TY - JOUR
T1 - Measurement of mitochondrial Ca2+ transport mediated by three transport proteins
T2 - VDAC1, the NA+/CA2+ exchanger, and the CA2+ uniporter
AU - Ben-Hail, Danya
AU - Palty, Raz
AU - Shoshan-Barmatz, Varda
PY - 2014/2/1
Y1 - 2014/2/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84893247772&partnerID=8YFLogxK
U2 - 10.1101/pdb.top066241
DO - 10.1101/pdb.top066241
M3 - Article
AN - SCOPUS:84893247772
SN - 1940-3402
VL - 2014
SP - 161
EP - 166
JO - Cold Spring Harbor Protocols
JF - Cold Spring Harbor Protocols
IS - 2
ER -