Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.
Background: There is ongoing controversy concerning the location and mobility of the N-terminal ␣-helix in VDAC1 during voltage gating. Results: mVDAC1 with the N-terminal ␣-helix cross-linked to -strand 11 forms typical voltage-gated channels. Conclusion:The N-terminal domain of VDAC1 does not move independently during voltage gating. Significance: This study dramatically alters the current view of voltage gating dynamic in VDAC1.The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 -strands and an N-terminal ␣-helix located near the midpoint of the pore. The position of this ␣-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal ␣-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the ␣-helix to the wall of the -barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal ␣-helix is located inside the pore of VDAC in the open state and remains associated with -strand 11 of the pore wall during voltage gating.The voltage-dependent anion channel (VDAC) 4 serves as the primary conduit between the mitochondria and the rest of the cell, facilitating free exchange of ions and metabolites across the outer mitochondrial membrane. In addition to its metabolic and energetic functions, VDAC has a more complex role, serving as a receptor for molecules and proteins that modulate the organelle's permeability and thereby its function (1-3). This multifunctional channel has been implicated in the metabolic stresses of cancer, cardiovascular disease, and mitochondrialdependent cell death (4 -8); thus, understanding the structure and function of VDAC constitutes a critical objective for basic as well as medical research.Single channel conductance experiments on VDAC1 at low membrane potential (Ͻ30 mV) reveal a high conductance (4.1 Ϯ 0.1 nanosiemens in 1 M KCl) indicative of a large pore, usually referred to as the "open" state of the channel (1). This conformer facilitates the passage of 10 6 ATP molecules (9) and displays a preference for monovalent anions over cations with the anion-to-cation permeability ratio of 2:1 in high salt (10) and 4:1 in physiological salt concentration (11). As voltage is increased (Ͼ30 mV) in either a positive or a negative direction, the channel switches into the lower conducting states (around 2 nanosiemens in 1 M KCl), termed as the "closed" state(s). These states are cation-selective wi...
Background: VDAC voltage gating depends on pH. Results: Acidification asymmetrically and reversibly enhances VDAC closure. Conclusion: pH-sensitive formation of stable salt bridges in the cytosolic side of VDAC explains its asymmetrical response to acidification. Significance: The pronounced sensitivity of the cytosolic side of VDAC to acidification provides new insights into the protective effect of cytosolic acidification during ischemia.
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