Measurement of mitochondrial Ca2+ transport mediated by three transport proteins: VDAC1, the Na+/Ca2+ exchanger, and the Ca2+ uniporter.
Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) 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 Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) 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 Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel;
Danya
D
Ben-Hail
Raz
R
Palty
Varda
V
Shoshan-Barmatz
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D050995:Voltage-Dependent Anion Channel 1 / Q000378:metabolism*
D015220:Calcium Channels; D019831:Sodium-Calcium Exchanger; C034703:mitochondrial calcium uniporter; D050995:Voltage-Dependent Anion Channel 1; D002118:Calcium