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Journal of Bioenergetics and Biomembranes

, Volume 45, Issue 3, pp 189–202 | Cite as

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

  • Christoph A. Blomeyer
  • Jason N. Bazil
  • David F. Stowe
  • Ranjan K. Pradhan
  • Ranjan K. Dash
  • Amadou K. S. Camara
Article

Abstract

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.

Keywords

Mitochondria Ca2+ uniporter Na+/Ca2+ exchanger Ca2+ buffering Bioenergetics 

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Christoph A. Blomeyer
    • 1
  • Jason N. Bazil
    • 2
  • David F. Stowe
    • 1
    • 3
  • Ranjan K. Pradhan
    • 2
  • Ranjan K. Dash
    • 2
  • Amadou K. S. Camara
    • 1
  1. 1.Department of AnesthesiologyMedical College of WisconsinMilwaukeeUSA
  2. 2.Biotechnology and Bioengineering Center and Department of PhysiologyMedical College of WisconsinMilwaukeeUSA
  3. 3.Research ServiceZablocki Veterans Affairs Medical CenterMilwaukeeUSA

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