A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

作者：Hagen Brian M; Boyman Liron; Kao Joseph P Y; Lederer W Jonathan^*

来源：Cell Calcium, 2012, 52(2): 170-181.

DOI：10.1016/j.ceca.2012.05.010

• Fluo-2MA
• Fluo-3
• Fluo-4
• Loading method
• Rat ventricular myocytes
• Cardiac
• Calcium dissociation constants
• Calcium transients
• Fractional shortening
• Compartmentalization
• Loading rate

The fluo family of indicators is frequently used in studying Ca2+ physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms the acetoxymethyl (AM) ester and the K+ salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca2+ transients and fractional shortening kinetics. Loading the K+ salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns suggesting differential loading into organelles. Ca2+ dissociation constants (K-d,K-Ca), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K-d,K-Ca (decrease in Ca2+ affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca2+ affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.

• 出版日期2012-8