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Results for “Voltage-Dependent Anion Channels”

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At least 19 recordsLinked to original sources

PubMed1992

Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier.

The mitochondrial benzodiazepine receptor (mBzR) has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized. mBzR comprises immunologically distinct protein subunits of 18-, 30-, and 32-kDa. The 18-kDa protein is labeled by the isoquinoline carboxamide mBzR ligand [3H]PK14105, whereas the 30- and 32-kDa subunits are labeled by the benzodiazepine (Bz) ligands [3H]flunitrazepam and [3H]AHN-086. Selective antibodies and reagents identify the 32- and 30-kDa proteins as the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ADC), respectively. While isoquinoline carboxamide and Bz ligands target different subunits, they interact allosterically, as the binding of Bz and isoquinoline carboxamide ligands is mutually competitive at low nanomolar concentrations. Moreover, eosin-5-maleimide and mercuric chloride inhibit [3H]PK11195 binding to the intact receptor via sulfhydryl groups that are present in ADC. VDAC and ADC, outer and inner mitochondrial membrane channel proteins, respectively, together with the 18-kDa subunit, may comprise mBzR at functionally important transport sites at the junction of two mitochondrial membranes.

Affinity Labels↗

PubMed1976

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria.

We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained from Paramecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0 M salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl- than to K+ and is impermeable to Ca++. The procedure for obtaining VDAC; AND THE PROPERTIES OF THE CHANNEL ARE HIGHLY REPRODUCIBLE. VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl- permeability suggest that there may indeed be a voltage-dependent Cl- permeability in mitochondria. The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.

Animals↗

PubMed1992

Ionic permeability of the mitochondrial outer membrane.

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM], derived from mouse liver, showed the presence of currents corresponding to low conductances (less than 50 pS), as well as of four distinct conductances of 99 pS, 152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).

Animals↗

PubMed2026

Defective RNA Polymerase III sensing of mitochondrial DNA in pulmonary epithelial cells impairs type I IFN immunity to SARS-CoV-2.

The clinical spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection ranges from asymptomatic cases to critical COVID-19 pneumonia. To investigate the role of host genetics in susceptibility to critical COVID-19 and identify pathophysiological mechanisms and pathways, we analyzed whole-exome and whole-genome sequencing data from the COVID Human Genetic Effort. We identified 10 rare, monoallelic predicted loss-of-function variants in 18 patients in POLR3A and POLR3C encoding two subunits of RNA polymerase III (POL III), a nuclear multisubunit enzyme, which has been implicated in cytosolic DNA sensing. These variants were deleterious for expression of full-length POLR3A and POLR3C proteins. We demonstrate that human pulmonary A549-hACE2 cells with reduced POLR3A or POLR3C expression exhibit impaired type I IFN responses to transfected mitochondrial DNA (mtDNA) or SARS-CoV-2 infection, together with increased viral replication. Mechanistically, we show that SARS-CoV-2 induces cellular mtDNA release via oligomerization of the mitochondrial voltage-dependent anion channel under virus-induced oxidative stress, enabling POL III-mtDNA interaction. These findings establish POL III as a sensor of endogenous mtDNA released during viral infection and indicate that autosomal dominant POL III haploinsufficiency may predispose individuals to critical COVID-19.

Humans↗

PubMed1992

Studies on human porin. VIII. Expression of "Porin 31HL" channels in the plasmalemma of the acute-lymphoblastic-leukemia cell line KM3 as revealed by light- and electron-microscopy.

First electron microscopy data on the expression of the VDAC "Porin 31HL" in the plasmalemma of a eucaryotic cell are presented. In a light and electron microscopic study we demonstrate the expression of the porin channel in the outer cell membrane of the pre-B lymphocyte type acute-lymphoblastic-leukemia cell line KM3. Monoclonal mouse anti-"Porin 31HL" antibodies were applied in indirect immunofluorescence or immunogold labelling experiments. The results confirm our early topological data on the expression of porin channels in the cytoplasmic membrane of different human cell types as revealed on the light microscopy level. Interestingly, only a pre-embedding immunoreaction approach was successful for gold particle labelling of the plasmalemma. This again is in agreement with our recent data on the accessibility of the acetylated N-terminal part of "Porin 31HL" molecules on the outer cell surface.

Antibodies, Monoclonal↗

PubMed2026

Integrative Proteomics and Ubiquitomics Reveal on-Targets and off-Targets of PROTAC dBET1.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that induce selective degradation of target proteins by hijacking the ubiquitin-proteasome system (UPS). Despite their transformative potential in eliminating disease-associated proteins, comprehensively identifying off-target degradation events remains technically challenging. Here, we employed an integrated proteomic and ubiquitinomic strategy to systematically profile the degradation landscape of the PROTAC molecule dBET1 in Jurkat T cells. By capturing the upstream ubiquitination events─which serve as earlier and more sensitive indicators than total protein abundance─our approach enabled the identification of previously overlooked off-target candidates. While dBET1 efficiently degraded its canonical BET family targets, our data also revealed the mitochondrial outer membrane protein VDAC1 as a putative off-target, evidenced by its depletion and increased multisite ubiquitination. Notably, our analysis framework enabled site-specific resolution of degradation events within BRD3, revealing preferential ubiquitination at functionally essential bromodomains, suggesting that degron-enriched regions may underlie domain-selective degradation. Additionally, dBET1 treatment was associated with mitochondrial depolarization and calcium homeostasis disruption, defects that we hypothesize may be functionally linked to the observed VDAC1 depletion. Together, this study demonstrates that integrating ubiquitomics provides a superior sensitivity layer for PROTAC safety assessment, capable of uncovering mechanism-based liabilities that escape conventional global proteomic screening.

Humans↗

PubMed2025

VDAC2 and Bak scarcity in liver mitochondria enables targeting hepatocarcinoma while sparing hepatocytes.

Differences between normal tissues and invading tumors that allow tumor targeting while saving normal tissue are much sought after. Here we show that scarcity of VDAC2, and the consequent lack of Bak recruitment to mitochondria, renders hepatocyte mitochondria resistant to permeabilization by truncated Bid (tBid), a Bcl-2 Homology 3 (BH3)-only, Bcl-2 family protein. Increased VDAC2 and Bak is found in most human liver cancers and mitochondria from tumors and hepatic cancer cell lines exhibit VDAC2- and Bak-dependent tBid sensitivity. Exploring potential therapeutic targeting, we find that combinations of activators of the tBid pathway with inhibitors of the Bcl-2 family proteins that suppress Bak activation enhance VDAC2-dependent death of hepatocarcinoma cells with little effect on normal hepatocytes. Furthermore, in vivo, combination of S63845, a selective Mcl-1 inhibitor, with tumor-nectrosis factor-related, apoptosis-induncing ligand (TRAIL) peptide reduces tumor growth, but only in tumors expressing VDAC2. Thus, we describe mitochondrial molecular fingerprint that discriminates liver from hepatocarcinoma and allows sparing normal tissue while targeting tumors.

Voltage-Dependent Anion Channel 2↗

PubMed1992

A new scheme of symbiosis: ligand- and voltage-gated anion channels in plants and animals.

Anion channels in the plasma membrane of both plant and animal cells participate in a number of important cellular functions such as volume regulation, trans-epithelial transport, stabilization of the membrane potential and excitability. Only very recently attention has turned to the presence of anion channels in higher plant cells. A dominant theme among recent discoveries is the role of Ca2+ in activating or modulating channel current involved in signal transduction. The major anion channel of stomatal guard cell protoplasts is a 32-40 pS channel which is highly selective for anions, in particular NO3-, Cl- and malate. These channels are characterized by a steep voltage dependence. Anion release is elicited upon depolarization and restricted to a narrow voltage span of -100 mV to the reversal potential of anions. During prolonged activation the current slowly inactivates. A rise in cytoplasmic calcium in the presence of nucleotides evokes activation of the anion channels. Following activation they catalyse anion currents 10-20 times higher than in the inactivated state thereby shifting the resting potential of the guard cell from a K(+)-conducting to an anion-conducting state. Patch-clamp studies have also revealed that growth hormones directly affect voltage-dependent activity of the anion channel in a dose-dependent manner. Auxin binding resulted in a shift of the activation potential towards the resting potential. Auxin-dependent gating of the anion channel is side- and hormone-specific. Its action is also channel-specific as K+ channels coexisting in the same membrane patch were insensitive to this ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Mapping the binding site on ankyrin for the voltage-dependent sodium channel from brain.

Erythrocyte ankyrin is a member of a family of proteins that mediate the linkage between membrane proteins and the underlying spectrin-actin-based cytoskeleton. Ankyrin has been shown to interact with a variety of integral membrane proteins such as the anion exchanger, the Na+K(+)-ATPase, and the voltage-dependent sodium channel (NaCh) in brain. To understand how ankyrin interacts with these proteins and maintains its specificity and high affinity for the voltage-dependent NaCh, we have mapped the binding site on ankyrin for the NaCh by examining the binding of purified ankyrin subfragments, prepared by proteolytic cleavage, to the purified rat brain NaCh incorporated into liposomes. 125I-Labeled ankyrin and the radiolabeled 89- and 43-kDa fragments of ankyrin bind to the NaCh with high affinities and with Kd values of 34, 22, and 63 nM, respectively, and have stoichiometries of approximately 1 mol/mol NaCh. The 72-kDa spectrin binding domain is inactive and does not bind to the NaCh. Dissection of ankyrin reveals that the 43-kDa domain retains all the binding properties of native ankyrin to the NaCh. Analysis of the primary structure reveals that the NaCh binding site is confined to a domain of ankyrin consisting entirely of the 11 terminal 33-amino acid repeats and is distinct from the ankyrin domains that interact with spectrin and the Na+K(+)-ATPase.

Animals↗

PubMed1992

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1979

A voltage-gated anion channel from the electric organ of Torpedo californica.

Addition of membrane vesicles prepared from the electric organ of Torpedo californica to the aqueous phase of a planar phospholipid bilayer system results in a large (up to 3 orders of magnitude) stepwise increase in membrane conductance. This increased conductance consists of two components: an ohmic background "leak" and a voltage-dependent, ideally anion-selective conductance. The anion conductance is low at voltages greater than +10 mV, rises sharply as the voltage becomes negative, and then saturates as the voltage becomes highly negative. (The trans side of the bilayer, to which vesicles are not added, is defined as ground.) Under high amplification, the anion conductance shows single channel behavior with a voltage-independent, single channel conductance of 13.9 +/- 0.1 pmho in 0.1 M Cl-. Furthermore, the anion channel, but not the background conductance, is inhibited by submillimolar concentrations of SITS and DIDS, two well known anion transport inhibitors. The inhibition is seen only when SITS or DIDS is added to the cis side. No cholinergic agents tested have any effect on the channel.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1992

Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae.

Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na+ or K+ equally well, and Ca2+ to a lesser extent. Its open probability (Po) is voltage-dependent, peaking at about -80 mV (cytoplasm negative), and falling to near zero at +80 mV. Elevated cytoplasmic Ca2+, alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K+ over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca2+, this channel is activated by positive going membrane voltages: mean Po is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg2+ or Na+, block the yeast plasma-membrane K+ channel in a similar but less pronounced manner.

Calcium↗

PubMed1992

Control of the hyperpolarization-activated cation current by external anions in rabbit sino-atrial node cells.

1. Effects of varying concentrations of anions on the hyperpolarization-activated current (I(f)) were studied in myocytes isolated from the rabbit sino-atrial node. Substituting Cs+ for the intracellular K+ clearly separated I(f) from the delayed rectifier K+ current. Control properties, including gating kinetics and ion selectivity, similar to previous studies were obtained. 2. Substitution of extracellular Cl- with larger anions including isethionate, glutamate, acetate, and aspartate, reduced the amplitude of I(f) without changing the reversal potential. Substitution with small anions such as iodide or nitrate supported an intact I(f). These effects were reproduced in the excised outside-out patch conformation. 3. The conductance for I(f) was a saturating function of the extracellular Cl- concentration ([Cl-]o) with an equilibrium binding constant (K1/2) of 11 mM and a slope factor of about 1 when substituted with large anions. Total removal of small anions completely abolished I(f). 4. The voltage-dependent gating of I(f) was not affected by changing ([Cl-]o), suggesting that Cl- modulates conductance properties of I(f). 5. The results indicate that I(f) conductance is unique in that it is dependent on an extracellular anion (Cl-), yet it is carried exclusively by cations, K+ and Na+. These effects are independent of any measurable voltage-dependent gating parameters.

Animals↗

PubMed1992

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.

Anions↗

PubMedDate not supplied

Relationships between pHi and tension in isolated rat mesenteric resistance arteries.

Investigations into the relationship between pHi and tension were carried out in rat mesenteric resistance arteries. Acute acidosis, induced by ammonium chloride pre-pulse, led to variable and transient tension development, but simultaneous removal of extracellular sodium led to a sustained rise in tension associated with maintained intracellular acidification. Dependence of tension and pHi recovery from acute acidosis on Na/H exchange and anion exchange pathways was demonstrated using pharmacological inhibitors. Additionally, removal of HCO3 suggested the anion pathway involved was Na-dependent HCO3 transport. Removal of extracellular calcium, or pharmacological inhibition of voltage-dependent calcium channels, prevented the tension development in response to NH4Cl pre-pulse in an Na-free medium, but did not affect pHi. Intracellular acidosis resulting from elevation of the PCO2 resulted in initial vasoconstriction followed by profound vasodilatation of arteries pre-contracted with noradrenaline (NA). The response to alkalosis induced by NH4Cl or by lowering the PCO2 led to initial dilatation followed by potentiation of NA-induced tension.

Ammonium Chloride↗

PubMed1992

Anionic currents of chick sensory neurons are affected by a phospholipase A2 purified from the venom of the taipan snake.

A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.

Amino Acid Sequence↗

PubMed1992

Characterization of high-conductance anion channels in rat bile duct epithelial cells.

We have utilized patch clamp recording techniques to identify a high-conductance anion channel in the plasma membrane of rat bile duct epithelial cells. Cells were isolated from the intrahepatic bile duct 2-6 wk after bile duct ligation. Channels were present in 27% (28/102) of excised patches, and, with 150 mM Cl- in bath and pipette solutions, the slope conductance of the fully open level was approximately 364 +/- 18 pS (n = 8) with current reversal = 0 +/- 1 mV. Channel characteristics were not affected by substitution of K+ for Na+ in the pipette solution; but substitution of HCO3-, gluconate, or increased NaCl caused a shift in the reversal potential toward the new equilibrium potential for Cl-. The permeability ratios were PHCO3-/PCl- = 0.51 +/- 0.03 (n = 5), Pgluconate/PCl- = 0.12 +/- 0.04 (n = 7), and PNa+/PCl- = 0.11 +/- 0.02 (n = 3). Current transitions to a subconductance level at 72% of the fully open level were present in most studies. Channel open probability was greatest near 0 mV and decreased rapidly outside of -20 to +20 mV because of voltage-dependent channel closure. The time course for current relaxation of summed single channel currents could be described by a single exponential with more rapid channel closure as the magnitude of the voltage step away from 0 mV increased. In the cell-attached configuration, the channel was rarely open (4/35, 11%) but opening could be induced by negative pipette pressure (5/14, 35%). Possible physiological roles for this channel are discussed.

Animals↗

PubMed1979

Characteristics of the anion channel in the sino-atrial node cell of the rabbit.

1. The anion permeability of the sino-atrial node cell membranes was determined by substituting various anions for Cl and observing the resultant transient changes in membrane potential. The permeability sequence was found to be in the following order: thiocyanate greater than NO3 greater than I Br greater than Cl greater than acetate. 2. The membrane resistance in Cl solution was compared with that in various anion solutions by the voltage-clamp method. The conductance sequence for the sino-atrial node cell membrane was observed to be the same as the permeability sequence. 3 The potential generated by the Na-K pump was partly short-circuited in normal bathing solution, and thus the pump could be responsible in part for the generation of the sin-atrial node resting membrane potential. 4 When Cl was replaced by acetate, a less permeable ion, the inward-going rectification disappeared. Thus, the inward-going rectification might be due partly to a time- and voltage-dependent Cl current.

Acetates↗