Abstract
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.
Original language | English (US) |
---|---|
Pages (from-to) | 497-505 |
Number of pages | 9 |
Journal | Cell Calcium |
Volume | 38 |
Issue number | 5 |
DOIs | |
State | Published - Nov 2005 |
Externally published | Yes |
Keywords
- Calcium-induced calcium release
- Cardiac muscle
- Confocal microscopy
- Ryanodine receptor
- Sarcoplasmic reticulum
- Skeletal muscle
ASJC Scopus subject areas
- Cell Biology
- Endocrinology