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The figures in this presentation were taken from: wadsworth/

The figures in this presentation were taken from: http://www.wadsworth.org/

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The figures in this presentation were taken from: wadsworth/

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  1. 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.

  2. Mitochondria and Cellular Metabolism

  3. 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

  4. a mitochondrion... work of Carmen Mannella from: http://www.wadsworth.org/BMS/SCBlinks/channels2.htm http://www.wadsworth.org/rvbc/tomography.html

  5. Images of VDAC Biophysics of Channels Associated with Apoptosis Frozen-hydrated specimen by Carmen Mannella from: http://www.wadsworth.org /BMS/SCBlinks/vdac.htm 2D-crystals in mitochon-drial outer membrane averaged surface view

  6. A N K I Y Q K V N K K L E T A V N L S K I F T A T E K K G W L K G T G K A A I G F R T N S N G A T G W T G F Y Y N N Y E Q E T P K T I A V P P G T D S R G D K T N L W N F E Y E D P L P A Y A E E R T S H G G N D T G Q D L S F Y L S I L W V A Q I F G K K T K G D N V L N C T K E S A T S T T F L L A T F Y L L R D K S E E T G A G H S G T G V F L G L I L C G Y L A L L L T K D S S T K D R Q Q K G S G G Y L A G V L M I M F V I A L G F K N T E G D S N E N L L K G I T V D R F P F D N S L H L E K Q A D G E T S D G L I A T T T K G G A Y K A V R S K G N N T V Q S N F A V K111 A F G N S Y Y H K V N S Q V E A G S K A A R L T A H S189 W N L T68 N F F K T A V E L G V T N G T K S G V T K V Y N T A Q S R P Q P S L I E252 A V P N37 K T L N N D R G A N V D W F F H E144 D P L V S282 F S P A T N S H G G T V L T E D I T F F T F G V F A S V L F A K N K K A I R I L T F N G T S A S V G N G A A A I V Y V F N D K T E A E F D G A K A G S L L V G L L A F I A A G I A T N K E K A E K D N S S K A S G D F I S G T L L A A Y I A F V Y H T T S K G L T G T N V D K L G H T V K K P Y P D G T H A G A D V E A D A R Q T K M L V H K A A N I A A K Q Y L Q D D T G G Y S A A V human VDAC1 N. crassa VDAC

  7. 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

  8. Chamber for making planar membranes Top view Side view Cross section 0.1 mm hole Saran partition

  9. Formation of Membrane from Monolayers 0.1 mm

  10. syringe cis trans 0.1 mm stir bar electrode V - + Planar Membrane Setup

  11. 200 pA addition of VDAC return to 10 mV switch from 10 to 40 mV time (sec) Insertion and voltage-dependent closure of VDAC

  12. The voltage dependence of VDAC

  13. 50 100 150 200 250 Time (minutes)

  14. VDAC Selectivity Change Upon Closure 2-

  15. applied voltage current recording

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

  17. Point mutations made in disputed strands

  18. (25 mM)

  19. Biotin modification + Avidin binding biotin

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