Mitochondria:More Than Just ATP Cows

Paul S. Brookes, PhD.Anesthesiology, University of Rochester Medical Center

Resurgence in Mitochondrial Research

Papers/Yr.(PubMed: mitochondri*)

0

2000

4000

6000

8000

10000

12000

1975 1980 1985 1990 1995 2000 2005

0

500

1000

1500

2000

1975 1980 1985 1990 1995 2000 2005

Papers/Yr.(PubMed: cytochrome c)

Largely attributable to a single paper…

Liu et al. (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86: 147-57

Knyazeva et al. (1975) Solubilization of cytochrome cin ischemic liver tissue. Vopr Med. Khim. 21: 481-85

But who observed it first ?

“Text Book” Views of Mitochondria

1850: Mitochondria coined by microscopists, from Greek mitos: thread, chondros: grain

Frey & Mannella TIBS (2000) 25: 319-24

Updated Views - 3D electron tomography

Mitochondrial DNA (mtDNA)

Complex I II III IV VMw (~kDa) 900 140 250 200 600Subunits (mtDNA) 46(7) 4(0) 11(1) 13(3) 10(2)

Parseghian & Luhrs (2006) Biochem. Cell Biol. 84: 589-604Mitochondria contain histones…

CxICxI

CxIIICxIII

cc

CxIVCxIVCxIICxII

O2e- CxVCxV

H+

H+ H+

H+

ADPATP

YY CC NNAAWW

II

MMQQ

DDKK

GG

RR

LL

HHSS

EETT PP

Ori

12SrRNA 16SrRNA

FFVV

LL

COX1

ATP6/8

ND1

Cyt-b1D

Loop

ND2

ND3N

D4

ND5

ND6

COX2

COX3

HumanmtDNA

(16.6 kB)

The Endosymbiont Theory of Mitochondrial Origin…

“Mother” cell w/oaerobic metabolism

“Daughter”cell withaerobic

metabolism

Cell with mitochondriaas “ATP Cows”

A long, long time ago (about 1.8 billion years)…

ATP

Was it for ATP, or to stop the toxic effects of rising atmospheric [O2]?2 books by Nick Lane…- Oxygen: The Molecule that made the World- Power, Sex, Suicide: Mitochondria & the Meaning of Life

H+ H+ H+

Q

O2 H2Oe-

e-

H+

ADP ATPFADH2

NADH

NAD+

FAD

TCACycle

H+

LEAK

Ca2+

ROSNO•

NO•

Outline

Model System: Cardiac Ischemia-Reperfusion Injury (IR)

Langendorff perfused rat heart:

0

50

100

150

10 min.

LVP

(mm

Hg)

PT Pore Opening↑ROS

↑[Ca2+]m

Murry et al. (1986) Circulation 74: 1124-36

0

50

100

150

& Ischemic Preconditioning (IPC)

Critical & Convergent Roles of NO• & Mitochondria in IPC

Diazoxide XenonVAs

plasma membrane

K+ATP

GSK-3βP

eNOS Akt

PKG(inactive)

PI3K

MAPK

PLC/PLD

GPCR

Adenosine, Angiotensin -II,Bradykinin,Acetylcholine,

Endothelin, Opioids

DAG

PDK-1

GFRs

Insulin,GF, IL-6

ETC

PKCε

Nitrite,NO• donors

Ca2+ PT

Pore

ROS

UCP

Diazoxide XenonVAs

plasma membrane

K+ATP

GSK-3βPP

eNOS AktAkt

PKG(inactive)

PI3K

MAPK

PLC/PLD

GPCR

Adenosine, Angiotensin -II,Bradykinin,Acetylcholine,

Endothelin, Opioids

DAG

PDK-1

GFRs

Insulin,GF, IL-6

ETC

PKCεPKCε

Nitrite,NO• donors

Ca2+

Ca2+ PT

Pore

ROS

UCP

mitochondrion

NO•

Jones & Bolli (2006)J. Mol. Cell. Cardiol. 40: 16-23

PDE5 Inhibitors eNOSDietary factorsACE Inhibitors

AT1 receptor blockadeNOS gene therapy

Statins, Exercise

Zaugg & Schaub (2003)J. Muscle Res. Cell Motil. 24: 219-49

Na+

H+H+

O2

ADPATP

Δψm+

–e-

Ca2+

MCUCa2+

Ca2+

Na+/Ca2+

Exch’

RaMNa+

H+

PTP

mRyRNa+/H+

Exch’

respiratorychain

Mitochondrial Ca2+ Uptake & Efflux Pathways

MCU: Mitochondrial Ca2+ Uniporter (high [Ca2+], quite slow)

RaM: Rapid Mode Ca2+ uptake (low [Ca2+], very fast)

mRyR: Mitochondrial Ryanodine Receptor

No protein identified

Found in excitable tissues

Succinate

Citrate

Isocitrate

α-KetoglutarateFumarate

Malate

Oxaloacetate

O-O

OO-

O-O

OO-

O-O

OO-

OH

O-O

OO-

O OH

OO-

O

-OO

O-

OHO

O-

O

-OO

O-

OO-

OO-

O

O

OO-

SS-CoA

OO

O-

Pyruvate

OO-

S-CoAAcetyl-CoA

SDH

MDH

CS

FH

Acon

ICDH

αKGDH

SCS

NADH, CO2

NADHCO2

NADH

NADH

GTP

FADH2

PDH

TCA Cycle… recent insights

OO-

OO-

Malonate

CO2

H2O2

H2O2

CO2

Fedotcheva et al. (2006)Free Rad. Biol Med. 41: 56-64

α-keto acids react readily with H2O2

HIF prolyl-hydroxylases use α-KG as a substrate and are inhibited by succinate

–OH

Degradation Transcription

Tumor Survival

CO2 O2PHD

Hif-1α Hif-1α

Selak et al. (2005) Cancer Cell. 7: 77-85

Succinate α-Ketoglutarate

Complex IImportant source of ROS in reverse direction

St-Pierre et al. (2002) JBC 277: 44784-44790

Apoptotic protein GRIM-19 is a CxI subunitFearnley et al. (2001) JBC 276: 38345-38348

Brandt (2006) Ann. Rev.Biochem. 75: 69-92.

Clementi (1998) PNAS 95: 7631-36Hsu (2005) J. Neurochem. 92: 1091-1103

Brown (2004) Biochim. Biophys. Acta 1658: 44-49Jekabsone (2003) J. Mol. Cell. Cardiol. 35: 803-09

Riobo (2001) Biochem. J. 359: 139-45Dahm (2006) J. Biol. Chem. 281:10056-65

Borutaite (2006) Biochim. Biophys. Acta. 1757: 562-6

Indirect evidence for S-nitrosation

Inhibited

Light,DTT RSNO (mM)

Active

75kDa subunit cleaved by caspasesRicci et al. (2004) Cell 117: 773-786

Can Complex I be S-nitrosated ?

I

VIII

IV

SNO (pmols/fraction)0 1 2 3 4 5 6

Blue-native gel SNO CL

NO•+ O3 hv

Superose 6 SNO CL

0

50

100

150

200

0 10 20 30 40 50Fraction #

SNO

SNO Blot

Ctrl.SNO

SDS-PAGE

Wholemito’s

100755037

2520

15

MALDI-TOF

Biotin Switch AssayCx Iband

75 kDasubunit

Burwell et al. (2006)Biochem. J. 394: 627-34

Yang et al. (2003) Free Rad. Res. 37: 1-10

Jaffrey (2005) Meth.Enzymol. 396: 105-18

Is Complex I S-nitrosation Physiologically Important?

0

5

10

15

20

25

IPC

pmol

s S

NO

/mg

prot

ein

Ctrl.

ND = notdetectable

ND

Ctrl. IPC

25

37

50

75100

Mitochondria from IPC hearts contain SNO

0

10

20

30

40

50

0 250 500[GSNO] (µM)

mol

SN

O/m

ol C

x I (

)

0

20

40

60

80

100

% C

ompl

ex I

Activ

ity (

)

S-nitrosation vs. Inhibition

Burwell et al. (2006) Biochem. J. 394: 627-34

Recovery from IR is enhanced in the dark

LVP

(mm

Hg)

10 min.

Light (8.7±1.7%)

Dark (24.6±2.3%)

100

0

100

0

100

0

100

00

20

40

60

80

100

Cel

l via

bilit

y (%

of c

ontro

l)

**

#*

HRIPC

GSNO(µM)

10 20 10 20

#* #

*

†#*

Nor

mox

ic C

ontro

l

mSNO(µM)

“mito-SNO” protects myocytes from IR injury

+ O

DQ

20

Metabolic Shutdown as a Protective Mechanism

Nitric OxideJ. Biol. Chem. (2000) 275, 20474-9

PNAS (2004) 101, 13683-88J. Clin. Invest. (2005) 115, 1232-40

Nitric OxideBiochem. J. (2006) 394, 627-34

AmobarbitalJ. Pharmacol. Exp. Therap. (2006) 316, 200-07

RanolazineBiochem. Pharmacol. (1995) 50, 1599-1606

CapsaicinEur. J. Pharm. (1995) 272, 269-78

BBA (1996) 1273, 21-30

CxICxI

CxIIICxIII

cc

CxIVCxIVCxIICxII

O2

GlycolysisGlycolysis

Nitric OxideJ. Biol. Chem. (1992) 267, 24929-32

IPCJ. Biol. Chem. (2002) 277, 24411-9

3-NitropropionateJ. Cereb. Blood Flow Metab. (1997) 17, 257-64

DiazoxidePNAS (2004) 101, 11880-11885

Hydrogen SulfideJ. Mol. Cell. Cardiol. (2006) 40, 119-30

Toxicol. App. Pharmacol. (1990) 103, 482-90

e-

Complex II

• mitoK+ATP channel opening ischemic preconditioning

• Drugs that open K+ATP (e.g. diazoxide) are Cx II inhibitors

• Cx II inhibitors (e.g. malonate) can mimic preconditioning• Splice variant of Cx II subunit 3 is within the SUR gene

PiTX

ATPK+

Ardehali et al. (2004) PNAS 101: 11880-5

Cx VCx II

ANT

mABC1InnerMembrane

Wohllk et al. (1998) Mol. Genet. Metab. 65: 187-90

Schafer et al. (1969) Biochem. Pharmacol. 18: 2678-81

Kir 6.1vs. SUR

ATPK+

H+ H+

O2

ADP

+

– CxII

FADH2

H+

Acetyl-CoA

Pyruvate

NADHNAD+

CxI

CxIII

CxIV CxV

PDH

ATP

e-

UCPH+

FAD

Q

QH2

QH•

H+

e-

cyt-c

Δψm

Q-cycle

α-KG

Citrate

Isocitrate

Succinate

OAA

CS

α-KGDH

ICDH

NADH

Enoyl-CoA

Acyl-CoA

Acyl-CoA DH

ETFQOR

Acyl-CoA -C2

NADH β-ox

Mito’ Sources of ROS – more than just CxI & CxIII

MonoamineOxidase B

DHODH

MDH

SDH

e-

Dihydro-Orotate Orotate

R–CH2–NH2 R–CH=O

H2O2

FAD FADH2

ROS Sources• Complex I• Complex III• Pyruvate Dehydrogenase• α-ketoglutarate Dehydrogenase• Acyl-CoA Dehydrogenase• Isocitrate Dehydrogenase• Dihydroorotate Dehydrogenase• β-oxidation e- Transfer Flavoprotein

(quinone oxidoreductase)• Monoamine Oxidase B

ROS Metalloproteases

Upstream signals(Ca2+, NO●, TNF etc.)

Phosphatases

PT poreapoptosis

NF-κB

CREB

Keap-1

Cell cycle,proliferation

Hif-1α

HO-1

MAPKs

Antioxidants(GSH, SOD etc)

Mitochondrial ROS & Cell Signaling

Mitochondrial ROS Generation & Hypoxia

Guzy et al. (2005) Cell Metab 1: 401-8

Bell et al. (2005) Mitochondrion 5: 322-32

Chandel et al. (2000) J. Biol. Chem. 275: 25130-8

Waypa & Schumacker (2002) Respir. Physiol. Neurobiol. 132: 81-91

Hypoxia

↑ROS Hif-1α

Current Dogma:

O2 (%)

Mito

RO

SG

ener

atio

n

Nor

mox

ia

Hyp

erox

ia

Hyp

oxia

1-5 20 50

H+

O2

H+

e-

H+

II

IIIIII

ccIVIV

Proposed Mechanism:

Q

H2O

H+

O2

H+

e-

H+

II

IIIIII

ccIVIV

QH•

NormoxiaElectrons flow all the way to O2 at complex IV, and fully

reduce it to H2O

O2O2

•-

HypoxiaLack of O2 at complex IV

causes a “back-up” of electrons in the chain, increasing lifetime of

semiquinone radical (QH•), which donates electrons

to O2, forming O2•-

Mitochondrial ROS Generation & Hypoxia

Prediction: Mitochondrial hypoxic signaling response is an inherent property of the respiratory chain. Mitochondria function autonomously in hypoxic signaling.

DCF Fluorescence

Chandel et al. (2000)J. Biol. Chem. 275: 25130-8

(results from Hep-3B cells)

Hif-1α Stabilization

But: ROS generation by isolated mitochondria at hypoxic [O2] (1% = 10μM) has never been measured.

And: kM of cytochrome c oxidase is ~1μM O2, but kM of the ROS generating system is unknown.

Probe specificity (esp. DCF)

Inhibitor specificity (rotenone, AA)

Probe location (mitoSOX Red)

Open Flow Respirometer

.

Air N2

Gas Inlet

Gas Exhaust

Stirrer motor& Stopper

StirringPropeller

O2 Electrode

HumidifierMass-flowControllers

Photomultiplier Tube

Fiber Optic Fluorimeter

O2

Cole et al. (1982) J. App. Physiol. 53: 1116-24

Brookes et al. (2003) J. Biol. Chem. 278: 31603-9

Hoffman et al. (2007) Am. J. Physiol. Heart. In-Press

Open-flow respirometry chamber: O2 consumed by mitochondria is replaced by O2 from headspace gas. This allows incubation at steady-state values of [O2] as low as 0.1uM. Simultaneous cytochrome spectral measurements inform on redox state of respiratory chain:

μM O2

% R

educ

tion

0

20

40

60

80

100

120

0 50 100 150 200 250

020406080

100120

0 2 4 6 8 100 2 4 6 8 10

Conventional respirometry chamber: [O2] is constantly changing, as mitochondria are consuming O2. Below 10% O2, the only way to maintain a relatively constant [O2] is to use very small amounts of mitochondria (imcompatible with ROS measurements).

[O2]

(μM

)

Time

All the interesting stuff happens in the last 10 sec. of the experiment!

0

200

Mitochondrial ROS Generation Decreases in HypoxiaH

2O2

(pm

ol/m

in/m

g pr

otei

n)

State 3

State 40

20

40

60

80

100

120

0 50 100 150 200 250

020406080

100120

0 2 4 6 8 10

0

20

40

60

80

100

50 100 150 200 250

020406080

0 2 4 6 8 10

100

0 2 4 6 8 10

0

μM O2

• Apparent kM of the ROS system ~0.5 μM O2

• Apparent kM for respiration ~2μM O2

• ROS generation can occur even at [O2] thatinhibits respiration, but ROS cannot increase !

Isolated liver mitochondria, glutamate plus malate as substrates (i.e. electrons fed into complex I). Amplex red/HRP fluorescent measurement of H2O2.

Hoffman et al. (2007)Am. J. Physiol. Heart. In-Press

Conclude:Hypoxic ROS generation is not inherent

to the mito’ respiratory chain

In cells, another signaling factor is probably responsible for elevated ROS:

NO•, Ca2+, Phosphorylation

Ca2+

Ca2+

OxidantsAttractylosidePiRSSRATP + ADP DepletionNAD(P)+ > NAD(P)H

VDAC

ANT

Cyp-D

Hexokinase

Creatine

Kinase

Bclfamilyproteins

pH<7Mg2+

Cyclosporin ABongrekic AcidRSHATP + ADPNAD(P)H > NAD(P)+

++

– –

PBR

Permeability Transition Pore (classical view)

Brookes et al. (2004) Am. J. Physiol. 287: C817-C833

Opening of the PT pore is mechanistically linked to cytochrome crelease. Cyt-c release can occur without pore opening, andOMM swelling/rupture does not appear to play a major role

Mechanism by which cytochrome c exitsMitochondria during apoptosis is still elusive

Swelling ≠ Cytochrome c Release

Brookes et al. (2000) J. Biol. Chem. 275: 20474-9

5 min.

Ca2+

10%

ΔA

bs @

540n

m v

s. in

itial C

ytochrome C

release (% of m

aximum

)0

20

40

60

80

100

5 min.

Ca2+Ca2+

10%

ΔA

bs @

540n

m v

s. in

itial C

ytochrome C

release (% of m

aximum

)0

20

40

60

80

100

0

20

40

60

80

100

Swelling (% of maximum)

Cyt

-C R

elea

se (%

of m

axim

um)

0

20

40

60

80

100

0 20 40 60 80 100Swelling (% of maximum)

Cyt

-C R

elea

se (%

of m

axim

um)

0

20

40

60

80

100

0 20 40 60 80 1000

20

40

60

80

100

0 20 40 60 80 100

Components of the PT Pore: knockout mice data

ANT Kokoszka et al. (2004) Nature 427(6973): 461-5 Non-essential

Halestrap (2003) Curr. Med. Chem. 10: 1507-25

Maybe other MCF proteins can sustitute?Palmieri (2004) Pflugers Arch. 447: 689-709

Or maybe it is essential?

VDAC Krauskopf et al. (2006) Biochim. Biophys. Acta 1757: 590-5 Non-essential

Cyp-D Nakagawa et al. (2005) Nature 434(7033): 652-8

Baines et al. (2005) Nature 434(7033): 658-62

Essential for PT pore, but pore is only important in necrosis, not apoptosis

Complex IV (Cytochrome c Oxidase) – NO• Inhibition

O2 O2•- OH•H2O2 H2O

e- e- e- e-

Fe2+

Fe3+ Cu

Cu2+

+

Koivisto et al. (1997)FEBS Lett. 417: 75-80

0

20

40

60

80

100

0.0 0.2 0.4 0.6 0.8 1.0[NO•] (µM)

Sta

te 3

Res

pira

tion

(% o

f max

’)

IC50 ~120nM@ 10μM [O2]

Brookes et al. (2003)JBC. 278: 31603-9

Nisoli et al. (2003) Science 299: 896-9

NO• Stimulates Mitochondrial Biogenesis

↑PGC-1α(PPARγ

coactivator 1α)↑NRF-1

↑mtTFA

nDNA subunits

mtDNA subunitsNO• cGMP

eNOS-/- mice have less mitochondria

Mitochondrial H+ Leak

H+

ATPADP

RespiratoryChain

CaCa2+2+

H+

O2

H+

e-

H+

H2O

ROS

+- ΔψCxICxI

CxIIICxIIIcc

CxIVCxIV

CxVCxV

H+

UCPUCP

Δψm

Res

pira

tion

MoreLeaky

SMR(kJ/kg/day)

1

10

100

1000

10 100 1000

H+

Leak

(nm

ols/

min

/mg

prot

ein)

r = 0.727P = 0.001

Brookes et al. (1998) Comp. Biochem. Physiol. 119B: 269-72Brookes et al. (1997) Biochim. Biophys. Acta 1330: 157-64

H+ Leak: a Component of Basal Metabolic Rate

Uns

atira

tion

Inde

x

10

100

1000

1 10 100 1000H+ Leak (nmols/min/mg protein)

r2=0.50p<0.05

H+ Leak Mechanisms & Modulators

Uncouplingproteins

UCP

H+

GDP,Genipin

ROS,e- philes

Lipidbilayer

H+

Non-specificprotein boundary

H+

AMP ANTallosterism

ANT

H+AMP

CATR

TransientPT pore

ANT

VDAC

Cyp-D

CATR CsA,SfA

H+

Uncoupling Proteins: Location & Homology

UCP-2Fleury et al. (1997) Nature Genetics 15: 269-72

UCP-4

UCP-3Boss et al. (1997) FEBS Lett. 408: 39-42

UCP-1 Brown adipose “thermogenin”, 10% of protein in BAT mito’

Leukocytes, Brain, Kidney, Adipose, Sk-M, Heart

Sk-M, BAT, T-cells, Macrophages

Brain

PUMP Plants

Nicholls & Rial E (1999) J. Bioenerg. Biomembr. 31: 399-406

Bouillaud et al. (2001) Biochim. Biophys. Acta 1504: 107-19

Sanchis et al. (1998) J. Biol. Chem. 273: 34611-5 UCP-5 Brain

Jezek et al. (1996) J. Biol. Chem. 271: 32743-8

Stuart et al. (1999) Biochim. Biophys. Acta. 1413: 50-4 fishUCP

59% = UCP1

57% = UCP1, 71% = UCP2

32% = UCP1-3

38% = UCP1-3

60% = UCP1, 82% = UCP2

H+ Leak in IPC

Nadtochiy et al. (2006) Biochem. J. 395: 611-18

Membrane Potential (mV)

Res

pira

tion

(nm

ols

O2/m

in/m

g pr

otei

n)

CON

IPC+GDPIPC

25

37UCP2

50

0

10

20

30

40

75 100 125 150 175

UCP2 mRNA↑ in delayed IPCJ. Biol. Chem. (2005) 280: 33470-6

ROS, RNS, e-philes ↑H+ Leak/UCPs

Nature (2002) 415: 96-9

J. Biol. Chem. (2003) 278: 48534-45

J. Neurochem. (1998) 70: 2195-202

Car

diac

VO

2/ R

PP Con IPC

0.0

0.5

1.0

Pre 1st

IPC

*

2nd

IPC

*

3rd

IPC

*

Circ. Res. (2003) 93: 192–200

DNP/FCCP/Tg ↑UCP cardioprotection

Cardiovasc. Res. (2000) 47: 68-73

J. Mol. Cell. Cardiol. (2003) 35: 749-59

Circulation (2004) 110: 528-33

ROS / RNS are essential for IPCAntiox. Redox. Sig. (2004) 6: 393-404

Cardiovasc Res. (2006) 70: 231-9

Δψ (mV)

Res

pira

tion

(nm

ols

O2/m

in/m

g pr

otei

n) Ctrl

1µM LNO2

LNO2 + GDP

0

10

20

30

40

0 25 50 75 100 125 150 175

Added LNO2 protectsmyocytes from IR

20

40

60

80

100

Ctrl.

IR 500nMLN

O2

Cel

l via

bilit

y (%

)

0

*

Ctrl.

10.06

9 10 11 12 13t (min.) →

9.95 10.06 IPC

LNO2 is formed insidemitochondria during IPC

O

O- NO2

O

O- NO29,10 12,13

ROSRNS

LipidOxidation

UCPActivation

Mild H+ Leak(↓Δψm)

↓ROS↓[Ca2+]m

PT PoreIPC

*O

O- NO2

& Nitration

Not complicated enough? Add in Mito Fusion & Fission

Youle et al. (2005) Nature Rev. Mol. Cell. Biol. 6: 657-63

Functions:• Required for cell division• Getting energy (ATP) to the right place• Regulation of apoptosis• Exchange of mtDNA• Dilution/concentration of damaged protein/lipid• Ssignaling along tubules?

Chan DC (2006) Cell 125: 1241-52

Yoon Y (2005) Science STKE Apr 19 2005(280):pe18

Tubular

Fragmented

Mitochondrial Morphology: a Tightly Regulated Process

OPA-1 (mammalian homolog of Mgm-1p) Mito inner membrane GTPase, remodeling of IMM

Fragmentation essential for apoptosis Youle et al. (2001) Dev. Cell 1: 515-25

hFis-1 (human homolog of yeast Fis1p) 17 kDa mito’ outer membrane protein, recruits DLP1

Overexpress hFis1 fragmentation apoptosis Sugioka et al. (2004) JBC. 279: 52726-34

Mfn-1/2 (mitofusin, mammalian homolog of Fzo-1p) 86 kDa GTPase, tethers 2 mitochondria for fusion

Yu et al. (2005) J. Cell Sci. 118: 4141-51Overexpress Fzo-1p inhibits apoptosis

DLP-1 (dynamin-like protein, DRP-1, Dnm1-p) 80 kDa cytosolic GTPase, pinchase

Fragmentation essential for high glucose-induced ROS Yu et al. (2006) PNAS 103: 2653-8

H+

O2ADP

Δψm+

–

Cx II

NADH

NAD+

Cx I

Cx V

ATP

ANT

Cx III Cx IVCyt-c

Ca2+

UniporterCa2+/Na+

Exch’ UCP

FADH2FAD

TCACycle

H2O

ADP ATPH+Ca2+

VDAC

R+H+

H+ H+

Ca2+ Na+

H+

Rotenone,Amytal

Piericidin, MPP+

DNP

CCCP

FCCP

H+

H+

DIDS

Valinomycin K+

NigericinK+

H+

Attractyloside,Bongkrekic Acid

PAO, NEM

GDP,Genipin

Ru RedRu-360

CGP37157

TTFA, 3-NP,Malonate,Atpenins

Antimycin A,MyxothiazolStigmatellin

Cyanide,Azide,

CO, NO• Oligomycin

K+ATPChannel

K+

Diazoxide,Pinacidil,Nicorandil

5-HD,ATP

Glibenclamide

+

PT Pore

Cyp-DVDAC ANT

Cyclosporin A,N-methyl-val-CsA

Sanglifehrin A

Chloramphenicol

mRNA

MitochondrialProtein Synthesis

MitochondrialMitochondrialInhibitorsInhibitors

Abbreviations… 3-NP: 3-nitro propionic acid, 5-HD: 5-hydroxydecanoate, ANT: adenine nucleotide translocase, CCCP:carbonylcyanide m-chlorophenylhydrazone, Cx I: respiratory complex I etc., Cyp-D: cyclophilin D, Cyt-c: cytochrome c, DNP: dinitrophenol, DIDS: 4,4-diisothiocyanato-stilbene-2,2'disulphonate, FCCP: carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone, MPP+: 1-methyl-4-phenylpyridinium, NEM: N-ethylmaleimide, PAO: phenylarsine oxide, TTFA: thenoyl-trifluoroacetone, UCP: uncoupling protein, VDAC: volatege dependent anion channel.

Arsenite,Ketomethylvalerate

Mersalyl,NEM

PhosphateTransporter

Pi

Trans-aminase

Amino-oxyacetate Glutamate

Oxaloacetate

Ro-68-3400

α-CHC PyruvateTransporter

Pyr