Low-conductance Channels Research Articles

Formation of High-Conductive C Subunit Channels upon Interaction with Cyclophilin D.

The c subunit of the ATP synthase is an inner mitochondrial membrane (IMM) protein. Besides its role as the main component of the rotor of the ATP synthase, c subunit from mammalian mitochondria exhibits ion channel activity. In particular, c subunit may be involved in one of the pathways leading to the formation of the permeability transition pore (PTP) during mitochondrial permeability transition (PT), a phenomenon consisting of the permeabilization of the IMM due to high levels of calcium. Our previous study on the synthetic c subunit showed that high concentrations of calcium induce misfolding into cross-β oligomers that form low-conductance channels in model lipid bilayers of about 400 pS. Here, we studied the effect of cyclophilin D (CypD), a mitochondrial chaperone and major regulator of PTP, on the electrophysiological activity of the c subunit to evaluate its role in the functional properties of c subunit. Our study shows that in presence of CypD, c subunit exhibits a larger conductance, up to 4 nS, that could be related to its potential role in mitochondrial toxicity. Further, our results suggest that CypD is necessary for the formation of c subunit induced PTP but may not be an integral part of the pore.

Effective suppression of conductance in multichannel molecular wires

Although quantum effects have introduced fascinating properties to molecular circuits, exploring new features for molecular circuits remains a challenge. In this work, effective suppression of conductance in the parallel intramolecular circuit is identified, going beyond the well-known superposition effect. By introducing oligo(phenylene ethynylene) (OPE) as a high-conductivity channel (HCC) and [cis-Pt(PEt3)2]2+ coordinated with m-phenylethynyl as a low-conductivity channel (LCC), the single-molecule conductance measured by the scanning tunneling microscopy break junction (STM-BJ) technique demonstrates that, in the multichannel molecular wires, the conductance decreases progressively with the addition of the LCC. A theoretical investigation indicates that all channels participate in electron transport. The conductance suppression in the multichannel structures is ascribable to the higher contribution of the LCC than of the HCC to the conductive orbitals. This finding improves the understanding of electron transport theory at the molecular scale and provides a strategy to design parallel intramolecular circuits.

Bisindolylpyrrole Induces a Cpr3- and Porin1/2-Dependent Transition in Yeast Mitochondrial Permeability in a Low Conductance State via the AACs-Associated Pore.

Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2–4 h after treatment of cells with 5 μM BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators.

Fabrication of a Spinose Structure LiFePO4 Cathode Via Surface Etching to Improve the Rate Capability and Cycling Stability of Li-Ion Batteries

Lithium iron phosphate (LiFePO4, LFP) has been demonstrated as a suitable cathode material for Li-ion batteries due to its relatively high theoretical capacity (170 mAh·g-1), cost effectiveness, long cycle life, good thermal stability, and environmental friendliness, However, key limitations of LFP are its low intrinsic electronic conductivity (10-9-10-10 S·cm-1) and limited lithium ion diffusion channel (one-dimensional path along the b-axis of the olivine structure), leading to poor rate capabilities and cycling stability in LFP batteries. To improve the rate capability of LFP cathode, the low electronic conductivity can be resolved by conductive material coating, heteroatomic doping, and downsizing of active materials. To improve the rate capability and cycling stability of LFP battery, we propose the surface treatment of LFP cathode material to increase the surface area. The surface area of LFP is controlled by the concentration of etching agents (0.5 M to 2.0 M), etching time (10 min to 360 min), and reaction temperatures (25 °C to 80 °C). The morphology of the etched LFP surface is characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The specific surface area and crystal structure of surface etched LFP are characterized by Brunauer-Emmett-Teller (BET) and X-ray diffraction (XRD), respectively. To determine battery performance, coin-cells are assembled with cathodes containing various surface areas. Cycling testing is performed using an Arbin tester to measure the rate capability at different C-rates and cycling stability at same C-rate.

ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore

SUMMARYPermeability transition (PT) is an increase in mitochondrial inner membrane permeability that can lead to a disruption of mitochondrial function and cell death. PT is responsible for tissue damage in stroke and myocardial infarction. It is caused by the opening of a large conductance (~1.5 nS) channel, the mitochondrial PT pore (mPTP). We directly tested the role of the c-subunit of ATP synthase in mPTP formation by measuring channel activity in c-subunit knockout mitochondria. We found that the classic mPTP conductance was lacking in c-subunit knockout mitochondria, but channels sensitive to the PT inhibitor cyclosporine A could be recorded. These channels had a significantly lower conductance compared with the cyclosporine A-sensitive channels detected in parental cells and were sensitive to the ATP/ADP translocase inhibitor bongkrekic acid. We propose that, in the absence of the c-subunit, mPTP cannot be formed, and a distinct cyclosporine A-sensitive low-conductance channel emerges.

Effect of Temporal Current Modulation on the Tracking Force Acting on a Relativistic Electron Beam in an Ohmic Plasma Channel

The effect of the current rise time in a relativistic electron beam pulse on the tracking force exerted by the low-conductivity Ohmic plasma channel on the beam has been analyzed using the “hard” beam model. It is shown using numerical analysis that an increase in the beam current rise time substantially reduces the tracking force considered here.

The Calculation of the Tracking Force in the Evolution of the Resistive Hose Instability of a Relativistic Electron Beam

We have analyzed the tacking force exerted by a low-conductivity Ohmic plasma channel on a relativistic electron beam. It has been shown that, for the given type of evolution of the resistive hose instability of the beam along its pulse, this force substantially depends on the frequency and the increment of the increase in the given instability.

Analysis of neuronal nicotinic acetylcholine receptor α4β2 activation at the single-channel level

The neuronal nicotinic acetylcholine receptor α4β2 forms pentameric proteins with two alternate stoichiometries. The high-sensitivity receptor is related to (α4)2(β2)3 stoichiometry while the low-sensitivity receptor to (α4)3(β2)2 stoichiometry. Both subtypes share two binding sites at the α4(+)/β2(−) interface with high affinity for agonists. (α4)3(β2)2 has an additional binding site at the α4(+)/α4(−) interface with low affinity for agonists. We investigated activation kinetics of both receptor subtypes by patch-clamp recordings of single-channel activity in the presence of several concentrations of acetylcholine (0.5 to 300μM). We used kinetic software to fit these data with kinetic models. We found that the high-sensitivity subtype correlates with the low-conductance channel (g−70=29pS) and does not activate with high efficacy. On the contrary, the low-sensitivity subtype correlated with a high-conductance channel (g−70=44pS) and exhibited higher activation efficacy. Opening events of individual nAChRs at high agonist concentrations occurred in clusters, which allowed us to determine kinetic constants for the activation of the triliganded receptor. Our kinetic modeling identified an intermediate state, between resting and open conformation of the receptor. Binding of the third molecule increases the efficacy of receptor activation by favoring the transition between resting and intermediate state around 18 times. The low rate for this transition in the diliganded receptor explains the action of acetylcholine as partial agonist when it binds to the high-affinity sites. The presence of the third binding site emerges as a potent modulator of nicotinic receptor α4β2 activation which may display different functions depending on agonist concentration.

CFTR: Structural Mishap by Mutation

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein is a member of the ABC (ATP‐binding cassette) family, which functions as a low conductance channel regulating the flow of certain anions (especially chloride and thiocyanate) across membranes of epithelial cells, allowing for osmosis‐based regulation of mucus consistency. The proper function of CFTR depends on the amino acid phenylalanine at position 508, which mediates a tertiary interaction between the surface of the N‐terminal nucleotide‐binding domain and cytoplasmic loop‐4 in the C‐terminal membrane‐spanning domain. Miscoding caused by the most common identified mutation, ΔF508, results in loss of phenylalanine by deletion of base pairs G, A, and A from the sequence “T‐A‐G” at position 507 and adjacent “A‐A‐A”, resulting in a codon made for isoleucine, but not for phenylalanine. Inheritance of two copies of the mutation causes part of the CFTR protein to misfold and degrade, not becoming fully processed in the endoplasmic reticulum. This mutation, among others, leads to a class II mutation of cystic fibrosis. The misfolded CFTR is either not transported to the surface of the membrane or is only transported in residual amounts, preventing the regulation of mucus consistency and causes cystic fibrosis. The mechanism of the ATP‐dependent gating mutation in CFTR protein can be further understood through modeling. Understanding the mutation that causes the misfolding of CFTR can help develop new treatments for those afflicted with cystic fibrosis. Walton High School SMART (Students Modeling A Research Topic) Team has designed a model of CFTR using 3D printing technology to investigate structure‐function relationships.

In situ catalytic synthesis of high-graphitized carbon-coated LiFePO4 nanoplates for superior Li-ion battery cathodes.

The low electronic conductivity and one-dimensional diffusion channel along the b axis for Li ions are two major obstacles to achieving high power density of LiFePO4 material. Coating carbon with excellent conductivity on the tailored LiFePO4 nanoparticles therefore plays an important role for efficient charge and mass transport within this material. We report here the in situ catalytic synthesis of high-graphitized carbon-coated LiFePO4 nanoplates with highly oriented (010) facets by introducing ferrocene as a catalyst during thermal treatment. The as-obtained material exhibits superior performances for Li-ion batteries at high rate (100 C) and low temperature (-20 °C), mainly because of fast electron transport through the graphitic carbon layer and efficient Li(+)-ion diffusion through the thin nanoplates.

Morphology control of lithium iron phosphate nanoparticles by soluble starch-assisted hydrothermal synthesis

Lithium iron phosphate (LiFePO4) is a potentially high efficiency cathode material for lithium ion batteries, but the low electronic conductivity and one-dimensional diffusion channel for lithium ions require small particle size and shape control during the synthesis. In this paper, well-crystallized and morphology-controlled LiFePO4 cathode material for lithium-ion batteries is successfully synthesized via a soluble starch-assisted hydrothermal method at 180 °C for 5 h, followed by calcining with phenolic resin at 750 °C for 6 h. In this study, we investigate the effect of five different concentrations of starch solution on controlling morphology of LiFePO4. Interestingly, the nano-sized LiFePO4 particles obtained in 0.075 mol L−1 starch solution exhibit a spheroidal microstructure, while the platelet shape LiFePO4 particles are synthesized in lower or higher concentration of starch solution. The mechanism and process of forming such spheroidal microstructure is discussed. These unique structural and morphological properties of LiFePO4 lead to high specific capacity and stable cycling performance. Analysis of the electrochemical impedance spectroscopy reveals that nano-sized carbon/polyacene coated LiFePO4 cathode materials play an critical role in achieving excellent electrochemical performance.

Forces acting on a particle in a concentration gradient under an externally applied oscillating electric field

We report a force field on a particle in a concentration (conductivity) gradient under an externally applied oscillating electric field. The conductivity gradient was established through integrated microcapillaries bridging high- and low-conductivity streams in dedicated microchannels. Particles in low-conductivity electrolyte were observed to experience a strong force with the application of an oscillating field and pulled to the microcapillary openings where they were held against the flow. Particle trapping was accompanied by a concurrent electrolyte injection from high- to low-conductivity channel, triggered with the externally applied field and further contributed to the conductivity gradient near the trapping sites. We experimentally evaluated the force dependence on the magnitude and frequency of the excitation field for 10 μm polystyrene particles immersed at various conductivity levels. The experiments suggest that the observed force cannot be simply explained by dielectrophoresis or diffusiophoresis alone and further requires the consideration of a so-called concentration polarization force. This force has been rather recently postulated based on a theoretical treatment and yet to be experimentally validated. Using the theoretical treatment of this force, together with fluidic drag and diffusiophoresis, we correctly predicted trapping trajectories of particles based on a simultaneous solution of Poisson-Nernst-Planck and Stokes equations. The predicted and measured trapping velocities were found in reasonable agreement (within a factor of <1.6), suggesting that the consideration of the concentration polarization force is necessary for describing the observed particle behavior.

Chlorotoxin-Fc Fusion Inhibits Release of MMP-2 from Pancreatic Cancer Cells

Chlorotoxin (CTX) is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion) venom, which inhibits low-conductance chloride channels in colonic epithelial cells. It has been reported that CTX also binds to matrix metalloproteinase-2 (MMP-2), membrane type-1 MMP, and tissue inhibitor of metalloproteinase-2, as well as CLC-3 chloride ion channels and other proteins. Pancreatic cancer cells require the activation of MMP-2 during invasion and migration. In this study, the fusion protein was generated by joining the CTX peptide to the amino terminus of the human IgG-Fc domain without a hinge domain, the monomeric form of chlorotoxin (M-CTX-Fc). The resulting fusion protein was then used to target pancreatic cancer cells (PANC-1) in vitro. M-CTX-Fc decreased MMP-2 release into the media of PANC-1 cells in a dose-dependent manner. M-CTX-Fc internalization into PANC-1 cells was observed. When the cells were treated with chlorpromazine (CPZ), the internalization of the fusion protein was reduced, implicating a clathrin-dependent internalization mechanism of M-CTX-Fc in PANC-1 cells. Furthermore, M-CTX-Fc clearly exhibited the inhibition of the migration depending on the concentration, but human IgG, as negative control of Fc, was not affected. The M-CTX-Fc may be an effective instrument for targeting pancreatic cancer.

Size and shape control of LiFePO4 nanocrystals for better lithium ion battery cathode materials

Lithium iron phosphate (LiFePO4) is a potential high efficiency cathode material for lithium ion batteries, but the low electronic conductivity and single diffusion channel for lithium ions require good particle size and shape control during the synthesis of this material. In this paper, six LiFePO4 nanocrystals with different size and shape have been successfully synthesized in ethylene glycol. The addition sequence Fe-PO4-Li helps to form LiFePO4 nanocrystals with mostly {010} faces exposed, and increasing the amount of LiOH leads to a decrease in particle size. The electrochemical performance of the six distinct LiFePO4 particles show that the most promising LiFePO4 nanocrystals either have predominant {010} face exposure or high specific area, with little iron(II) oxidation.

Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei.

BackgroundGlycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.Methods/Principal FindingsWe hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl−∼0.31), while the other two types of channels are slightly selective for cations (P K+/P Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore.Conclusions/SignificanceThese results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.

Stable lipid bilayers based on micro- and nano-fabrication

In this review, we will discuss our recent approaches for improving the mechanical stability of free-standing bilayer lipid membranes (BLMs) by combining with BLM formation and micro- and nano-fabrication techniques. BLMs were prepared across a microaperture fabricated in silicon (Si) chips or nanoporous alumina films, and their mechanical stability and electric properties were investigated. BLMs spanned over the porous alumina showed background noise currents small enough for recording activities of low-conductance channels, though further stability enhancement of porous alumina films was necessary. BLMs suspended in a thin Si3N4 septum showed a dramatic improvement of BLM stability. The BLMs were resistant to a voltage of ± 1 V and the membrane lifetime was 15–43 h with and without incorporated channels. The membrane containing gramicidin channel exhibited tolerance to repetitive solution exchanges, though the electric properties of the BLMs are necessary to be improved. The realisation of BLMs having both mechanical stability and proper electric properties will open up a variety of applications including highly sensitive biosensors and high-throughput drug screenings for ion channels.

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca2+ channels in non-excitable cells has been studied. Receptor- and store-operated Ca2+ influxes have been measured using the fluorescent method of detection of cytosolic Ca2+ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca2+ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels Imax and non-selective channels Ins in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance Imin channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.

Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1

In this study, we compared the channel intrinsic stability of the bacterial K +-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at ∼45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.

Regulation Of The Single Channel Conductance Of K2p10.1 (Trek2) By The Amino-terminus

K2P2.1 (TREK-1) and K2P10.1 (TREK-2), when expressed in mammalian cell lines, show several single channel conductance levels. Recent studies show that the N-terminus of TREKs controls the unitary conductance levels via alternative translation initiation mechanism that produces isoforms with long and short N-termini. For TREK-2, the isoform with the full length N-terminus (residues 1-69) has a low conductance level, whereas that with the short one (residues 55-69 or 67-69) has a large conductance level. The role of the N-terminus and the putative slide helix region in the control of the unitary conductance was studied further using deletion and substitution TREK-2 mutants. Deletion of the N-terminus up to residue 36 (out of 69 residues) had no effect on conductance levels. Further deletions up to residues 40 (∼150-pS at −40 mV), 44 (∼90-pS), and 49 (>30-pS) produced channels with levels that were different from the levels observed in the wild type TREK-2. A mutation within the putative slide helix region (residues 47-55) to render it non-helical resulted in formation of mainly the low conductance channel, and greatly reduced the open probability. These TREK-2 mutants also inhibited the alternative translation initiation. These results suggest that the distal region of the N-terminus (residues 37-55) including the putative helical region (residues 37-54) controls the unitary conductance of TREK-2 in an unpredictable way. The data also indicate that the putative helical region is important for the control of channel activity.

Predictions Suggesting a Participation of β-Sheet Configuration in the M2 Domain of the P2X 7 Receptor: A Novel Conformation?

Scanning experiments have shown that the putative TM2 domain of the P2X 7 receptor (P2X 7R) lines the ionic pore. However, none has identified an α-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a β-sheet conformation in the TM2 domain of P2X 2. These data led us to investigate a new architecture within the P2X receptor family. P2X 7R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (∼10 pS) and a large pore (>400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X 7R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X 7R (hP2X 7R). We provide evidence for a β-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X 7R is partially folded in a β-sheet conformation, and may play an important role in channel/pore formation associated with P2X 7R activation. It is important to note that most nonselective large pores have a transmembrane β-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X 7R field and/or raise new questions about this issue.