title: Studying mitochondrial Ca(2+) uptake - a revisit.
abstract: Mitochondrial Ca(2+) sequestration is a well-known process that is involved in various physiological and pathological mechanisms. Using isolated suspended mitochondria one unique mitochondrial Ca(2+) uniporter was considered to account ubiquitously for the transfer of Ca(2+) into these organelles. However, by applying alternative techniques for measuring mitochondrial Ca(2+) uptake evidences for molecularly distinct mitochondrial Ca(2+) carriers accumulated recently. Herein we compared different methodical approaches of studying mitochondrial Ca(2+) uptake. Patch clamp technique on mitoplasts from endothelial and HeLa cells revealed the existence of three and two mitoplast Ca(2+) currents (I(CaMito)), respectively. According to their conductance, these channels were named small (s-), intermediate (i-), large (l-) and extra-large (xl-) mitoplast Ca(2+) currents (MCC). i-MCC was found in mitoplasts of both cell types whereas s-MCC and l-MCC or xl-MCC were/was exclusively found in mitoplasts from endothelial cells or HeLa cells. The comparison of mitochondrial Ca(2+) signals, measured either indirectly by sensing extra-mitochondrial Ca(2+) or directly by recording changes of the matrix Ca(2+), showed different Ca(2+) sensitivities of the distinct mitochondrial Ca(2+) uptake routes. Subpopulations of mitochondria with different Ca(2+) uptake capacities in intact endothelial cells could be identified using Rhod-2/AM. In contrast, cells expressing mitochondrial targeted pericam or cameleon (4mtD3cpv) showed homogeneous mitochondrial Ca(2+) signals in response to cell stimulation. The comparison of different experimental approaches and protocols using isolated organelles, permeabilized and intact cells, pointed to cell-type specific and versatile pathways for mitochondrial Ca(2+) uptake. Moreover, this work highlights the necessity of the utilization of multiple technical approaches to study the complexity of mitochondrial Ca(2+) homeostasis.
authors.affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, A-8010 Graz, Austria.
authors.forename: Claire
authors.initials: C
authors.lastname: Jean-Quartier
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Bondarenko
authors.forename: Muhammad Rizwan
authors.initials: MR
authors.lastname: Alam
authors.forename: Michael
authors.initials: M
authors.lastname: Trenker
authors.forename: Markus
authors.initials: M
authors.lastname: Waldeck-Weiermair
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
mesh_terms: D002118:Calcium / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006367:HeLa Cells; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*
chemical_list: D002118:Calcium
references.citation: Nature. 2010 Sep 16;467(7313):291-6
references.pmid: 20693986
references.citation: Nature. 1992 Jul 23;358(6384):325-7
references.pmid: 1322496
references.citation: Cell Calcium. 2009 Aug;46(2):122-9
references.pmid: 19631981
references.citation: J Biol Chem. 1982 Nov 10;257(21):13028-33
references.pmid: 6290489
references.citation: FEBS Lett. 2010 May 17;584(10):1948-55
references.pmid: 20096690
references.citation: Cell Calcium. 2008 Mar;43(3):250-9
references.pmid: 17628663
references.citation: Proc Natl Acad Sci U S A. 1982 Jan;79(1):123-6
references.pmid: 6275382
references.citation: EMBO J. 1999 Jan 4;18(1):96-108
references.pmid: 9878054
references.citation: Nature. 2008 Dec 4;456(7222):605-10
references.pmid: 19052620
references.citation: Nature. 2004 Jan 22;427(6972):360-4
references.pmid: 14737170
references.citation: Methods. 2008 Nov;46(3):152-9
references.pmid: 18848629
references.citation: Annu Rev Genet. 2006;40:159-85
references.pmid: 16771627
references.citation: Int J Biochem Cell Biol. 2009 Oct;41(10):1972-6
references.pmid: 19481172
references.citation: Semin Cell Dev Biol. 2009 May;20(3):337-45
references.pmid: 19530305
references.citation: EMBO J. 2009 Oct 21;28(20):3074-89
references.pmid: 19745815
references.citation: Methods Enzymol. 1990;182:203-25
references.pmid: 2156127
references.citation: Cell Tissue Res. 1984;236(2):471-5
references.pmid: 6733773
references.citation: Cell. 2008 Dec 26;135(7):1165-7
references.pmid: 19109886
references.citation: Arch Biochem Biophys. 1979 Jul;195(2):468-77
references.pmid: 112926
references.citation: J Biol Chem. 2003 May 23;278(21):19062-70
references.pmid: 12660243
references.citation: Cell Calcium. 2010 May;47(5):433-40
references.pmid: 20403634
references.citation: J Biol Chem. 2003 Mar 21;278(12):10807-15
references.pmid: 12529366
references.citation: J Cardiovasc Pharmacol. 1993 Apr;21(4):595-9
references.pmid: 7681905
references.citation: Arch Int Physiol Biochim. 1969 Dec;77(5):916-34
references.pmid: 4190879
references.citation: Cell Metab. 2011 May 4;13(5):601-11
references.pmid: 21531342
references.citation: Biochim Biophys Acta. 2011 May;1813(5):1003-13
references.pmid: 21146562
references.citation: Curr Mol Med. 2008 Mar;8(2):119-30
references.pmid: 18336292
references.citation: Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3197-202
references.pmid: 11248055
references.citation: Nature. 2011 Jun 19;476(7360):336-40
references.pmid: 21685888
references.citation: Pflugers Arch. 2007 Dec;455(3):375-96
references.pmid: 17611770
references.citation: Circulation. 2009 May 12;119(18):2435-43
references.pmid: 19398664
references.citation: Cell Calcium. 2008 Jul;44(1):1-5
references.pmid: 18378306
references.citation: Science. 2003 Apr 4;300(5616):65-7
references.pmid: 12677047
references.citation: Methods Enzymol. 2003;360:202-25
references.pmid: 12622151
references.citation: Science. 2009 Oct 2;326(5949):144-7
references.pmid: 19797662
references.citation: Nat Biotechnol. 2002 Jan;20(1):87-90
references.pmid: 11753368
references.citation: Chem Biol. 2006 May;13(5):521-30
references.pmid: 16720273
references.citation: Nat Rev Mol Cell Biol. 2000 Oct;1(1):11-21
references.pmid: 11413485
references.citation: FEBS Lett. 2010 May 17;584(10):1942-7
references.pmid: 20074570
references.citation: Nat Cell Biol. 2008 Nov;10(11):1235-7; author reply 1237-40
references.pmid: 18978830
references.citation: Am J Physiol. 1994 Aug;267(2 Pt 1):C313-39
references.pmid: 8074170
references.citation: Biochim Biophys Acta. 2005 Nov 10;1717(1):1-10
references.pmid: 16246297
references.citation: Mol Interv. 2005 Apr;5(2):112-27
references.pmid: 15821159
references.citation: Mol Cell Biochem. 2000 Oct;213(1-2):29-35
references.pmid: 11129955
references.citation: Mol Cell. 2004 Oct 8;16(1):59-68
references.pmid: 15469822
references.citation: Proc Natl Acad Sci U S A. 1961 Nov 15;47:1744-50
references.pmid: 13885269
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2135-40
references.pmid: 10051607
references.citation: Curr Biol. 2010 Oct 26;20(20):R888-91
references.pmid: 20971432
references.citation: J Biol Chem. 1949 Jun;179(2):957-72
references.pmid: 18150026
references.citation: Cell Calcium. 2010 Jul;48(1):61-9
references.pmid: 20667591
references.citation: J Biol Chem. 2011 Sep 16;286(37):32533-41
references.pmid: 21775425
references.citation: J Biol Chem. 2011 Aug 12;286(32):28444-55
references.pmid: 21613221
references.citation: Science. 2004 Aug 6;305(5685):858-62
references.pmid: 15297672
references.citation: Proc Natl Acad Sci U S A. 1983 Jun;80(12):3734-7
references.pmid: 6407019
references.citation: Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):907-12
references.pmid: 20079335
references.citation: Curr Biol. 2006 Jul 25;16(14):R551-60
references.pmid: 16860735
references.citation: Nature. 1997 Aug 28;388(6645):882-7
references.pmid: 9278050
references.citation: Cell Calcium. 2010 Aug-Sep;48(2-3):168-75
references.pmid: 20832113
references.citation: Nat Rev Mol Cell Biol. 2003 Jul;4(7):517-29
references.pmid: 12838335
references.citation: Cell Calcium. 2008 Jul;44(1):51-63
references.pmid: 18242694
references.citation: J Biol Chem. 1993 Jan 15;268(2):938-47
references.pmid: 8419373
references.citation: Mol Cell Biochem. 1998 Jul;184(1-2):81-100
references.pmid: 9746314
references.citation: Biochem Biophys Res Commun. 1991 Oct 15;180(1):209-15
references.pmid: 1930217
references.citation: Cell Calcium. 2010 Nov;48(5):288-301
references.pmid: 21047682
references.citation: Nat Protoc. 2007;2(2):287-95
references.pmid: 17406588
references.citation: Nat Cell Biol. 2007 Apr;9(4):445-452
references.pmid: 17351641
references.citation: Cell Calcium. 2005 Feb;37(2):129-36
references.pmid: 15589993
references.citation: Nature. 2011 Jun 19;476(7360):341-5
references.pmid: 21685886
references.citation: Cell Calcium. 2005 Sep-Oct;38(3-4):213-22
references.pmid: 16122797
references.citation: Annu Rev Cell Biol. 1988;4:155-81
references.pmid: 3058159
references.citation: Essays Biochem. 2010;47:115-37
references.pmid: 20533904
references.citation: Biochem J. 2006 May 15;396(1):173-82
references.pmid: 16466345
references.citation: Int Rev Cell Mol Biol. 2010;284:1-65
references.pmid: 20875628
references.citation: J Biol Chem. 2011 Jun 17;286(24):21324-9
references.pmid: 21524998