Voltage-dependent Anion Channel Phosphorylation Research Articles

Extracellular Signal-Regulated Kinase1 (ERK1)-Mediated Phosphorylation of Voltage-Dependent Anion Channel (VDAC) Suppresses its Conductance.

ERK1 is one of the members of the mitogen-activated protein kinases that regulate important cellular functions. VDAC is located at the outer membrane of mitochondria. Here, an interaction between VDAC and ERK1 has been studied on an artificial planar lipid bilayer using in vitro electrophysiology experiments. We report that VDAC is phosphorylated by ERK1 in the presence of Mg2+-ATP and its single-channel currents are inhibited on the artificial bilayer membrane. Treatment of Alkaline phosphatase on ERK1 phosphorylated VDAC leads to partial recovery of the single-channel VDAC currents. Later, phosphorylation of VDAC was demonstrated by Pro-Q diamond dye. Mass Spectrometric studies indicate phosphorylation of VDAC at Threonine 33, Threonine 55, and Serine 35. In a nutshell, phosphorylation of VDAC leads to the closure of the channel.

Flavonoid GL-V9 induces apoptosis and inhibits glycolysis of breast cancer via disrupting GSK-3β-modulated mitochondrial binding of HKII

Energy metabolism plays important roles in the growth and survival of cancer cells. Here, we find a newly synthesized flavonoid named GL-V9, which inhibits glycolysis and induces apoptosis of human breast cancer cell lines, and investigate the underlying mechanism. Results show that hexokinase II (HKII) plays important roles in the anticancer effects of GL-V9. GL-V9 not only downregulates the expression of HKII in MDA-MB-231 and MCF-7 cells, but also induces dissociation of HKII from voltage-dependent anion channel (VDAC) in mitochondria, resulting in glycolytic inhibition and mitochondrial-mediated apoptosis. The dissociation of mitochondrial HKII is attributed to GSK-3β-induced phosphorylation of mitochondrial VDAC. Our in vivo experiments also show that GL-V9 significantly inhibits the growth of human breast cancer due to activation of GSK-3β and inactivation of AKT. Thus, GL-V9 induces cytotoxicity in breast cancer cells via disrupting the mitochondrial binding of HKII. Our works demonstrate the significance of metabolic regulators in cancer growth and offer a fresh insight into the molecular basis for the development of GL-V9 as a candidate for breast carcinoma treatment.

GSK3β and VDAC Involvement in ER Stress and Apoptosis Modulation during Orthotopic Liver Transplantation

We investigated the involvement of glycogen synthase kinase-3β (GSK3β) and the voltage-dependent anion channel (VDAC) in livers subjected to cold ischemia–reperfusion injury (I/R) associated with orthotopic liver transplantation (OLT). Rat livers were preserved in University of Wisconsin (UW) and Institute Georges Lopez (IGL-1) solution, the latter enriched or not with trimetazidine, and then subjected to OLT. Transaminase (ALT) and HMGB1 protein levels, glutamate dehydrogenase (GLDH), and oxidative stress (MDA) were measured. The AKT protein kinase and its direct substrates, GSK3β and VDAC, as well as caspases 3, 9, and cytochrome C and reticulum endoplasmic stress-related proteins (GRP78, pPERK, ATF4, and CHOP), were determined by Western blot. IGL-1+TMZ significantly reduced liver injury. We also observed a significant phosphorylation of AKT, which in turn induced the phosphorylation and inhibition of GSK3β. In addition, TMZ protected the mitochondria since, in comparison with IGL-1 alone, we found reductions in VDAC phosphorylation, apoptosis, and GLDH release. All these results were correlated with decreased ER stress. Addition of TMZ to IGL-1 solution increased the tolerance of the liver graft to I/R injury through inhibition of GSK3β and VDAC, contributing to ER stress reduction and cell death prevention.

Regulation of Mitochondrial Signaling and Quality Control by the 18KDA Translocator Protein (TSPO)

The 18-kDa translocator protein (TSPO) is localized to the outer mitochondrial membrane (OMM) where it is found in complex with the voltage dependent anion channel (VDAC). TSPO expression is elevated in cancer and inflammatory conditions and it is well known for its ligand binding properties and application in PET imaging as a marker of neuroinflammation. TSPO is implicated in multiple mitochondrial functions including steroidogenesis, apoptosis and ATP production although precise molecular mechanisms remain to be elucidated. We recently demonstrated that increased TSPO expression limits mitochondrial ubiquitination and mitophagy through a reactive oxygen species (ROS) -dependent process, which leads to changes in mitochondrial morphology and biomass.Here we have corroborated this pathway by establishing an interplay with the protein kinase A (PKA) signaling complex, which is recruited to mitochondria by TSPO via acyl-CoA binding domain containing 3 (ACBD3). Using targeted luminescent protein chimeras, we show that the combined accumulation of TSPO and PKA alters subcellular Ca2+ dynamics and metabolism, which we propose arises from increased VDAC phosphorylation and subsequent changes in channel activity. Of note, this pathway is activated under glutamate-induced cell stress in the neuronal cell line, SH-SY5Y.This work highlights an important role for the OMM in fine-tuning mitochondrial signaling and mitophagy. As an inducible factor, TSPO represents a critical physiological element that facilitates mitochondrial adaptation to altered cellular metabolic requirements under stress conditions.

Phosphorylation of voltage-dependent anion channel by c-Jun N-terminal Kinase-3 leads to closure of the channel

Stress activated c-Jun N-terminal Kinase-3 (JNK3) has been reported to act on mitochondrion to promote neuronal cell death. Phosphorylation of mitochondrial Voltage-Dependent Anion Channel (VDAC) plays an important role in mitochondria-mediated cell death. Keeping these in view phosphorylation of rat brain VDAC by JNK3 has been studied in vitro. Pro Q Diamond phospho-protein staining experiment demonstrates VDAC is phosphorylated by JNK3. Bilayer electrophysiological experiments show that single-channel conductance of VDAC phosphorylated by JNK3 is significantly lower than that of the native VDAC at a membrane potential. The opening probability of VDAC undergoes massive reduction due to phosphorylation by JNK3. These indicate closure of VDAC due to phosphorylation by JNK3. Treatment of phosphorylated VDAC with alkaline phosphatase reversed the VDAC functional activity as shown by single-channel current and opening probability. The physiological consequence of closure of VDAC as a result of phosphorylation has been attributed to JNK3 dependent mitochondria-mediated apoptosis.

Magic angle spinning nuclear magnetic resonance characterization of voltage-dependent anion channel gating in two-dimensional lipid crystalline bilayers.

The N-terminus of the voltage-dependent anion channel (VDAC) has been proposed to contain the mechanistically important gating helices that modulate channel opening and closing. In this study, we utilize magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the location and structure of the N-terminus for functional channels in lipid bilayers by measuring long-range 13C–13C distances between residues in the N-terminus and other domains of VDAC reconstituted into DMPC lipid bilayers. Our structural studies show that the distance between A14 Cβ in the N-terminal helix and S193 Cβ is ∼4–6 Å. Furthermore, VDAC phosphorylation by a mitochondrial kinase at residue S193 has been claimed to delay mitochondrial cell death by causing a conformational change that closes the channel, and a VDAC-Ser193Glu mutant has been reported to show properties very similar to those of phosphorylated VDAC in a cellular context. We expressed VDAC-S193E and reconstituted it into DMPC lipid bilayers. Two-dimensional 13C–13C correlation experiments showed chemical shift perturbations for residues located in the N-terminus, indicating possible structural perturbations to that region. However, electrophysiological data recorded on VDAC-S193E showed that channel characteristics were identical to those of wild type samples, indicating that phosphorylation of S193 does not directly affect channel gating. The combination of NMR and electrophysiological results allows us to discuss the validity of proposed gating models.

S.27.01 Mitochondria and Aβ metabolism

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm, and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. For example, one of the long-standing puzzles was that in permeabilized cells, adenine nucleotide translocase is less accessible to cytosolic ADP than in isolated mitochondria. Still another puzzle was that, according to channel-reconstitution experiments, voltage regulation of VDAC is limited to potentials exceeding 30 mV, which are believed to be much too high for MOM. We have solved these puzzles and uncovered multiple new functional links by identifying a missing player in the regulation of VDAC and, hence, MOM permeability — the cytoskeletal protein tubulin. We have shown that, depending on VDAC phosphorylation state and applied voltage, nanomolar to micromolar concentrations of dimeric tubulin induce functionally important reversible blockage of VDAC reconstituted into planar phospholipid membranes. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective “gating charge” of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology are. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity. The findings, obtained in a channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Taken together, these results suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC interaction with tubulin at the mitochondria–cytosol interface. Immediate physiological implications include new insights into serine/threonine kinase signaling pathways, Ca2 + homeostasis, and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. In the present review, we speculate how these findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Redox-sensitive glycogen synthase kinase 3β-directed control of mitochondrial permeability transition: rheostatic regulation of acute kidney injury

Mitochondrial dysfunction plays a pivotal role in necroapoptotic cell death and in the development of acute kidney injury (AKI). Evidence suggests that glycogen synthase kinase (GSK) 3β resides at the nexus of multiple signaling pathways implicated in the regulation of mitochondrial permeability transition (MPT). In cultured renal tubular epithelial cells, a discrete pool of GSK3β was detected in mitochondria. Coimmunoprecipitation assay confirmed that GSK3β physically interacts with cyclophilin F and voltage-dependent anion channel (VDAC), key MPT regulators that possess multiple GSK3β phosphorylation consensus motifs, suggesting that GSK3β has a direct control of MPT. Upon a strong burst of reactive oxygen species elicited by the pro-oxidant herbicide paraquat, the activity of the redox-sensitive GSK3β was drastically enhanced. This was accompanied by augmented phosphorylation of cyclophilin F and VDAC, associated with MPT and cell death. Inhibition of GSK3β by either the selective inhibitor 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) or forced expression of a kinase-dead mutant obliterated paraquat-induced phosphorylation of cyclophilin F and VDAC, prevented MPT, and improved cellular viability. Conversely, ectopic expression of a constitutively active GSK3β amplified the effect of paraquat on cyclophilin F and VDAC phosphorylation and sensitized cells to paraquat-induced MPT and death. In vivo, paraquat injection elicited marked oxidant stress in the kidney and resulted in acute kidney dysfunction and massive tubular apoptosis and necrosis. Consistent with in vitro findings, the activity of GSK3β was augmented in the kidney after paraquat injury, associated with increased phosphorylation of cyclophilin F and VDAC and sensitized MPT. TDZD-8 blocked GSK3β activity in the kidney, intercepted cyclophilin F and VDAC phosphorylation, prevented MPT, attenuated tubular cell death, and ameliorated paraquat-induced AKI. Our data suggest that the redox-sensitive GSK3β regulates renal tubular injury in AKI by controlling the activity of MPT regulators.

VDAC phosphorylation impairment causes mitochondrial membrane permeability during lipid accumulation

Non‐alcoholic steatosis is a chronic disease characterized by lipid accumulation in the cytoplasm of hepatocytes involving early mitochondrial impairment and late hepatocyte cell death. The voltage‐dependent anion channel (VDAC) is a mitochondrial membrane channel mediating the flux of metabolites, calcium and water across the mitochondrial outer membrane. Upon lethal stress, VDAC can mediate mitochondrial membrane permeabilization (MMP) and cell death. Combination of four experimental models of steatosis (human biopsies, isolated mitochondria from ob/ob obese mice, high fat diet‐fed mice or immortalized cell lines) displayed, in steatotic livers, favored calcium‐induced MMP induction and permeability of VDAC. Thus, lipid accumulation increased the influx of water and calcium into mitochondria and sensitized the organelle to matrix swelling, depolarization and cytochrome c release. Moreover, NADH oxidase activity and channel properties of VDAC are sensitized to calcium regulation in steatosis models. These findings are associated with the hypophosphorylation of VDAC on a threonine residue and the loss of its interaction with the anti‐apoptotic Bcl‐XL and GSK3 kinase. In conclusion, VDAC acts as an early sensor of lipid toxicity and its GSK3‐mediated phosphorylation status controls outer mitochondrial membrane permeabilization in steatosis.

Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation

Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early mitochondrial impairment and late hepatocyte cell death. However, mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that outer mitochondrial membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a membrane channel and NADH oxidase, as a cause of early mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls outer mitochondrial membrane permeabilization in hepatosteatosis.

Purificação e caracterização da VDAC de mitocôndrias corticais aviares: identificação de modificações pós-traducionais nas porinas neuronais murinas e aviares

A VDAC é uma porina presente na MME cuja função é crucial no metabolismo energético, sobrevivência e morte celular. A caracterização da VDAC torna-se importante para a compreensão das inter-relações da mitocôndria com os diferentes componentes citosólicos, tais como a HK. A ligação HK-VDAC favorece a utilização do ATP intramitocondrial em células neuronais, a HK cerebral pode interagir de formas diferentes com a VDAC, o que resulta em diferentes sítios de ligação (sítios A e B). Os variados papéis metabólicos das isoformas da VDAC podem ser explicados pela presença de alterações pós-traducionais. No presente trabalho purificamos a VDAC1 mitocondrial neuronal proveniente de cérebro aviar. Paralelamente, comprovamos que a presença de múltiplas formas das VDACs 1 e 2 em cérebros murino e aviar, seja devida à presença de modificações pós-traducionais, nomeadamente a fosforilação. A proteína isolada apresentou peso molecular de 30KDa. Quando submetida à eletroforese e posteriormente à coloração para a identificação de fosfoproteínas, a mesma mostrou-se desfosforilada. O conhecimento da presença, ou ausência de fosforilação das VDACs, reside na importância de estabelecer-se as bases moleculares ligadas à existência de sítios A e B nas mitocôndrias neuronais.

Attenuation of endoplasmic reticulum stress and mitochondrial injury in kidney with ischemic postconditioning application and trimetazidine treatment

BackgroundEndoplasmic reticulum (ER) and mitochondria have been implicated in the pathology of renal ischemia/reperfusion (I/R). In the present study, we investigated whether the use of ischemic postconditioning (IPostC) and trimetazidine (TMZ) separately or combined could reduce ER stress and mitochondria damage after renal ischemia.MethodsKidneys of Wistar rats were subjected to 60-min of warm ischemia followed by 120-min of reperfusion (I/R group, n = 6), or to 6 cycles of ischemia/reperfusion (10-s each cycle) just after 60-min of warm ischemia (IPostC group, n = 6), or to i.p. injection of TMZ (3 mg/kg) 30-min before ischemia (TMZ group, n = 6), or to the combination of both treatments (IPostC+TMZ group, n = 6). The results of these experimental groups were compared to those of a sham-operated group in which rat renal pedicles were only dissected. Sodium reabsorption rate, creatinine clearance lactate deshydrogenase (LDH) activity in plasma, and concentration of malonedialdehyde (MDA) in tissue were determined. In addition, Western blot analysis was performed to identify the amounts of cytochrome c, c-JunNH2-terminal kinase (JNK), voltage-dependent anion channel (VDAC), glycogen synthase kinase 3-beta (GSK3-β), and ER stress parameters.ResultsIPostC or/and TMZ significantly decreased cytolysis, oxidative stress and improved renal function in comparison to I/R group. IPostC but not TMZ significantly attenuated ER stress parameters versus I/R group. Indeed, it down-regulated the glucose-regulated protein 78 (GRP78), the activating transcription factor 4 (ATF4), the RNA activated protein kinase (PKR)-like ER kinas (PERK), the X box binding protein-1 (XBP-1) and the caspase12 protein levels. TMZ treatment significantly augmented GSK3-β phosphorylation and reduced levels of cytochrome c and VDAC phosphorylation in comparison to IPostC application. The combination of both treatments gave a synergetic effect. It significantly improved the survival rate, attenuated cytolysis, oxidative stress and improved renal function.ConclusionThis study revealed that IPostC protects kidney from I/R injury by suppressing ER stress while the beneficial effects of TMZ are mediated by mitochondria protection. The combination of both treatments ameliorated functional recovery.

VDAC Phosphorylation in Control of Mitochondrial Respiration

Tubulin has been recently identified as a powerful regulator of voltage dependent anion channel (VDAC). Dimeric tubulin induces reversible blockage of VDAC reconstituted into planar lipid membranes and blocks the flux of ATP/ADP across the mitochondrial outer membrane. Here we show that phosphorylation of VDAC by cytosolic kinases protein kinase A (PKA) and glycogen synthase kinase-3β (GSK3β) increases the on-rate of tubulin binding by orders of magnitude, but only for tubulin at the cis (or cytosolic) side of the membrane. This and the fact that the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 are in agreement with our findings that phosphorylation state of VDAC determines its blockage by tubulin. When HepG2 cells were treated with the microtubule-destabilizing agent colchicine that is known to increase free tubulin, mitochondrial potential, as measured by tetramethylrhodamine methyester (TMRM) uptake, decreased. Inhibition of PKA activity with H89 increased mitochondrial potential and, furthermore, reversed mitochondrial depolarization induced by colchicine. To further explore a role VDAC-tubulin interaction in cell proliferation and survival, we studied the effect of erastin, the selective anti-tumour agent involved in the RAS-RAF-MEK signalling pathway, on VDAC blockage by tubulin. We found that erastin reverses tubulin blockage of VDAC isolated from HepG2 cells, promoting the unblocked state of the channel. Our findings suggest a novel functional link between serine/threonine kinase signalling pathways, mitochondrial respiration, and highly dynamic microtubule network which is characteristic of carcinogenesis and cell proliferation.

Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.

Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.

VDAC Phosphorylation Regulates Interaction with Tubulin

Mitochondria and mitochondrial bioenergetics are believed to be involved in glycogen synthase kinase (GSK3β)-related cardioprotection. Recently it was suggested that cardioprotection could be achieved through the preservation of mitochondrial binding of hexokinase II (HXKII), or/and through GSK3β phosphorylation of voltage dependent anion channel (VDAC) (Pastorino et al., Cancer Res., 2005; Das et al., Circ. Res., 2008). VDAC, the most abundant channel in the mitochondria outer membrane (MOM), is known to be responsible for most of the metabolite and ATP/ADP fluxes across MOM. Recently we have found that dimeric αβ-tubulin regulates mitochondrial respiration by directly blocking VDAC and hence, permeability of MOM for ATP/ADP (Rostovtseva et al., PNAS, 2008). Here, using mammalian VDAC reconstituted into planar lipid membrane, we show that tubulin-VDAC interaction appears to be very sensitive to the state of VDAC phosphorylation. When VDAC is phosphorylated in vitro by either GSK3β or protein kinase A (PKA), the on-rate of tubulin binding increases up to 100 times compared with untreated VDAC. Importantly, the basic properties of VDAC, such as single-channel conductance, selectivity, and voltage gating, remain almost unaltered after phosphorylation. Nonspecific alkaline phosphatase and tyrosine kinase inhibitor PP2A dephosphorylate VDAC, which results in decreased tubulin binding. Gel analysis and subsequent phospho-staining confirm that VDAC contains motifs recognized by both GSK3β and PKA. Phosphorylation causes a pronounced asymmetry of tubulin binding to VDAC. These findings allow us to point to the tentative GSK3β and PKA serine/threonine phosphorylation sites positioned on the cytosolic loops of VDAC. The results show that VDAC phosphorylation enhances tubulin-induced VDAC closure and thus could reduce MOM permeability and mitochondria respiration. We suggest that GSK3β cardioprotective effect is more complex that was initially thought because along with HXKII it involves tubulin as a potent regulator of VDAC and hence, cellular respiration.

Governing Respiration: Tubulin's C-Terminus Interaction with VDAC

Mitochondria have long been known to localize within and move along the tubulin-microtubule network. It was shown that tubulin binds to isolated mitochondria with high-affinity and specifically associates with the mitochondrial voltage-dependent anion channel (VDAC). We found that nanomolar concentrations of dimeric tubulin vastly increase VDAC sensitivity to voltage allowing for VDAC blockage at low transmembrane potentials. Tubulin interaction with VDAC requires the presence of anionic C-terminal tails (CTT) on the intact protein. Tubulin with proteolytically cleaved CTTs does not block the channel. Actin, also an acidic protein but lacking CTTs, does not induce VDAC blockage. Two synthetic peptides with the sequences of mammalian α and β brain tubulin CTT do not induce detectable channel closure up to micromolar concentrations. These results suggest a completely new role for dimeric tubulin and its charged CTT. We propose a model for tubulin-VDAC channel interaction in which the tubulin CTT penetrates into the channel lumen, potentially reaching through the channel and interacting with a positively charged domain of VDAC. We found that tubulin/VDAC interaction is greatly dependent on the state of VDAC phosphorylation. Remarkably, phosphorylated VDAC is more than an order of magnitude more sensitive to tubulin-induced closure than dephosphorylated VDAC.Tubulin addition to isolated mitochondria increases Km for ADP and lowers oxygen consumption, most likely by restricting flux of ADP through VDAC. Thus interaction of tubulin CTT with VDAC blocks nucleotide passage into and out of mitochondria, thereby regulating oxidative phosphorylation. Examination of the evolution of CTT sequences in mitochondria-containing cells reveals high conservation of charge and length of the tails. Considering the known conservation of VDAC folding pattern throughout mitochondria-containing eukaryotes, we propose that this interaction is widespread and ancient.

Anti-hypertrophic effect of NHE-1 inhibition involves GSK-3β-dependent attenuation of mitochondrial dysfunction

Although Na +–H + exchanger 1 (NHE-1) inhibition has been demonstrated to have anti-hypertrophic effect indirectly through mitochondria, the detailed cellular mechanisms mediating this effect remain elusive. In this study we sought to determine whether NHE-1 inhibition exerts an anti-hypertrophic effect by modulating the mitochondrial permeability transition pore (mPTP) opening through the AMP-activated protein kinase (AMPK)/glycogen synthase kinase 3β (GSK-3β) pathway during hypertrophy in cardiomyocytes. An in vivo model of hypertrophy was induced in male Sprague–Dawley rats by subjecting them to 3, 7 or 28 days of coronary artery ligation (CAL). To induce hypertrophy in vitro, cardiomyocytes isolated from hearts of neonatal (1–3 days) Sprague–Dawley rats were exposed to endothelin-1 (ET-1, 10 nM) in the presence or absence of various treatments. The results demonstrate that CAL affected both AMPKα and GSK-3β phosphorylation in a time-dependent manner. In cultured cardiomyocytes, ET-1 increased phosphorylation of AMPKα 1/α 2 Ser485/Ser491 and GSK-3β Ser9 by 80% ( P < 0.05) and 225% ( P < 0.05) respectively, both of which were significantly blunted by the NHE-1 inhibitor AVE-4890 (5 μM). ET-1-induced phosphorylation of GSK-3β Ser9 was attenuated by inhibitors of phosphatidylinositol 3-kinase (LY294002), Akt (Akt inhibitor VIII), ERK1/2 (PD98059) and by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β- d-ribofuranoside (AICAR). Prevention of GSK-3β Ser9 phosphorylation was also accompanied by suppression of ET-1-induced increases in cell surface area, ANP and α-skeletal actin gene expression. Co-immunoprecipitation studies revealed that GSK-3β interacts with components of the mPTP, voltage-dependent anion channel (VDAC) and adenine nucleotide translocase. Furthermore, ET-1 reduced phosphorylation of VDAC, which was associated with both mPTP opening and mitochondrial membrane depolarization. These effects were mimicked by the GSK-3β inhibitor SB216763, thus showing that modulation of mPTP formation is GSK-3β-dependent. In conclusion, anti-hypertrophic effect of NHE-1 inhibition can be mediated through activation of GSK-3β which in turn induces inhibition of mPTP opening due to VDAC phosphorylation.

Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II

Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H(2)O(2). However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase Cepsilon (PKCepsilon)-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCepsilon.

Mitochondrial VDAC Can Be Phosphorylated by Cyclic AMP-Dependent Protein Kinase

The voltage dependent anion channel (VDAC) of the outer membrane of mitochondria is thought to play a role in transport of metabolites including ATP across mitochondrial membrane and modulate mitochondrial functions such as respiration. However, regulation of this anion channel is only poorly understood. In this paper we demonstrate that VDAC purified from rat liver mitochondria can be phosphorylated by the catalytic subunit of cAMP dependent protein kinase (PKA). PKA phosphorylates VDAC linearly up to fifteenfold in sixty minutes. The level of VDAC phosphorylation increases to twofold and sevenfold of control value after ten and thirty minutes of reaction, respectively. Data presented here suggest the possibilty that voltage dependent anion channel membrane of mitochondria may be a target of PKA in vivo.