Dokument

Summary:
With about 10 000 copies per cell the voltage-dependent anion channels (VDACs) are the most abundant proteins of the mitochondrial outer membrane and are known to be involved in mitochondrial processes such as ATP-, calcium or ROS-transport. Beside this, they were identified as key players of mitochondrial physiology such as being involved in the mitochondrial related apoptotic pathway. Because of their strategic location as well as interaction with pro- and anti-apoptotic proteins, VDACs are involved in various diseases like Alzheimer, Down syndrome, cancer, stroke, and amylotrophic lateral sclerosis. Because of this multifunctionality, VDACs are important targets for medical approaches. After their discovery, VDACs have been extensively studied in terms of their structural organisation and their gating mechanism. The N-terminal region in the pores interior fuelled further debates about the gating mechanism of VDACs. VDACs reply to an applied voltage in a symmetric manner showing one open and several closed states. In this work I classified the closed states into at least three major states. With these quantitative electrophysiological measurements I demonstrated for the first time that a conformational variability of the N-terminus is essential for VDACs function. Through engineering of double-cysteine mVDAC1 variants affixing the N-terminal segment at the bottom and midpoint of the pore I verified that the N-terminus is the major trigger of VDAC´s gating. Additionally, it was shown that channel transitions are not solely dependent on the N-terminus. By analysing EMP as minimal model system for β-barrel gating, I revealed that a loop-independent gating exists, similar to that observed in the double-cysteine mVDAC1 variants. Given VDACs that interact with a plethora of effector proteins, I focused on the pro-apoptotic VDAC-effector Bid/tBid. Recent studies imply that Bid is a key player in neuronal cell death pathways. Accordingly, Bid seems to promote mitochondrial demise by release of death promoting proteins in the cytosol and the acceleration of oxidative stress. Furthermore, Bid-deficient neurons are highly resistant to cell death stimuli including oxygen-glucose deprivation (OGD) and glutamate-induced excitotoxicity in vitro and show reduced damage after cerebral ischemia and brain trauma in vivo. Here I could show a direct interaction between mVDAC1 and Bid/tBid and characterised the electrophysiological influence on VDAC in a quantitative manner. Biophysical analyse by SRCD, SROCD and EPR measurements give first insights into the structure of the VDAC-tBid complex. These data highlight the critical role for VDAC1 as a mitochondrial receptor for Bid thus providing a major control point of neuronal demise.

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