Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes

Abstract

The mitochondrial calcium uniporter is a multisubunit Ca²⁺ channel that mediates mitochondrial Ca²⁺ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca²⁺ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca²⁺-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca²⁺ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca²⁺ > Sr²⁺ > Ba²⁺, Mn²⁺, and Mg²⁺ Patch clamp recordings further reveal macroscopic and single-channel Ca²⁺ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca²⁺-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.