Pore-Forming VDAC Proteins of the Outer Mitochondrial Membrane: Regulation and Pathophysiological Role.

Voltage-dependent anion channels (VDAC1-3) of the outer mitochondrial membrane are a family of pore-forming ß-barrel proteins that carry out controlled "filtration" of small molecules and ions between the cytoplasm and mitochondria. Due to the conformational transitions between the closed and open states and interaction with cytoplasmic and mitochondrial proteins, VDACs not only regulate the mitochondrial membrane permeability for major metabolites and ions, but also participate in the control of essential intracellular processes and pathological conditions. This review discusses novel data on the molecular structure, regulatory mechanisms, and pathophysiological role of VDAC proteins, as well as future directions in this area of research.

VDAC in Retinal Health and Disease.

The retina, a tissue of the central nervous system, is vital for vision as its photoreceptors capture light and transform it into electrical signals, which are further processed before they are sent to the brain to be interpreted as images. The retina is unique in that it is continuously exposed to light and has the highest metabolic rate and demand for energy amongst all the tissues in the body. Consequently, the retina is very susceptible to oxidative stress. VDAC, a pore in the outer membrane of mitochondria, shuttles metabolites between mitochondria and the cytosol and normally protects cells from oxidative damage, but when a cell's integrity is greatly compromised it initiates cell death. There are three isoforms of VDAC, and existing evidence indicates that all three are expressed in the retina. However, their precise localization and function in each cell type is unknown. It appears that most retinal cells express substantial amounts of VDAC2 and VDAC3, presumably to protect them from oxidative stress. Photoreceptors express VDAC2, HK2, and PKM2-key proteins in the Warburg pathway that also protect these cells. Consistent with its role in initiating cell death, VDAC is overexpressed in the retinal degenerative diseases retinitis pigmentosa, age related macular degeneration (AMD), and glaucoma. Treatment with antioxidants or inhibiting VDAC oligomerization reduced its expression and improved cell survival. Thus, VDAC may be a promising therapeutic candidate for the treatment of these diseases.

LRRC8/VRAC volume-regulated anion channels are crucial for hearing.

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC­K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

Glutathionylation of a glycolytic enzyme promotes cell death and vigor loss during aging of elm seeds.

Seed deterioration during storage is a major problem in agricultural and forestry production and for germplasm conservation. Our previous studies have shown that a mitochondrial outer membrane protein VOLTAGE-DEPENDENT ANION CHANNEL (VDAC) is involved in programmed cell death-like viability loss during the controlled deterioration treatment (CDT) of elm (Ulmus pumila L.) seeds, but its underlying mechanism remains unclear. In this study, we demonstrate that the oxidative modification of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) is functioned in the gate regulation of VDAC during the CDT of elm seeds. Through biochemical and cytological methods and observations of transgenic material [Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and yeast (Saccharomyces cerevisiae)], we demonstrate that cysteine S-glutathionylated UpGAPDH1 interacts with UpVDAC3 during seed aging, which leads to a mitochondrial permeability transition and aggravation of cell death, as indicated by the leakage of the mitochondrial proapoptotic factor cytochrome c and the emergence of apoptotic nucleus. Physiological assays and inductively coupled plasma mass spectrometry analysis revealed that GAPDH glutathionylation is mediated by increased glutathione, which might be caused by increases in the concentrations of free metals, especially Zn. Introduction of the Zn-specific chelator TPEN [(N,N,N',N'-Tetrakis (2-pyridylmethyl)ethylenediamine)] significantly delayed seed aging. We conclude that glutathionylated UpGAPDH1 interacts with UpVDAC3 and serves as a proapoptotic protein for VDAC-gating regulation and cell death initiation during seed aging.

Targeting VDAC: A potential therapeutic approach for mitochondrial dysfunction in Alzheimer's disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of Alzheimer's disease, a neurodegenerative disorder characterized by progressive cognitive decline. Voltage-dependent anion channel (VDAC), a protein located in the outer mitochondrial membrane, plays a critical role in regulating mitochondrial function and cellular energy metabolism. Recent studies have identified VDAC as a potential therapeutic target for Alzheimer's disease. This article aims to provide an overview of the role of VDAC in mitochondrial dysfunction, its association with Alzheimer's disease, and the potential of targeting VDAC for developing novel therapeutic interventions. Understanding the involvement of VDAC in Alzheimer's disease may pave the way for the development of effective treatments that can restore mitochondrial function and halt disease progression.

Effect of VBIT-4 on the functional activity of isolated mitochondria and cell viability.

VBIT-4 is a new inhibitor of the oligomerization of VDAC proteins of the outer mitochondrial membrane preventing the development of oxidative stress, mitochondrial dysfunction, and cell death in various pathologies. However, as a VDAC inhibitor, VBIT-4 may itself cause mitochondrial dysfunction in healthy cells. The article examines the effect of VBIT-4 on the functional activity of rat liver mitochondria and cell cultures. We have demonstrated that high concentrations of VBIT-4 (15-30 µM) suppressed mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. VBIT-4 induced depolarization of organelles fueled by substrates of complex I but not complex II of the respiratory chain. VBIT-4 has been found to inhibit the activity of complexes I, III, and IV of the respiratory chain. Molecular docking demonstrated that VBIT-4 interacts with the rotenone-binding site in complex I with similar affinity. 15-30 µM VBIT-4 caused an increase in H2O2 production in mitochondria, decreased the Ca2+ retention capacity, but increased the time of Ca2+-dependent mitochondrial swelling. We have found that the incubation of breast adenocarcinoma (MCF-7) with 30 µM VBIT-4 for 48 h led to the decrease of the mitochondrial membrane potential, an increase in ROS production and death of MCF-7 cells. The mechanism of action of VBIT-4 on mitochondria and cells is discussed.

Structural insights into human EMC and its interaction with VDAC.

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved, multi-subunit complex acting as an insertase at the ER membrane. Growing evidence shows that the EMC is also involved in stabilizing and trafficking membrane proteins. However, the structural basis and regulation of its multifunctionality remain elusive. Here, we report cryo-electron microscopy structures of human EMC in apo- and voltage-dependent anion channel (VDAC)-bound states at resolutions of 3.47 Å and 3.32 Å, respectively. We discovered a specific interaction between VDAC proteins and the EMC at mitochondria-ER contact sites, which is conserved from yeast to humans. Moreover, we identified a gating plug located inside the EMC hydrophilic vestibule, the substrate-binding pocket for client insertion. Conformation changes of this gating plug during the apo-to-VDAC-bound transition reveal that the EMC unlikely acts as an insertase in the VDAC1-bound state. Based on the data analysis, the gating plug may regulate EMC functions by modifying the hydrophilic vestibule in different states. Our discovery offers valuable insights into the structural basis of EMC's multifunctionality.

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development.

The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.

Bombyx mori voltage-dependent anion-selective channel induces programmed cell death to defend against Bombyx mori nucleopolyhedrovirus infection.

BACKGROUND: Voltage-dependent anion-selective channels (VDACs) serve as pore proteins within the mitochondrial membrane, aiding in the regulation of cell life and cell death. Although the occurrence of cell death is crucial for defense against virus infection, the function played by VDAC in Bombyx mori, in response to the influence of Bombyx mori nucleopolyhedrovirus (BmNPV), remains unclear. RESULTS: BmVDAC was found to be relatively highly expressed both during embryonic development, and in the Malpighian tubule and midgut. Additionally, the expression levels of BmVDAC were found to be different among silkworm strains with varying levels of resistance to BmNPV, strongly suggesting a connection between BmVDAC and virus infection. To gain further insight into the function of BmVDAC in BmNPV, we employed RNA interference (RNAi) to silence and overexpress it by pIZT/V5-His-mCherry. The results revealed that BmVDAC is instrumental in developing the resistance of host cells to BmNPV infection in BmN cell-line cells, which was further validated as likely to be associated with initiating programmed cell death (PCD). Furthermore, we evaluated the function of BmVDAC in another insect, Spodoptera exigua. Knockdown of the BmVDAC homolog in S. exigua, SeVDAC, made the larvae more sensitive to BmNPV. CONCLUSION: We have substantiated the pivotal role of BmVDAC in conferring resistance against BmNPV infection, primarily associated with the initiation of PCD. The findings of this study shine new light on the molecular mechanisms governing the silkworm's response to BmNPV infection, thereby supporting innovative approaches for pest biocontrol. © 2024 Society of Chemical Industry.

The small yeast GTPase Rho5 requires specific mitochondrial outer membrane proteins for translocation under oxidative stress and interacts with the VDAC Por1.

Yeast Rho5 is a small GTPase which mediates the response to nutrient and oxidative stress, and triggers mitophagy and apoptosis. We here studied the rapid translocation of a GFP-tagged Rho5 to mitochondria under such stress conditions by live-cell fluorescence microscopy in the background of strains lacking different mitochondrial outer membrane proteins (MOMP). Fun14, Msp1 and Alo1 were found to be required for efficient recruitment of the GTPase, whereas translocation of Dck1 and Lmo1, the subunits of its dimeric GDP/GTP exchange factor (GEF), remained unaffected. An influence of the voltage-dependent anion channel (VDAC) Por1 on the association of GFP-Rho5 with mitochondria under oxidative stress conditions appeared to be strain-dependent. However, epistasis analyses and bimolecular fluorescence complementation (BiFC) studies indicate a genetic and physical interaction. All four strains lacking a single MOMP were investigated for their effect on mitophagy.

Solute Transport through Mitochondrial Porins In Vitro and In Vivo.

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article.

Neuronal Protection by Ha-RAS-GTPase Signaling through Selective Downregulation of Plasmalemmal Voltage-Dependent Anion Channel-1.

The small GTPase RAS acts as a plasma membrane-anchored intracellular neurotrophin counteracting neuronal degeneration in the brain, but the underlying molecular mechanisms are largely unknown. In transgenic mice expressing constitutively activated V12-Ha-RAS selectively in neurons, proteome analysis uncovered a 70% decrease in voltage-dependent anion channel-1 (VDAC-1) in the cortex and hippocampus. We observed a corresponding reduction in the levels of mRNA splicing variant coding for plasma membrane-targeted VDAC-1 (pl-VDAC-1) while mRNA levels for mitochondrial membrane VDAC-1 (mt-VDAC-1) remained constant. In primary cortical neurons derived from V12-Ha-RAS animals, a decrease in pl-VDAC-1 mRNA levels was observed, accompanied by a concomitant reduction in the ferricyanide reductase activity associated with VDAC-1 protein. Application of MEK inhibitor U0126 to transgenic cortical neurons reconstituted pl-VDAC-1 mRNA to reach wild-type levels. Excitotoxic glutamate-induced cell death was strongly attenuated in transgenic V12-Ha-RAS overexpressing cortical cultures. Consistently, a neuroprotective effect could also be achieved in wild-type cortical cultures by the extracellular application of channel-blocking antibody targeting the N-terminus of VDAC-1. These results may encourage novel therapeutic approaches toward blocking pl-VDAC-1 by monoclonal antibody targeting for complementary treatments in transplantation and neurodegenerative disease.

Prediction and biological analysis of yeast VDAC1 phosphorylation.

The mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), is the major permeability pathway for the flux of metabolites and ions between cytosol and mitochondria. In yeast, several Por1 phosphorylation sites have been identified. Protein phosphorylation is a major modification regulating a variety of biological activities, but the potential biological roles of Por1 phosphorylation remains unaddressed. In this work, we analysed 10 experimentally observed phosphorylation sites in yeast Por1 using bioinformatics tools. Two of the residues, T100 and S133, predicted to reduce and increase pore permeability, respectively, were validated using biological assays. In accordance, Por1T100D reduced mitochondrial respiration, while Por1S133E phosphomimetic mutant increased it. Por1T100A expression also improved respiratory growth, while Por1S133A caused defects in all growth conditions tested, notably in fermenting media. In conclusion, we found phosphorylation has the potential to modulate Por1, causing a marked effect on mitochondrial function. It can also impact on cell morphology and growth both in respiratory and, unpredictably, also in fermenting conditions, expanding our knowledge on the role of Por1 in cell physiology.

Mitochondrial VOLTAGE-DEPENDENT ANION CHANNEL 3 regulates stomatal closure by abscisic acid signaling.

In Arabidopsis (Arabidopsis thaliana), stomatal closure mediated by abscisic acid (ABA) is redundantly controlled by ABA receptor family proteins (PYRABACTIN RESISTANCE 1 [PYR1]/PYR1-LIKE [PYLs]) and subclass III SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2 (SnRK2s). Among these proteins, the roles of PYR1, PYL2, and SnRK2.6 are more dominant. A recent discovery showed that ABA-induced accumulation of reactive oxygen species (ROS) in mitochondria promotes stomatal closure. By analyzing stomatal movements in an array of single and higher order mutants, we revealed that the mitochondrial protein VOLTAGE-DEPENDENT ANION CHANNEL 3 (VDAC3) jointly regulates ABA-mediated stomatal closure with a specialized set of PYLs and SnRK2s by affecting cellular and mitochondrial ROS accumulation. VDAC3 interacted with 9 PYLs and all 3 subclass III SnRK2s. Single mutation in VDAC3, PYLs (except PYR1 and PYL2), or SnRK2.2/2.3 had little effect on ABA-mediated stomatal closure. However, knocking out PYR1, PYL1/2/4/8, or SnRK2.2/2.3 in vdac3 mutants resulted in significantly delayed or attenuated ABA-mediated stomatal closure, despite the presence of other PYLs or SnRK2s conferring redundant functions. We found that cellular and mitochondrial accumulation of ROS induced by ABA was altered in vdac3pyl1 mutants. Moreover, H2O2 treatment restored ABA-induced stomatal closure in mutants with decreased stomatal sensitivity to ABA. Our work reveals that VDAC3 ensures redundant control of ABA-mediated stomatal closure by canonical ABA signaling components.

Voltage-dependent anion channel 3 (VDAC3) mediates P. syringae induced ABA-SA signaling crosstalk in Arabidopsis thaliana.

Pathogen severely affects plant mitochondrial processes including respiration, however, the roles and mechanism of mitochondrial protein during the immune response remain largely unexplored. The interplay of plant hormone signaling during defense is an outcome of plant pathogen interaction. We recently discovered that the Arabidopsis calcineurin B-like interacting protein kinase 9 (AtCIPK9) interacts with the voltage-dependent anion channel 3 (AtVDAC3) and inhibits MV-induced oxidative damage. Here we report the characterization of AtVDAC3 in an antagonistic interaction pathway between abscisic acid (ABA) and salicylic acid (SA) signaling in Pseudomonas syringae -Arabidopsis interaction. In this study, we observed that mutants of AtVDAC3 were highly susceptible to Pseudomonas syringae infection as compared to the wild type (WT) Arabidopsis plants. Transcripts of VDAC3 and CIPK9 were inducible upon ABA application. Following pathogen exposure, expression analyses of ABA and SA biosynthesis genes indicated that the function of VDAC3 is required for isochorisimate synthase 1 (ICS1) expression but not for Nine-cis-epoxycaotenoid dioxygenase 3 (NCED3) expression. Despite the fact that vdac3 mutants had increased NCED3 expression in response to pathogen challenge, transcripts of ABA sensitive genes such as AtRD22 and AtRAB18 were downregulated even after exogenous ABA application. VDAC3 is required for ABA responsive genes expression upon exogenous ABA application. We also found that Pseudomonas syringae-induced SA signaling is downregulated in vdac3 mutants since overexpression of VDAC3 resulted in hyperaccumulation of Pathogenesis related gene1 (PR1) transcript. Interestingly, ABA application prior to P. syringae inoculation resulted in the upregulation of ABA responsive genes like Responsive to ABA18 (RAB18) and Responsive to dehydration 22 (RD22). Intriguingly, in the absence of AtVDAC3, Pst challenge can dramatically increase ABA-induced RD22 and RAB18 expression. Altogether our results reveal a novel Pathogen-SA-ABA interaction pathway in plants. Our findings show that ABA plays a significant role in modifying plant-pathogen interactions, owing to cross-talk with the biotic stress signaling pathways of ABA and SA.

Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase.

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

A novel CPT1A covalent inhibitor modulates fatty acid oxidation and CPT1A-VDAC1 axis with therapeutic potential for colorectal cancer.

Colorectal cancer (CRC) is a common and deadly disease of the digestive system, but its targeted therapy is hampered by the lack of reliable and specific biomarkers. Hence, discovering new therapeutic targets and agents for CRC is an urgent and challenging task. Here we report that carnitine palmitoyltransferase 1A (CPT1A), a mitochondrial enzyme that catalyzes fatty acid oxidation (FAO), is a potential target for CRC treatment. We show that CPT1A is overexpressed in CRC cells and that its inhibition by a secolignan-type compound, 2,6-dihydroxypeperomin B (DHP-B), isolated from the plant Peperomia dindygulensis, suppresses tumor cell growth and induces apoptosis. We demonstrate that DHP-B covalently binds to Cys96 of CPT1A, blocks FAO, and disrupts the mitochondrial CPT1A-VDAC1 interaction, leading to increased mitochondrial permeability and reduced oxygen consumption and energy metabolism in CRC cells. We also reveal that CPT1A expression correlates with the survival of tumor-bearing animals and that DHP-B exhibits anti-CRC activity in vitro and in vivo. Our study uncovers the molecular mechanism of DHP-B as a novel CPT1A inhibitor and provides a rationale for its preclinical development as well as a new strategy for CRC targeted therapy.

Seasonal expressions of the translocator protein (18 kDa), voltage-dependent anion channel, and steroidogenic acute regulatory protein in the scent glands of muskrats (Ondatra zibethicus).

Steroidogenesis machinery involves the steroidogenic acute regulatory protein (StAR), which regulates cholesterol transfer within the mitochondria, and the transport of cholesterol via a channel composed of 18-kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC) plus some accessory proteins. In this study, we investigated the immunolocalizations and expressions of StAR, TSPO, VDAC and cytochrome P450 side chain cleavage enzyme (P450scc, CYP11A1) in the scent glands of muskrats (Ondatra zibethicus) during the breeding and non-breeding periods. StAR, TSPO, VDAC and CYP11A1 were immunolocalized in the scent glandular, interstitial and epithelial cells in both breeding and non-breeding seasons with stronger immunostaining in the breeding season. The mRNA expression levels of StAR, TSPO, VDAC and CYP11A1 were higher in the scent glands of the breeding season than those of the non-breeding season. The circulating follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) as well as scent glandular T and dihydrotestosterone (DHT) concentrations were also significantly higher in the breeding season. Additionally, the transcriptomic study in the scent glands identified that differentially expressed genes might be related to the lipid metabolic process, integral component of membrane, and steroid hormone receptor activity and hormone activity using GO analysis. Further in vitro study verified that StAR, TSPO, VDAC and CYP11A1 expression levels increased significantly after human chorionic gonadotropin, hCG/FSH treatment compared with the control group. The KEGG pathway enriched by differentially expressed genes detected to be involved in endocrine system or amino acid metabolism. These findings suggested that the scent glands of the muskrats have ability to synthesize steroids de novo, and that the steroid hormones may have an important regulatory role in the scent glandular function via an autocrine/paracrine pathway.

Longitudinal diffusion barriers imposed by myofilaments and mitochondria in murine cardiac myocytes.

Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases from 30 to 2,000, roughly as expected for pores with porin channel dimensions. Bodipy-FL ATP diffuses >40-fold slower than in free water at 25°C. From several fluorophores analyzed, bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. Unbound fluorophores diffuse at 0.5-8 × 10-7 cm2/s (5-80 µm2/s). Analysis of Na/K pump and veratridine-modified Na channel currents suggests that Na diffusion is nearly unrestricted at 35°C (time constant for equilibration with the pipette tip, ∼20 s). Using multiple strategies, we estimate that at 35°C, ATP diffuses four to eight times slower than in free water. To address whether restrictions are caused more by protein or membrane networks, we verified first that a protein gel, 10 g% gelatin, restricts diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is less restricted than in intact myocytes. Notably, myofilaments extracted similarly from skeletal (diaphragm) myocytes are less restrictive. Solute diffusion in myocytes with sarcolemma permeabilized by ß-escin (80 µM) is similar to diffusion in intact myocytes. Restrictions are strain-dependent, being twofold greater in BL6 myocytes than in CD1/J6/129svJ myocytes. Furthermore, longitudinal diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, VDAC1, than in WT CD1/J6/129svJ myocytes. Thus, mitochondria networks restrict long-range diffusion while presumably optimizing nucleotide transfer between myofilaments and mitochondria. We project that diffusion restrictions imposed by both myofilaments and the outer mitochondrial membrane are important determinants of total free cytoplasmic AMP and ADP (∼10 µM). However, the capacity of diffusion to deliver ATP to myofilaments remains ∼100-fold greater than ATP consumption.

Human VDAC pseudogenes: an emerging role for VDAC1P8 pseudogene in acute myeloid leukemia.

BACKGROUND: Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. RESULTS: We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). CONCLUSIONS: Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.