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Increases ATP productionCa2+ homeostasisFatigue/disease/apoptosis/cell
deathProtein synthesisSignals to new sites e.g. RYR, IP3
Mitochondria and Ca2+
Why bother?
2Rizzuto et al., Oncogene (2003) 22, 8619-8627.
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Maternally transmitted
Polarised -150 – 200 mV
More cristae = more ATP
Epithelial cells, few
Muscle, up to 1000 per cell
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How to tackle mitochondria?
Since 1961, isolate them from tissue
alter function, lose regulatory factors
Since early 1990’s, study them in situ– O2 consumption, intrinsic fluorescence,
– Use fluorescent dyes (Ca2+, ROS, Memb. pot.)
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CGP-37157
Mitochondrial Ca2+ entry and release
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Blocking Ca2+ entry and exit
Depolarise mitochondria: FCCP
Inhibit Ca2+ uniporter: Ru360
Na+-Ca2+ exchanger: CGP37157
H+-Ca2+ exchanger ?
MTP: Cyclosporin A ??
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, Na+, K+
Mitochondria selectively take up Ca2+ butno other physiologically relevant ion.
Kirichok et al., Nature (2004) 427, 360-4.
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Mitochondrial mysteries
• Structures of all Ca2+ transport related proteins are unknown.
• Structure of mitochondrial transition pore (MTP, PTP, MPTP) also unknown.
• Mitochondrial myopathies.
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Mouse muscle
•10–15% fibre volume
•Denser at surface
•Fixed positions withdesmin + plectin
•Close to Ca2+ release site
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In mammalian muscle, mitochondria are near transverse tubules
11Csordás et al., Trends Cardiovasc Med. 2001 11 269-275
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General model of Ca2+ stimulation of ROS production
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Measure mitochondrial Ca2+ and ROS simultaneously
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In motor neurones, mitochondria limit increase in cytosolic Ca2+
Cytosol Mitochondria
David et al. J Physiol 1998 509, 59-65
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Useful links for confocal users
• http://rsb.info.nih.gov/ij
• http://www.tsienlab.ucsd.edu/
• http://www.invitrogen.com/site/us/en/home/support/Tutorials.html