Quantitative assays to measure the transport activity of the mitochondrial calcium uniporter in cell lines or Xenopus oocytes

The mitochondrial calcium uniporter, which mediates mitochondrial Ca(2+) uptake, regulates key cellular functions, including intracellular Ca(2+) signaling, cell-fate determination, and mitochondrial bioenergetics. Here, we describe two complementary strategies to quantify the uniporter’s transport activity. First, we detail a mitochondrial Ca(2+) radionuclide uptake assay in cultured cell lines. Second, we describe electrophysiological recordings of the uniporter expressed in Xenopus oocytes. These approaches enable a detailed kinetic analysis of the uniporter to link its molecular properties to physiological functions. For complete details on the use and execution of this protocol, please refer to Tsai and Tsai (2018) and Phillips et al. (2019).