Dynamics of mitochondrial [Ca2+] measured with the low-Ca2+-affinity dye rhod-5N

Available methods to measure mitochondrial [Ca2+] ([Ca2+]M) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca2+]M values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca2+-affinity dye rhod-5N provides [Ca2+]M values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca2+-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5 mM. Addition of Ca2+ buffers containing between 4.5 and 10 μM [Ca2+] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca2+]M up to the 100 μM-1 mM range, which were dependent on mitochondrial membrane potential. Ca2+ release from mitochondria was largely dependent on [Na+]. We have then used rhod-5N loaded cells to investigate the [Ca2+]M response to agonist stimulation at the single-cell and subcellular level. The [Ca2+]M peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25 μM. In the presence of the Ca2+ uniporter stimulator kaempferol, the [Ca2+]M peaks induced by histamine were also highly variable, and the mean [Ca2+]M peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca2+] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca2+]M peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca2+]M increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.