increases atp production ca 2+ homeostasis fatigue/disease/apoptosis/cell death protein synthesis

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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