The figures in this presentation were taken from:http://www.wadsworth.org/Blachly-Dyson, E., Peng, S. Z., Colombini, M. and Forte, M. 1989. Probing the structure of the mitochondrial channel, VDAC, by site-directed mutagenesis: a progress report. Journal of Bioenergetics and Biomembranes, 21:471-483.Blachly-Dyson, E., Peng, S. Z., Colombini, M. and Forte, M. 1990. Alteration of the selectivity of the VDAC ion channel by site-directed mutagenesis: implications for the structure of a membrane ion channel. Science, 247: 1233-1236.Thomas, L., Kocsis, E., Colombini, M., Erbe, E., Trus, B.L. and Steven, A.C. 1991. Surface topography and molecular stoichiometry of the mitochondrial channel, VDAC, in crystalline arrays. Journal of Structural Biology, 106: 161-171.Peng, S., Blachly-Dyson, E., Colombini, M. and Forte, M. 1992. Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel. Biophysical Journal, 62: 123-153.Thomas, L., Blachly-Dyson, E., Colombini, M., and Forte, M. 1993. Mapping of residues forming the voltage sensor of the VDAC ion channel. Proceedings of the National Academy of Sciences U.S.A., 90: 5446-5449.Song, J., Midson, C., Blachly-Dyson, E., Forte, M., and Colombini, M. 1998. The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes. Biophysical Journal, 74: 2926-2944.Song, J., Midson, C., Blachly-Dyson, E., Forte, M., and Colombini, M. 1998. The topology of VDAC as probed by biotin modification. Journal of Biological Chemistry, 273:24406‑24413.Vander Heiden, M.G., Chandel, N.S., Li, X.X., Schumacker, P.T., Colombini, M., and Thompson, C.B. 2000. Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proceedings of the National Academy of Sciences U.S.A., 97: 4666‑4671.

Mitochondria and Cellular Metabolism

Voltage Dependent Anion-selective Channel (VDAC):

• 32kDa protein located in the mitochondrial outer membrane • Responsible for the metabolite permeation through the mitochondrial outer membrane of all eukaryotes

• Permeable to non-electrolytes up to 5 kDa but far more restrictive for charged molecules

from: http://www.wadsworth.org/BMS/SCBlinks/channels2.htm

http://www.wadsworth.org/rvbc/tomography.html

work of Carmen Mannella

a mitochondrion...

Biophysics of Channels Associated with Apoptosis

Images of VDAC

2D-crystals in mitochon-drial outer membrane

averaged surface view

Frozen-hydrated specimen

by Carmen Mannella

from: http://www.wadsworth.org

/BMS/SCBlinks/vdac.htm

human VDAC1

N. crassa VDAC

A V P

AF

SD

IA

KS A

ND

LL

NK

DF

YH

L

AA

G

T

I

E

V

K

S

N

T

P

N

N37

V

A

F

K

V

T

G

K

S

T

H

DK

V

T

S

G

A

L

E

G

K

F

T

D

K

P

N

G

L T68

V

T

Q

T

W

N

T

A

N

A

L

E

T

K

V

E

M

AD

N

L

A

K

G

L

K

A

E

G

I

F

S

F

L

P

A

T

N

A

R

GA

K111FN

L

H

F

K

Q

S

N

F

H

G

R

A

F

F

D

L

L

KG

P

T

A

N

I

D

A

I

V

G

H E144

G

F

L

A

G

A

S

A

G

Y

D

V

QKAAI

T

G Y S A A V

G

Y

H

A

P

T

Y

S

A

A

I

T

A

T

D

N

L

S

V

F

S189

AS Y Y H K V N S Q V E A G S K A

T

WN

SKTGNTVGLEVATK

Y

R

I

D

P

V

S

F

V

K

G

K

I

N

D

R

G

V

A

A

I

A

Y

N

V

L

LR

E252GV

T

L

G

V

G

A

S

F

D

T

Q

K

LD

Q

A

T

H

K

V

G

T

S

F

T

F

E

S282

A V P PTYA

D LGKS AR

D VFTK GYG F

G L IKLDLKTKSEN

GLEFTSSGSANTE

T TKVTGSLETKYRWTEYGLT

F TEKWNTDNTLGTEITVEDQ

LARGLKLTFDSSFSPNTGKKN

AKIKTGYKREHINLGCDMDFD

I AGPSIRGALVLGY E

GWLAGYQMNFETAKSRV

TQ S N F A V

GYKTDEFQLHTNVNDGTEFGGS I Y Q K V N K K L E T A V N L

AW

TAGNSNTRFGIAAKYQIDPDACFSAKVNN

SSLIGLGYTQTL

KPGIKLTLSALLDGKN

VNAGGHKLGLGLEFQA

The functional properties of VDAC were analyzed by reconstituting the channels into a planar phospholipid membrane

• Detergent-solubilized VDAC is dispersed into the aqueous phase

• VDAC spontaneously inserts into the membrane

Top view

Side view

Cross section

Saran partition0.1 mm hole

Chamber for making planar membranes

0.1 mm

Formation of Membrane from Monolayers

V

+

-

syringe

stir bar

electrode

cis trans

0.1 mm

Planar Membrane Setup

200 pA

switch from 10 to 40 mV

return to 10 mV

addition of VDAC

time (sec)

Insertion and voltage-dependent closure of VDAC

The voltage dependence of VDAC

Time (minutes) 50 100 150 200 250

Ion F

lux

(x105 io

ns/

sec )

0

1

2

3

4

5

6

7

Ion F

lux

( 106 io

ns/

sec )

0

2

4

6

8

10

12

14

16

VDAC openVDAC closed

Na+ PC succinate2- ATP4--2HPO4

VDAC Selectivity Change Upon Closure

2-

current recording

applied voltage

Solution structure of the integral human membrane protein VDAC-1 in detergent micelles; Hiller et al., Science. 321: 1206–1210 (2008).

Point mutations made in disputed strands

(25 mM)

Biotin modification + Avidin binding

biotin