Different subunit location of the inhibition and transport sites in the mitochondrial calcium uniporter

The mitochondrial calcium uniporter behaves as a cooperative mechanism, where the velocity is dependent on [Ca2+](ex). Transport kinetics follows a sigmoidal behavior with a Hill coefficient near 2.0, indicating the binding of at least two calcium molecules. Calcium transport in mitochondria is dependent on a negative inner membrane potential and is inhibited by policationic ruthenium compounds. In this study, calcium uptake activity was reconstituted into cytochrome oxidase vesicles by incorporating solubilized mitochondrial proteins. Calcium accumulation plotted against increasing Ca2+ concentrations followed a sigmoidal behavior with a Hill coefficient of 1.53. The uptake was sensitive to ruthenium policationic inhibitors, e. g. ruthenium red and Ru-360. After mitochondrial proteins were separated by preparative isoelectrofocusing and incorporated into cytochrome oxidase vesicles, two peaks of calcium uptake activity were recovered. One of the activities was inhibited by Ru-360, while the second activity was insensitive to Ru-360 and was associated with proteins focused at very acidic isoelectric points. By using a thiol-group crosslinker and radiolabeled Ru-360, we proposed a scheme of partial dissociation of the uniporter inhibitor-binding subunit under acidic conditions.