Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release
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In cardiac mitochondria, matrix free Ca2+ ([Ca2+]m) is primarily regulated by Ca2+ uptake and release via the Ca2+ uniporter (CU) and Na+/Ca2+ exchanger (NCE) as well as by Ca2+ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca2+ buffering affects these dynamics under various Ca2+ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca2+ buffering on the uptake and release of Ca2+, we monitored Ca2+ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca2+] ([Ca2+]e) and [Ca2+]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca2+]e and [Ca2+]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca2+-induced changes in mitochondrial bioenergetics. Our [Ca2+]e and [Ca2+]m measurements demonstrate that Ca2+ uptake and release do not show reciprocal Ca2+ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca2+ buffering system in the matrix compartment. The Na+- induced Ca2+ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca2+ uptake in the presence of large amounts of CaCl2, but not by Na+- induced Ca2+ release.
KeywordsMitochondria Ca2+ uniporter Na+/Ca2+ exchanger Ca2+ buffering Bioenergetics
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- Baysal K, Jung DW, Gunter KK, Gunter TE, Brierley GP (1994) Na+-dependent Ca2+ efflux mechanism of heart mitochondria is not a passive Ca2+/2Na+ exchanger. Am J Physiol 266(3 Pt 1):C800–C808Google Scholar
- Bazil JN, Blomeyer CA, Pradhan RK, Camara AK, & Dash RK (2012) Modeling the calcium sequestration system in isolated guinea pig cardiac mitochondria. J Bioenerg Biomembr, accepted for publicationGoogle Scholar
- Brand MD (1985) The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter. Biochem J 229(1):161–166Google Scholar
- Cox DA, Matlib MA (1993) A role for the mitochondrial Na+-Ca2+ exchanger in the regulation of oxidative phosphorylation in isolated heart mitochondria. J Biol Chem 268(2):938–947Google Scholar
- Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450Google Scholar
- Gunter TE, Pfeiffer DR (1990) Mechanisms by which mitochondria transport calcium. Am J Physiol 258(5 Pt 1):C755–C786Google Scholar
- Jung DW, Apel LM, Brierley GP (1992) Transmembrane gradients of free Na+ in isolated heart mitochondria estimated using a fluorescent probe. Am J Physiol 262(4 Pt 1):C1047–C1055Google Scholar
- Li W, Shariat-Madar Z, Powers M, Sun X, Lane RD, Garlid KD (1992) Reconstitution, identification, purification, and immunological characterization of the 110-kDa Na+/Ca2+ antiporter from beef heart mitochondria. J Biol Chem 267(25):17983–17989Google Scholar
- Santo-Domingo J, Demaurex N (2010) Calcium uptake mechanisms of mitochondria. Biochim Biophys Acta 1797(6–7):907–912Google Scholar