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Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release
Authors:Christoph A. Blomeyer  Jason N. Bazil  David F. Stowe  Ranjan K. Pradhan  Ranjan K. Dash  Amadou K. S. Camara
Affiliation:1. Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
2. Biotechnology and Bioengineering Center and Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
3. Research Service, Zablocki Veterans Affairs Medical Center, 5000 W. National Avenue, Milwaukee, WI, 53295, USA
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.
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