Changes in Mitochondrial Oxygen Consumption can Associate with Increases in Total Mitochondrial Calcium

Abstract

Several novel candidate proteins have recently been proposed to mediate Ca2+ transport across the mitochondrial inner membrane. In addition to elucidating the basic mitochondrial Ca2+ handling machinery, these discoveries may uncover unknown modes of regulation by Ca2+ of mitochondrial processes. During the course of our studies into the function of the newly identified candidate protein, MICU1, we were intrigued to find that intact MICU1 knockdown HeLa cells increased their oxygen consumption by ∼15% during store depletion-induced Ca2+ entry without any detectable rise in matrix free Ca2+. There was however a robust increase in total mitochondrial Ca2+, indicating effective buffering of the mitochondrial Ca2+ in knockdown cells. The oxygen consumption was then measured in both control and MICU1 knockdown HeLa cells at various [Ca2+], which caused a dose-dependent Ca2+ entry. At the lowest [Ca2+] (0.2mM CaCl2), the rise in oxygen consumption in knockdown cells exceeded that in control cells which showed an increase matrix [Ca2+]. With 1mM CaCl2 addition, the rise in oxygen consumption was similar in knockdown and control cells (∼30%). In control cells, oxygen consumption continued to rise with 10mM CaCl2 addition, whereas knockdown cells showed a tendency for a lower oxygen consumption response relative to that with 1mM CaCl2, suggesting dysfunction. For all Ca2+ concentrations, the oxygen consumption increases were entirely antimycin-sensitive, indicating a mitochondrial origin. Ca2+ addition to permeabilized knockdown cells triggered a ruthenium red-sensitive rise in oxygen consumption, confirming the mitochondrial origin and suggesting the lack of dependence on cytosolic and plasma membrane Ca2+ handling processes. Thus, mitochondrial Ca2+ influx by itself can cause stimulation of respiration. Alternatively, mitochondrial Ca2+ may be available to interact with Ca2+-sensitive proteins prior to binding to buffering species.