Confocal imaging of CICR events from isolated and immobilized SR vesicles.
The Ca2+ concentration inside the sarcoplasmic reticulum ([Ca2+]SR) is a difficult parameter to measure in ventricular cardiac myocytes. Interference from Ca2+-sensitive dye loading into cellular compartments other than the SR interferes with free Ca2+ measurement. In addition, the composition of the cytosol surrounding the SR in intact cells cannot be easily controlled. We have developed a method to measure localized [Ca2+]SR in immobilized membrane vesicles during rapid solution switches. Ca2+ uptake and release in rat SR membrane vesicles was monitored using confocal microscopy. Vesicles were immobilized on a coverslip using an agarose matrix. Perfusion with a Ca2+-containing solution supplemented with ATP initiated SR Ca2+ uptake, causing a rise in intravesicular fluorescence in vesicles containing the low-affinity Ca2+ indicator fluo-5N. Perfusion with caffeine caused SR Ca2+ release and a decrease in intravesicular flourescence. Although caffeine-dependent release was readily visible with extravesicular Ca2+-green, Ca2+ which leaked from the SR was detected only indirectly as eventless release. We conclude that SR Ca2+ uptake and release can be selectively measured in functional SR vesicles using a confocal microscope. Caffeine-dependent release is directly measurable though SR Ca2+ leak can only be inferred as subresolution events, presumably because channels in separate vesicles were not close enough to result in concerted Ca2+-induced Ca2+ release.
Shannon
T R
TR
Department of Physiology, Loyola University, 2160 South First Avenue, Maywood, IL 60153, USA.
Bers
D M
DM
Blatter
L A
LA
Niggli
E
E
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D018613:Microscopy, Confocal; D018482:Muscle, Skeletal / Q000648:ultrastructure; D032383:Myocytes, Cardiac / Q000648:ultrastructure; D011817:Rabbits; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D002110:Caffeine; D002118:Calcium