Loss of voltage dependent anion channel 1 affects mitochondrial bioenergetics in rat dopamine cells
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ABSTRACT:
PROVIDER: PRJNA486658 | ENA |
REPOSITORIES: ENA
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Project description:Voltage dependent anion channel 1 (VDAC1) is a multi-functional protein that regulates mitochondrial membrane potential, calcium regulation, and apoptosis. VDAC1 also interacts with a number of signaling pathways important for energy homeostasis and proteins involved in neurodegenerative diseases such as alpha-synuclein. To identify novel signaling pathways dependent upon VDAC1 protein in dopamine cells, we used CRISPR-Cas9 gene editing in rat immortalized dopaminergic N27 cells. Western blot confirmed that VDAC1 protein levels were reduced ~90%. Mitochondrial bioenergetics was assessed to determine if there was functional loss of mitochondrial function without VDAC1. Loss of VDAC1 resulted in lower ATP-linked and maximum respiration, and spare respiratory capacity. Transcriptomics was conducted in these cells to identify the pathways perturbed by loss of VDAC. This study sheds novel insight into the different regulatory roles mediated by VDAC1 in dopamine cells.
2019-07-31 | GSE118737 | GEO
Project description:Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this novel role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.
2021-03-09 | GSE168487 | GEO
Project description:Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis
| PRJNA707551 | ENA
Project description:In plant cells, the ALMTs are key plasma and vacuolar membranes anion channels regulating plant responses to the environment. Vacuolar ALMTs control stomata aperture and anion accumulation in guard cells. The activation of vacuolar ALMTs is voltage and malate dependent, but the underlying mechanisms remain elusive. Here we report the cryo-EM structure of ALMT9 from Arabidopsis thaliana (AtALMT9), a malate-activated vacuolar anion channel in various lipid-bound states. In all the states, lipids interact with the ion conduction pore of AtALMT9. We found lipid-bound states in plugged and unplugged conformations. Two unplugged states represent with distinct pore width profiles. Structural and functional analysis identified conserved residues involved in ion conduction and pore lipid plugging. Molecular dynamic simulations revealed a peculiar anion conduction mechanism in AtALMT9. We propose a voltage dependent activation mechanism based on the competition between pore-lipids and malate at the cytosolic entrance of the channel
2025-03-04 | PXD059651 | Pride
Project description:The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to "gate" between an open and several "closed" states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.
| S-EPMC4156087 | biostudies-literature
Project description:Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, VDAC3) are relevant component of the outer mitochondrial membrane and play crucial role in cellular processes, in regulation of metabolism and in survival pathways. Recently, we reported the characterization of the oxidation pattern of cysteines in rat and human VDACs and highlighted the key role of these residues in protein function. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, has so far remained undetermined. Herein we have investigated by nanoUHPLC/High Resolution nanoESI-MS/MS. the position of intramolecular disulfide bonds in the rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to avoid disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14 and the alternative disulfide bridges between cysteines 48, 77 and 104. A disulfide bond, very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features.
2024-06-16 | PXD044041 | Pride
Project description:Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin’s voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl- anion at a conserved binding site formed by amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl- binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion-binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin’s mechanical expansion. We observe helix fraying at prestin’s anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin’s fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and helps define prestin’s unique voltage-sensing mechanism and electromotility.
2023-12-06 | PXD046965 | Pride
Project description:The voltage-dependent anion-selective channels (VDACs), also known as eukaryotic porins, are pore-forming proteins which allow the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions regulating organelle and cellular metabolism. We have recently reported about the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing for the first time the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed us to extend the investigation to the human VDACs. Over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as certained also in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell.
2020-03-09 | PXD017482 | Pride
Project description:Voltage-Dependent Anion Channels, the most abundant proteins of the mitochondrial outer membrane, are responsible for exchange of ions and metabolites between cytosol and mitochondria. They participate in the control of glycolytic metabolism through interaction with numerous enzymes and play a key role in regulation of mitochondria-mediated apoptosis, cancer and neurodegenerative diseases. The structural characterization of VDACs presents difficulties because there is no established protocol for the isolation of a single VDAC isoform and/or separation from other membrane proteins, so they can only be studied as component of a mixture. Recently, we have showed that High-Resolution Mass Spectrometry coupled with UHPLC, represents a powerful tool for their structural characterization. Using this technique we discovered that cysteines in rat and human VDACs show a preferred oxidation state, ranging from reduced to trioxidized form, that is remarkably conserved between rat and human VDACs. More recently, characterization of intramolecular disulfide bonds in rVDAC2 was obtained. The successful characterization of disulfide bonds in rVDAC2, prompted us to use the same procedure for the investigation of intramolecular disulfide bonds in rVDAC3 and also to attempt a possible characterization of intermolecular disulfide bonds formed by this protein with other VDAC isoforms. As a result, three intramolecular and seven intermolecular disulfide bonds between rVDAC3 with rVDAC1 and rVDAC2 were uniquely characterized. Furthermore, evidence was obtained for the existence of two additional intramolecular disulfide bonds between Cys2/Cys8 with Cys36 and Cys122, although these identification were not supported by MS/MS spectra.
2025-09-08 | PXD064110 | Pride
Project description:Regulation of fin regeneration by a voltage-gated sodium channel
| PRJNA775662 | ENA