Expression Of Voltage-dependent Anion Channel Research Articles

Increased Expression of Voltage-Dependent Anion Channel and Adenine Nucleotide Translocase and the Sensitivity of Ca2+-Induced Mitochondrial Permeability Transition Opening Pore in Old Rat Heart

Increased Expression of Voltage-Dependent Anion Channel and Adenine Nucleotide Translocase and the Sensitivity of Ca2+-Induced Mitochondrial Permeability Transition Opening Pore in Old Rat Heart

Voltage-dependent anion channel (VDAC) is involved in apoptosis of cell lines carrying the mitochondrial DNA mutation

BackgroundThe mitochondrial voltage-dependent anion channel (VDAC) is increasingly implicated in the control of apoptosis. We have studied the effects the mitochondrial DNA (mtDNA) tRNAIle mutation on VDAC expression, localization, and apoptosis.MethodsLymphoblastoid cell lines were derived from 3 symptomatic and 1 asymptomatic members of a family with hypertension associated with the A4263G tRNAIle mutation as well as from control subjects. Mitochondrial potential (ΔΨm) and apoptosis were measured by flow cytometry; co-localization of VDAC and Bax was evaluated by confocal microscopy.ResultsExpression of VDAC and Bax in mtDNA cell lines was found to be increased compared to controls, while expression of the small conductance calcium-dependant potassium channel (sKCa) was unchanged. Confocal imaging revealed co-localization of VDAC/Bax on the outer mitochondrial membrane of A4263G cell lines but not from controls. Flow cytometry indicated that the mitochondrial potential was decreased by 32% in mutated cells versus controls while rates of apoptosis were increased (P < 0.05). The difference was attenuated by Cyclosporin A (CsA, 2 μM), a blocker of VDAC.ConclusionWe conclude that increased expression of mitochondrial VDAC and subcellular co-localization of VDAC/Bax increases mitochondrial permeability and apoptosis in cell lines carrying the mtDNA tRNAIle A4263G mutation.

Decreased Expression of the Voltage-Dependent Anion Channel in Differentiated PC-12 and SH-SY5Y Cells Following Low-Level Pb Exposure

Lead (Pb) has been shown to disrupt cellular energy metabolism, which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage-dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5 and 10microM Pb on VDAC expression in vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time reverse transcription-polymerase chain reaction demonstrated a significant decrease in messenger RNA levels for the VDAC1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5 and 10microM Pb reduces VDAC transcription and expression and is associated with reduced cellular ATP levels.

Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2) Is Involved in ABA-Mediated Early Seedling Development

The voltage-dependent anion channel (VDAC) is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana.

Modulation of Myocardial Mitochondrial VDAC/ MitoKATP Channels in Sepsis

In the present study, we tested the hypothesis that norepinephrine by modulating myocardial voltage‐dependent anion channel (VDAC)/mitoKATP channels can affect the release of cytochrome C and regulate intrinsic apoptotic cascade. Male Sprague‐Dawley rats (350‐400g) were made septic using 200mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. VDAC mRNA and protein expression was determined in the myocardium and isolated single adult rat ventricular myocytes (ARVMs) obtained from sham and septic rats. In addition, mitochondria and cytosolic fractions were isolated from ARVM treated with norepinephrine (NE, 10ìmoles) in the presence and absence of 5‐Hydroxydecanoic acid (5HD; 100ìmoles), a selective mitoKATP channel blocker. Sepsis produced a significant down‐regulation of VDAC mRNA and protein along with upregulation of Bax in both myocardium and ARVMs. Pretreatment of septic ARVMs with 5HD blocked NE‐induced decrease in VDAC expression and the release of cytochrome C. These results suggest that sepsis downregulates myocardial VDAC expression. It appears that NE modulates mitoKATP channels and increases the release of cytochrome C, which may play a crucial role in sepsis‐induced cardiomyocyte dysfunction.

Expression and localization of voltage-dependent anion channels (VDAC) in human spermatozoa

Voltage-dependent anion channels (VDAC), also known as mitochondrial porins, are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures. VDAC allow the passage of the metabolites across the mitochondrial outer membrane, and are involved in metabolite transport and signal transduction. Several recent studies have indicated the important roles of VDAC in maintaining normal structure and motility of mammalian spermatozoa. To study the expression and localization of VDAC in human spermatozoa, different experimental approaches were applied: (1) specific primers were designed and VDAC gene sequences were cloned by PCR amplification from human testis cDNA library; (2) recombinant VDAC proteins were produced in the expression vector Escherichia coli BL21 (DE3); (3) human sperm VDAC proteins were extracted, separated and analyzed by Western blotting; (4) the localization of VDAC in human spermatozoa were detected using immunofluorescence. The three gene sequences and recombinant VDAC proteins were obtained, respectively. VDAC proteins were detected to be located in human spermatozoa, especially in sperm flagella. Our study elucidated for the first time that VDAC were synthesized and secreted at the testis level and eventually became an integral part of sperm proteins.

Haloperidol disrupts Akt signalling to reveal a phosphorylation-dependent regulation of pro-apoptotic Bcl-XS function

The antipsychotic drug haloperidol is still used to treat psychosis and “agitation”, often with devastating consequences, particularly in geriatric and pre-demented patients. Cytotoxicity induced by haloperidol has been associated with induction of Bcl-XS, a pro-apoptotic member of the Bcl-2 family, as well as with modulation of the Akt pro-survival pathway. Using preneuronal PC12 and primary neuronal cultures, we show that haloperidol inactivates Akt. This induces the dephosphorylation of serine residues in Bcl-XS and promotes its association with the mitochondrial voltage-dependent anion channel (VDAC), as well as with cytochrome c- and caspase-3-dependent events. These events are sensitive to expression of constitutively active Akt. Mutation of Serine106 (Ser106), which is flanked by a putative Akt motif, hinders the association of the Bcl-XS protein with Akt, but promotes its association with VDAC. The dephosphorylation mimic, Bcl-XS(Ser106Ala), induces caspase-dependent PC12 and neuronal cell apoptosis. In contrast, Bcl-XS(Ser106Ala) induces a significant loss of VDAC expression, and cytochrome c- and caspase-independent toxicity in the non-neuronal HEK293A cells. We link haloperidol and Akt to Bcl-XS-sensitive toxicity via cell line-dependent mitochondrial events centering on VDAC. This clearly mitigates the chronic use of haloperidol in neuropsychiatric populations, but supports its use as a potential acute therapeutic in cancer, where apoptosis is desirable.

Mitochondrial modulation is involved in the hepatoprotection of Limonium sinense extract against liver damage in mice

Aim of the Study Limonium sinense (Girard) Ktze is a Chinese folk medicine used to treat fever, hemorrhage, hepatitis, and other disorders. The present research focused on the protective effects of L. sinense extracts (LSE) against liver damage. Materials and methods In this study the extract from the root of Limonium sinense was used. Aminotransferase activity detection, electron microscopy, mitochondrial function evaluation, RT-PCR and western blot were used to evaluate the hepatoprotection of LSE in LPS/ d-GalN-intoxicated mice. Results Pretreatment with 100, 200 or 400 mg/kg LSE significantly blocked the increase in both serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels induced by treatment with LPS plus d-GalN (LPS/ d-GalN). Ultrastructural observation by electron microscopy showed reduced hepatocyte nuclear condensation and less lipid deposition. The decrease in both the mitochondrial membrane potential (14.6%) and sensitivity to mitochondrial swelling induced by Ca 2+ (45.9%) observed in the liver of LPS/ d-GalN-treated mice were prevented by pretreatment with LSE. In addition, different doses of LSE increased both the transcription and the translation of voltage-dependent anion channels (VDAC), which was down-regulated by LPS/ d-GalN treatment. Conclusions In summary, LSE protects livers against LPS/ d-GalN-induced damage, possibly by mitochondrial mechanisms related to increased expression of VDAC.

Protective Effects of Asiatic Acid on Rotenone- or H2O2-Induced Injury in SH-SY5Y Cells

Parkinson's disease (PD) is a progressive neurodegenerative disorder with a prevalence of 1-2% in people over the age of 50. Mitochondrial dysfunction occurred in PD patients showing a 15-30% loss of activity in complex I. Asiatic acid (AA), a triterpenoid, is an antioxidant and used for depression treatment, but the effect of AA against PD-like damage has never been reported. In the present study, we investigated the protective effects of AA against H(2)O(2) or rotenone-induced cellular injury and mitochondrial dysfunction in SH-SY5Y cells. Mitochondrial membrane potential (MMP) and the expression of voltage-dependent anion channel (VDAC) were detected with or without AA pretreatment following cellular injury to address the possible mechanisms of AA neuroprotection. The results showed that pre-treatment of AA (0.01-100 nM) protected cells against the toxicity induced by rotenone or H(2)O(2). In addition, MMP dissipation occurred following the exposure of rotenone, which could be prevented by AA treatment. More interestingly, pre-administration of AA inhibited the elevation of VDAC mRNA and protein levels induced by rotenone(100 nM) or H(2)O(2) (300 microM).These data indicate that AA could protect neuronal cells against mitochondrial dysfunctional injury and suggest that AA might be developed as an agent for PD prevention or therapy.

Plasminogen Structural Domains Exhibit Different Functions When Associated with Cell Surface GRP78 or the Voltage-dependent Anion Channel

Both the voltage-dependent anion channel and the glucose-regulated protein 78 have been identified as plasminogen kringle 5 receptors on endothelial cells. In this study, we demonstrate that kringle 5 binds to a region localized in the N-terminal domain of the glucose-regulated protein 78, whereas microplasminogen does so through the C-terminal domain of the glucose-regulated protein 78. Both plasminogen fragments induce Ca(2+) signaling cascades; however, kringle 5 acts through voltage-dependent anion channel and microplasminogen does so via the glucose-regulated protein 78. Because trafficking of voltage-dependent anion channel to the cell surface is associated with heat shock proteins, we investigated a possible association between voltage-dependent anion channel and glucose-regulated protein 78 on the surface of 1-LN human prostate tumor cells. We demonstrate that these proteins co-localize, and changes in the expression of the glucoseregulated protein 78 affect the expression of voltage-dependent anion channel. To differentiate the functions of these receptor proteins, either when acting singly or as a complex, we employed human hexokinase I as a specific ligand for voltage-dependent anion channel, in addition to kringle 5. We show that kringle 5 inhibits 1-LN cell proliferation and promotes caspase-7 activity by a mechanism that requires binding to cell surface voltage-dependent anion channel and is inhibited by human hexokinase I.

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that naïve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in naïve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both naïve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human naïve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.

Expression of VDAC Regulated by Extracts of Limonium sinense Ktze root Against CCl4-induced Liver Damage

The expression of mitochondrial voltage-dependent anion channels (VDAC) mayunderlie the protective effects of Limonium sinense (Girard) Ktze root extracts (LSE) againstcarbon tetrachloride-induced liver damage. Pretreatment of mice with 100 mg/kg, 200mg/kg or 400 mg/kg LSE significantly blocked the carbon tetrachloride-induced increase inboth serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT)levels. Ultrastructural observations by electron microscope confirmed hepatoprotection,showing decreased nuclear condensation, ameliorated mitochondrial fragmentation of thecristae and less lipid deposition. Pretreatment with LSE prevented the decrease of thedisruption of mitochondrial membrane potential (15.3%) observed in the liver of the carbontetrachloride-insulted mice, further demonstrating the mitochondrial protection. In addition,LSE treatment (100-400 mg/kg) significantly increased both transcription and translation ofVDAC. The above data suggests that LSE mitigates the damage to liver mitochondriainduced by carbon tetrachloride, possibly through regulation of mitochondrial VDAC, one ofthe most important proteins in the mitochondrial outer membrane.

Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC ( PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one α-helix, 13 transmembrane β-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis.

VDAC‐1 May Interact with CFTR to Impart Important Cellular Function in Mouse Heart

The cystic fibrosis transmembrane-conductance regulator (CFTR) Cl− channels may play an important role in ischemic preconditioning (IPC)-mediated cardioprotection in mouse heart. To investigate the mechanisms for the role of CFTR in IPC we used proteomic approaches to analyze changes in the protein expression profiles of both plasma membrane (PM) and mitochondrial (MT) compartments caused by CFTR gene knockout. Proteins from heart tissue (10 hearts pooled as one sample) of either wild-type (FVB/NJ) or CFTR−/− (FABPCFTR) mice were isolated, purified, and fractionated. Marker enzyme assays demonstrated a 1.74-fold enrichment of PM in the PM fractions and a 4.4-fold enrichment of MT in MT fractions, and a strong reduction of other compartments. Proteomic analysis (2D-PAGE, MALDI-TOF/TOF mass spectrometry) unambiguously identified a significant (40%) decrease in the expression of the voltage-dependent anion channel 1 (VDAC-1) in cardiac PM proteins of the CFTR−/− mice. Immunocytochemistry confirmed VDAC-1 expression in PM of cardiac myocytes. Western blots on both 2D-PAGEs and 1D gels further confirmed the reduced expression of VDAC-1 in PM of CFTR−/− mouse heart. These results strongly suggest that VDAC-1 may be an important component of the CFTR subproteome and may interact with CFTR to impart orchestrated functions in IPC-mediated cardioprotection. (Supported by NCRR at NIH P20 RR15581 and NIH R01 HL63914)

Furanonaphthoquinones cause apoptosis of cancer cells by inducing the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel

The mitochondrial production of reactive oxygen species (ROS) has been implicated in the anticancer activity of furanonaphthoquinone. However, the mechanism of the activation remains elusive. In the current study, we found that treatment of HeLa cells with 2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13) induces mitochondrial swelling, followed by apoptosis. This toxic effect of FNQ13 was reduced by the radical scavengers α-tocopherol and trolox. Cytochemical experiments in isolated mitochondria showed that a combination of FNQ13 and NADH induces the production of H2O2 at the exterior mitochondrial membrane surface. This production of H2O2 was reduced by an antibody to the voltage-dependent anion channel (VDAC). Overexpression of the VDAC by transfection with vdac1 cDNA increased the production of H2O2 by HeLa cells, whereas transfection with a small interfering RNA to VDAC reduced FNQ13-induced H2O2 production and cell death due to an almost complete knockdown of VDAC expression. We also found significant correlations between the expression of VDAC and the induction of H2O2 production and cell death by FNQ13 in 11 human cancer cell lines. These results indicate that the anticancer activity of furanonaphthoquinones depends on the production of reactive oxygen species by mitochondrial permeability transition pores (MPTP) including the VDAC.

Acetyl- l-carnitine up-regulates expression of voltage-dependent anion channel in the rat brain

Acetyl- l-carnitine (ALC) exerts unique neuroprotective, neuromodulatory, and neurotrophic properties, which play an important role in counteracting various pathological processes, and have antioxidative properties, protecting cells against lipid peroxidation. In this study, suppression subtractive hybridization (SSH) method was applied for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts after treatment of rats with ALC. The technique generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes that are regulated after ALC treatment. In the present paper, we report the identification of the gene of mitochondrial voltage-dependent anion channel (VDAC) protein which is positively modulated by the ALC treatment. VDAC is a small pore-forming protein of the mitochondrial outer membrane. It represents an interesting tool for Ca 2+ homeostasis, and it plays a central role in apoptosis. In addition, VDAC seems to have a relevant role in the synaptic plasticity.

Genetic Demonstration That the Plasma Membrane Maxianion Channel and Voltage-dependent Anion Channels Are Unrelated Proteins

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide

The precise molecular mechanism underlying arsenic trioxide (As(2)O(3))-induced apoptosis is a subject of extensive study. Here, we show that clinically relevant doses of As(2)O(3) can induce typical apoptosis in IM-9, a multiple myeloma cell line, in a Bcl-2 inhibitable manner. We confirmed that As(2)O(3) directly induced cytochrome c (cyto c) release from isolated mouse liver mitochondria via the mitochondrial permeability transition pore, and we further identified the voltage-dependent anion channel (VDAC) as a biological target of As(2)O(3) responsible for eliciting cyto c release in apoptosis. First, pretreatment of the isolated mitochondria with an anti-VDAC antibody specifically prevented As(2)O(3)-induced cyto c release. Second, in proteoliposome experiments, VDAC by itself was sufficient to mediate As(2)O(3)-induced cyto c release, which could be specifically inhibited by Bcl-X(L). Third, As(2)O(3) induced mitochondria membrane potential (DeltaPsim) reduction and cyto c release only in the VDAC-expressing, but not in the VDAC-deficient yeast strain. Finally, we found that As(2)O(3) induced the increased expression and homodimerization of VDAC in IM-9 cells, but not in Bcl-2 overexpressing cells, suggesting that VDAC homodimerization could potentially determine its gating capacity to cyto c, and Bcl-2 blockage of VDAC homodimerization represents a novel mechanism for its inhibition of apoptosis.

Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria.

Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.

Mutations in the voltage-dependent anion channel of the mitochondrial outer membrane cause a dominant nonlethal growth impairment.

Point mutations at K234 and K236 in the yeast voltage-dependent anion channel 1 (VDAC1) of the mitochondrial outer membrane have been shown to markedly impair the membrane insertion of this protein (Smith et al., 1995; Angeles et al., 1998). Mutants of this type were expressed in vivo in a strain of yeast with a disruption in the VDAC1 gene. Expression of the various VDAC1 forms was under the control of a Gall promoter. Wild-type VDAC1 expression fully complemented the slow growth phenotype caused by the disruption. VDAC1 mutants in which K234 and K236 were replaced by arginine, glutamate, or glutamine caused a more severe negative effect on growth. This effect appeared to be dominant since the mutant VDAC1 forms suppressed growth in a yeast strain that retained its VDAC1 gene. This apparent dominant negative effect on growth did not seem to be specific for any stage of the cell cycle. However, the growth defect was not lethal as the affected cells still could accumulate the vital stain, FUN1. Expression of a mutant in which K234 had been replaced by glutamate had more serious negative growth effects than did a similar mutation at K236. Expression of delta71-116 VDAC1 complemented the VDAC1 disruption; however, expression of the same deletion mutant in which the lysines corresponding to K234 and K236 were mutated to glutamate severely impaired growth. These results have shown that a deficiency of lysine at positions 234 and 236 in VDAC I causes a nonlethal growth defect that is more severe than deletion of 45 amino acids from VDACI or disruption of the VDAC1 gene. They also indicate that there is a hierarchy in the importance of these lysines with mutations at K234 being the more serious.