca2+ signals that closely resembled the Ca2+ sparks occurring naturally, not only in amplitude and size, but also in their ability to trigger additional Ca2+ sparks or Ca2+ waves. 4. Surprisingly, at low photolytic power minuscule events with estimated Ca2+ release 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 RyRs exhibiting a different gating mode and may correspond to the elusive 'Ca2+ quark'. 5. The Ca2+ quark represents the fundamental Ca2+ release event of excitable cells implementing hierarchical Ca2+ signalling systems with Ca2+ release events of various but distinct amplitude levels (i.e. Ca2+ quarks, Ca2+ sparks and full cellular Ca2+ transients). 6. A graded recruitment of nanoscopic Ca2+ release domains (i.e. Ca2+ quarks) exhibiting variable degrees of spatial coherence and coupling may then build up intermediate Ca2+ signalling events (i.e. Ca2+ sparks). This mechanism suggests the existence of Ca2+ sparks caused by gating of a variable fraction of RyRs from within an individual cluster. Additional mobilization of a variable number of these Ca2+ sparks enables cardiac cells to show graded cellular Ca2+ transients. Similar recruitment processes may underlie regulation of Ca2+ signalling on the cellular level in general.
Lipp
P
P
Department of Physiology, University of Bern, Buhlplatz 5, 3012 Bern, Switzerland.
Niggli
E
E
D000085:Acetates; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000502:physiology; D020746:Calcium Channels, L-Type; D002614:Chelating Agents; D005029:Ethylenediamines; D006168:Guinea Pigs; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology*; D009124:Muscle Proteins / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D018408:Patch-Clamp Techniques; D010782:Photolysis; D017785:Photons; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology
D000085:Acetates; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002614:Chelating Agents; D005029:Ethylenediamines; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C057014:DM-nitrophen; D002118:Calcium