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
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AA
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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
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