Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
STAR Protoc. 2021 Nov 23;2(4):100979. doi: 10.1016/j.xpro.2021.100979. eCollection 2021 Dec 17.
The mitochondrial calcium uniporter, which mediates mitochondrial Ca uptake, regulates key cellular functions, including intracellular Ca 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 radionuclide uptake assay in cultured cell lines. Second, we describe electrophysiological recordings of the uniporter expressed in 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).
线粒体钙单向转运体介导线粒体钙摄取，调节关键的细胞功能，包括细胞内钙信号转导、细胞命运决定和线粒体生物能学。在这里，我们描述了两种互补的策略来量化单向转运体的转运活性。首先，我们详细描述了在培养的细胞系中进行线粒体钙放射性核素摄取测定的方法。其次，我们描述了在卵母细胞中表达的单向转运体的电生理记录。这些方法能够对单向转运体进行详细的动力学分析，将其分子特性与生理功能联系起来。如需了解本协议的使用和执行的详细信息，请参阅 Tsai 和 Tsai（2018）和 Phillips 等人（2019）的文献。
