Abstract 5420: Dephosphorylation of the Cardiac Mitochondrial Voltage-Dependent Anion Channel Prevents Inhibition by Hexokinase

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Qunli Cheng ◽  
Zeljko J Bosnjak ◽  
Wai-Meng Kwok
Keyword(s):  
Mitochondrial Membrane ◽  
Mitochondrial Permeability Transition ◽  
Adenine Nucleotide ◽  
Standard Procedure ◽  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Cyclophilin D ◽  
Voltage Dependent Anion Channel ◽  
Voltage Dependent ◽  
The Mean

The mitochondrial permeability transition pore (mPTP) has been implicated as the end effector in ischemic and pharmacological preconditioning. Though the molecular composition of the mPTP is thought to consist of cyclophilin D located in the mitochondrial matrix, adenine nucleotide translocase on the inner mitochondrial membrane, and the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane, recent studies have raised the possibility that VDAC may be a regulatory, rather than a major, component of mPTP. Nevertheless, VDAC is likely to be a critical component of the preconditioning signaling pathway since it is the main conduit for metabolite diffusion across the mitochondrial outer membrane. Yet, the direct measurements of cardiac VDAC activity and modulation have been limited. In the present study, we purified VDAC from rat hearts using standard procedure and investigated its modulation by phosphatase and hexokinase. VDAC was incorporated into planar lipid bilayer for measurements of channel activities. The channel exhibited the reported voltage-dependent gating. Several conductance states were identified, with the most prevalent between 1.5 to 2 nS in 0.5 M NaCl. Koenig’s polyanion, a VDAC blocker, triggered channel flickering and decreased the mean current by 78±6%. In the presence of phosphatase (1 unit/ml), the mean conductance significantly increased from 1.81±0.03 to 3.68±0.61 nS (n=9; mean±SEM). However, the addition of a recombinant hexokinase (5 units/ml; GenWay Biotech) had no significant effect on the phosphatase-enhanced VDAC current (n=4). In contrast, recombinant hexokinase alone significantly decreased the mean conductance from 1.75±0.05 to 0.79±0.19 nS (n=4). The addition of phosphatase reversed the inhibitory effect of hexokinase and further enhanced VDAC activity, increasing the mean conductance to 2.69±0.19 nS (n=4). Our results suggest that the dephosphorylation of VDAC prevents the inhibitory effects of hexokinase. Furthermore, VDAC activity suppressed by hexokinase can be reversed by dephosphorylation of the channel. In conclusion, we have reported on a novel observation at the functional level that basal phosphorylation of the cardiac VDAC may be required for its modulation by hexokinase.

The mitochondrial permeability transition pore

1999 ◽  
Vol 66 ◽  
pp. 167-179 ◽  
Author(s):  
Martin Crompton ◽  
Sukaina Virji ◽  
Veronica Doyle ◽  
Nicholas Johnson ◽  
John M. Ward
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Adenine Nucleotide ◽  
Permeability Transition Pore ◽  
Anion Channel ◽  
Permeability Transition ◽  
Adenine Nucleotide Translocase ◽  
Voltage Dependent Anion Channel ◽  
Inner Membrane ◽  
Membrane Pore ◽  
Voltage Dependent

This chapter reviews recent advances in the identification of the structural elements of the permeability transition pore. The discovery that cyclosporin A (CsA) inhibits the pore proved instrumental. Various approaches indicate that CsA blocks the pore by binding to cyclophilin (CyP)-D. In particular, covalent labelling of CyP-D in situ by a photoactive CsA derivative has shown that pore ligands have the same effects on the degree to which CsA both blocks the pore and binds to CyP-D. The recognition that CyP-D is a key component has enabled the other constituents to be resolved. Use of a CyP-D fusion protein as affinity matrix has revealed that CyP-D binds very strongly to 1:1 complexes of the voltage-dependent anion channel (from the outer membrane) and adenine nucleotide translocase (inner membrane). Our current model envisages that the pore arises as a complex between these three components at contact sites between the mitochondrial inner and outer membranes. This is in line with recent reconstitutions of pore activity from protein fractions containing these proteins. The strength of interaction between these proteins suggests that it may be a permanent feature rather than assembled only under pathological conditions. Calcium, the key activator of the pore, does not appear to affect pore assembly; rather, an allosteric action allowing pore flicker into an open state is indicated. CsA inhibits pore flicker and lowers the binding affinity for calcium. Whether adenine nucleotide translocase or the voltage-dependent anion channel (via inner membrane insertion) provides the inner membrane pore has not been settled, and data relevant to this issue are also documented.

scholarly journals Functions of BCL-XLat the Interface between Cell Death and Metabolism

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Judith Michels ◽  
Oliver Kepp ◽  
Laura Senovilla ◽  
Delphine Lissa ◽  
Maria Castedo ◽  
...  
Keyword(s):  
Cell Death ◽  
Mitochondrial Permeability Transition ◽  
Atp Synthesis ◽  
Anion Channel ◽  
Permeability Transition ◽  
Short Review ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent Anion Channel ◽  
Regulated Necrosis ◽  
Voltage Dependent

The BCL-2 homolog BCL-XL, one of the two protein products ofBCL2L1, has originally been characterized for its prominent prosurvival functions. Similar to BCL-2, BCL-XLbinds to its multidomain proapoptotic counterparts BAX and BAK, hence preventing the formation of lethal pores in the mitochondrial outer membrane, as well as to multiple BH3-only proteins, thus interrupting apical proapoptotic signals. In addition, BCL-XLhas been suggested to exert cytoprotective functions by sequestering a cytosolic pool of the pro-apoptotic transcription factor p53 and by binding to the voltage-dependent anion channel 1 (VDAC1), thereby inhibiting the so-called mitochondrial permeability transition (MPT). Thus, BCL-XLappears to play a prominent role in the regulation of multiple distinct types of cell death, including apoptosis and regulated necrosis. More recently, great attention has been given to the cell death-unrelated functions of BCL-2-like proteins. In particular, BCL-XLhas been shown to modulate a number of pathophysiological processes, including—but not limited to—mitochondrial ATP synthesis, protein acetylation, autophagy and mitosis. In this short review article, we will discuss the functions of BCL-XLat the interface between cell death and metabolism.

scholarly journals Biogenesis of Porin of the Outer Mitochondrial Membrane Involves an Import Pathway via Receptors and the General Import Pore of the Tom Complex

2001 ◽  
Vol 152 (2) ◽  
pp. 289-300 ◽  
Author(s):  
Thomas Krimmer ◽  
Doron Rapaport ◽  
Michael T. Ryan ◽  
Chris Meisinger ◽  
C. Kenneth Kassenbrock ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Membrane ◽  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Outer Mitochondrial Membrane ◽  
Voltage Dependent Anion Channel ◽  
Surface Receptors ◽  
Voltage Dependent ◽  
Surface Receptor

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro–imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

scholarly journals VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release

2001 ◽  
Vol 155 (6) ◽  
pp. 1003-1016 ◽  
Author(s):  
Muniswamy Madesh ◽  
György Hajnóczky
Keyword(s):  
Cytochrome C ◽  
Hepg2 Cells ◽  
Mitochondrial Membrane ◽  
Mitochondrial Permeability Transition ◽  
Anion Channel ◽  
Permeability Transition ◽  
Mitochondrial Outer Membrane ◽  
Inner Mitochondrial Membrane ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release

Enhanced formation of reactive oxygen species (ROS), superoxide (O2·−), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2·− elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2·− and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2·−-induced CCR. Furthermore, O2·− alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2·−-induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2·− exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2·−-induced CCR did not depend on Bax translocation to mitochondria. O2·−-induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2·− triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.

scholarly journals Identification of two voltage-dependent anion channel-like protein sequences conserved in Kinetoplastida

2012 ◽  
Vol 8 (3) ◽  
pp. 446-449 ◽  
Author(s):  
Nadine Flinner ◽  
Enrico Schleiff ◽  
Oliver Mirus
Keyword(s):  
Outer Membrane ◽  
Trypanosoma Brucei ◽  
Protein Sequences ◽  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent Anion Channel ◽  
Voltage Dependent

The eukaryotic porin superfamily consists of two families, voltage-dependent anion channel (VDAC) and Tom40, which are both located in the mitochondrial outer membrane. In Trypanosoma brucei , only a single member of the VDAC family has been described. We report the detection of two additional eukaryotic porin-like sequences in T. brucei . By bioinformatic means, we classify both as putative VDAC isoforms.

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