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Mitochondrial Ca2+ Uptake from Plasma Membrane Cav3.2 Protein Channels Contributes to Ischemic Toxicity in PC12 Cells*

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Abstract

Background: T-type Ca2+ (Cav3.2) channel inhibitors prevent neuronal death during ischemia, but the cytotoxic signals have not been resolved.

Results: Cav3.2 channels generate cytotoxic Ca2+ elevations in the cytosol and mitochondria of PC12 cells deprived of oxygen and glucose.

Conclusion: Ca2+ transfer from Cav3.2 channels to mitochondria contributes to ischemic toxicity.

Significance: Cav3.2 channels and the mitochondrial Ca2+ uniporter (MCU) are potential targets for the treatment of stroke.

Abstract

T-type Ca2+ channel inhibitors protect hippocampal CA1 neurons from delayed death after global ischemia in rats, suggesting that Cav3.1, Cav3.2, or Cav3.3 channels generate cytotoxic Ca2+ elevations during anoxia. To test this hypothesis, we measured the Ca2+ concentration changes evoked by oxygen and glucose deprivation (OGD) in the cytosol and in the mitochondria of PC12 cells. OGD evoked long-lasting cytosolic Ca2+ elevations that were reduced by Cav3.2 inhibition (50 μm Ni2+) and Cav3.1/Cav3.2 silencing and potentiated by Cav3.2 overexpression. The kinetics of the sustained cytosolic Ca2+ elevations occurring during OGD directly correlated to the extent of cell death measured 20 h after reoxygenation, which was decreased by Ni2+ and Cav3.1/Cav3.2 silencing and increased by Cav3.2 overexpression. Ni2+ and Cav3.1/Cav3.2 silencing delayed the decline of cellular ATP during OGD, consistent with a reduction in the Ca2+ load actively extruded by plasma membrane Ca2+ pumps. The cytosolic Ca2+ elevations were paralleled by mitochondrial Ca2+ elevations that were also increased by Cav3.2 overexpression and decreased by Ni2+ but not by Cav3.1/Cav3.2 silencing. Overexpression and silencing of the mitochondrial Ca2+ uniporter, the major mitochondrial Ca2+ uptake protein, revealed that the cytotoxicity was correlated to the amplitude of the mitochondrial, rather than the cytosolic, Ca2+ elevations. Selective activation of T-type Ca2+ channels evoked both cytosolic and mitochondrial Ca2+ elevations, but only the mitochondrial responses were reduced by Cav3.1/Cav3.2 silencing. We conclude that the opening of Cav3.2 channels during ischemia contribute to the entry of Ca2+ ions that are transmitted to mitochondria, resulting in a deleterious mitochondrial Ca2+ overload.

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Most cited references 39

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Regulation of cell death: the calcium-apoptosis link.

To live or to die? This crucial question eloquently reflects the dual role of Ca2+ in living organisms--survival factor or ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.
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A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial Ca2+ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival 1 , but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter 2 , was still unknown. We have identified in silico a protein (denominated MCU) that shares tissue distribution with MICU1, a recently characterized uniporter regulator 3 , coexists with uniporter activity in phylogeny and includes two trasmembrane domains in the sequence. siRNA silencing of MCU in HeLa cells drastically reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the [Ca2+]mt rise evoked by IP3-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein exhibited channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively-charged residues of the putative pore forming region were replaced, had no channel activity and reduced agonist-dependent [Ca2+]mt transients when overexpressed in HeLa cells. Overall, these data demonstrate that the identified 40 kDa protein is the channel responsible for Ruthenium Red-sensitive mitochondrial Ca2+ uptake, thus providing molecular basis for this process of utmost physiological and pathological relevance.
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Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.

Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter. Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1. All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call 'mitochondrial calcium uniporter' (MCU). MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca(2+) uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter. Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca(2+) uniporter.

Author and article information

Affiliations
From the Departments of []Cell Physiology and Metabolism,
[§]Fundamental Neuroscience, and
[]Clinical Neurosciences, University of Geneva, Geneva CH-1211, Switzerland
Author notes
[1] To whom correspondence should be addressed: Department of Cell Physiology and Metabolism, University of Geneva, Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland. Tel.: 41-22-379-5399; Fax: 41-22-379-5338; E-mail: Nicolas.Demaurex@123456unige.ch.
Journal
J Biol Chem
J. Biol. Chem
jbc
jbc
JBC
The Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A.)
0021-9258
1083-351X
3 May 2013
18 March 2013
18 March 2013
: 288
: 18
: 12459-12468
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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