Measurement of Mitochondrial Ca2+ Transport Mediated by Three Transport Proteins: VDAC1, the Na+/Ca2+ Exchanger, and the Ca2+ Uniporter

Abstract

Ca2+ is a ubiquitous cellular signal, with changes in intracellular Ca2+ concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca2+ uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca2+ signaling, energy metabolism and cell death. Ca2+ transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca2+ transport activity across both mitochondrial membranes. The first of these is Ca2+ transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca2+ uniporter (MCU) that transports Ca2+ and is powered by the steep mitochondrial membrane potential. In the event of Ca2+ overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na+-dependent mitochondrial Ca2+ efflux mediated by mitochondrial NCLX, a member of the Na+/Ca2+ exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca2+-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.