Dynamics of mitochondrial [Ca(2+)] measured with the low-Ca(2+)-affinity dye rhod-5N.

Available methods to measure mitochondrial [Ca(2+)] (Ca(2+)) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher Ca(2+) values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca(2+)-affinity dye rhod-5N provides Ca(2+) values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca(2+)-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5mM. Addition of Ca(2+) buffers containing between 4.5 and 10μM [Ca(2+)] to permeabilized cells loaded with rhod-5N induced increases in calibrated Ca(2+) up to the 100μM-1mM range, which were dependent on mitochondrial membrane potential. Ca(2+) release from mitochondria was largely dependent on [Na(+)]. We have then used rhod-5N loaded cells to investigate the Ca(2+) response to agonist stimulation at the single-cell and subcellular level. The Ca(2+) peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25μM. In the presence of the Ca(2+) uniporter stimulator kaempferol, the Ca(2+) peaks induced by histamine were also highly variable, and the mean Ca(2+) peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca(2+)] peaks showed little correlation among the heights of the peaks in both compartments. Studying the Ca(2+) peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the Ca(2+) increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.