Confocal imaging of CICR events from isolated and immobilized SR vesicles

T. R. Shannon, Donald M Bers, L. A. Blatter, E. Niggli

Research output: Contribution to journalArticle

3 Scopus citations

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 languageEnglish (US)
Pages (from-to)497-505
Number of pages9
JournalCell Calcium
Volume38
Issue number5
DOIs
StatePublished - Nov 2005
Externally publishedYes

Keywords

  • Calcium-induced calcium release
  • Cardiac muscle
  • Confocal microscopy
  • Ryanodine receptor
  • Sarcoplasmic reticulum
  • Skeletal muscle

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

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