Induced Ion Channel Research Articles

Host-guest interaction induced ion channels for accelerated OH− transport in anion exchange membranes

Anion exchange membranes (AEMs) with fast ion transport capability are highly desired in numerous industries. Improving the ion conductivity of AEMs through rational molecular structure design is of great importance. Inspired by the biological ion channels formed by the supramolecular assembly of protein chains, we herein develop a strategy for preparing high-performance AEMs by constructing ion conduction channels via crown ether-based supramolecular host-guest interaction. In relatively apolar solvent media, one dibenzo[24]crown-8 (DB24C8) ring tethers one dibenzylammonium salt (DBAS) guest molecule to form stable supramolecular assembly. Hence, by grafting DB24C8 rings and DBAS moieties onto the cationic side chains, the resulting host-guest interaction actuates cationic groups to assembly into connected ion conducting channels in AEM, as proved by small angle X-ray scattering and transmission electron microscopy analyses. The host-guest interaction induced ion channels boost the OH− conduction in AEM, thus the conductivity of the resultant AEM is twice that of the control membrane (without host-guest interaction) in spite of the same ion exchange capacity. Besides, a demonstration of the H2/O2 AEMFC using the host-guest interacted AEM offers almost two times higher peak power density than the control membrane. This design of supramolecular host-guest interaction induced ion channels provides a roadmap for developing AEMs with satisfactory ion conductivity.

Mimicking Cellular Signaling Pathways within Synthetic Multicompartment Vesicles with Triggered Enzyme Activity and Induced Ion Channel Recruitment

AbstractSubcellular compartmentalization of cells, a defining characteristic of eukaryotes, is fundamental for the fine tuning of internal processes and the responding to external stimuli. Reproducing and controlling such compartmentalized hierarchical organization, responsiveness, and communication is important toward understanding biological systems and applying them to smart materials. Herein, a cellular signal transduction strategy (triggered release from subcompartments) is leveraged to develop responsive, purely artificial, polymeric multicompartment assemblies. Incorporation of responsive nanoparticles—loaded with enzymatic substrate or ion channels—as subcompartments inside micrometer‐sized polymeric vesicles (polymersomes) allowed to conduct bioinspired signaling cascades. Response of these subcompartments to an externally added stimulus is achieved and studied by using confocal laser scanning microscopy (CLSM) coupled with in situ fluorescence correlation spectroscopy (FCS). Signal triggered activity of an enzymatic reaction is demonstrated in multicompartments through recombination of compartmentalized substrate and enzyme. In parallel, a two‐step signaling cascade is achieved by triggering the recruitment of ion channels from inner subcompartments to the vesicles' membrane, inducing ion permeability, mimicking endosome‐mediated insertion of internally stored channels. This design shows remarkable versatility, robustness, and controllability, demonstrating that multicompartment polymer‐based assemblies offer an ideal scaffold for the development of complex cell‐inspired responsive systems for applications in biosensing, catalysis, and medicine.

The MAPK pathway is involved in the regulation of rapid pacing-induced ionic channel remodeling in rat atrial myocytes

Alterations to the expression L‑type calcium channels (LTCCs) and Kv4.3 potassium channels form the possible basis of atrial electrical remodeling during rapid pacing. The mitogen‑activated protein kinase (MAPK) pathway is affected by increases in cytoplasmic Ca2+, and therefore represents an attractive candidate for the regulation and mediation of Ca2+‑induced ion channel remodeling. The present study aimed to investigate alterations to the ion channel‑MAPK axis, and to determine its influence on ion channel remodeling during atrial fibrillation. Rat atrial myocytes were isolated, cultured, and in vitro rapid pacing was established. Intracellular Ca2+ signals were monitored using the Fluo‑3/AM Ca2+ indicator. Verapamil, PD98058 and SB203580 were added to the culture medium of various groups at specific time‑points. The mRNA expression levels of LTCC‑α1c and Kv4.3 potassium channels were detected by reverse transcription‑polymerase chain reaction. Western blotting was performed to determine the expression levels of channel and signaling proteins. The results demonstrated that fast pacing significantly increased the intracellular Ca2+ concentration in atrial myocytes, whereas treatment with verapamil markedly inhibited this increase. In addition, verapamil significantly antagonized the rapid pacing‑induced activation of extracellular signal‑regulated kinase (ERK) and p38MAPK. These results indicated that the MAPK pathway may have an important role in the opening of LTCCs, and alterations to MAPK molecule expression could affect the expression and remodeling of ion channels.

Recording ion channels across soy-extracted lecithin bilayer generated by water-soluble quantum dots

We report on the quantum dot (QD)-induced ion channels across a soya-derived lecithin bilayer supported on a laser drilled of ~100 μm aperture of cellulose acetate substrate that separates two electrolytic chambers. Adequate current bursts were observed when the bilayer was subjected to a gating voltage. The voltage-dependent current fluctuation, across the bilayer, was attributed to the insertion of ~20 nm sized water-soluble CdSe QDs, forming nanopores due to their spontaneous aggregation. Apart from a closed state, the first observable conductance levels were found as 6.3 and 11 nS, as for the respective biasing voltages of −10 and −20 mV. The highest observable conductance states, at corresponding voltages were ~14.3 and 21.1 nS. Considering two simplified models, we predict that the non-spherical pores (dnspore) can be a better approximation over spherical nanopores (dspore) for exhibiting a definite conductance level. At times, even dnspore ≤ 4dspore and that the non-spherical nanopores were associated with a smaller No. of QDs than the case for spherical nanopores, for a definite conductance state. It seems like the current events are partly stochastic, possibly due to thermal effects on the aggregated QDs that would form nanopores. The dwell time of the states was predicted in the range of 384–411 μs. The ion channel mechanism in natural phospholipid bilayers over artificial ones will provide a closer account to understand ion transport mechanism in live cells and signaling activity including labelling with fluorescent QDs.

Taurine-magnesium Coordination Compound Attenuates Hypoxia/Reoxygenation Induced Ion Channel Dysfunction in Rat Ventricular Myocytes

Because of the known anti-arrhythmic effects of taurine-magnesium coordination compound (TMCC), the aim of the present study was to explore the electrophysiological effects of TMCC on hypoxia/reoxygenation (H/R)-induced arrhythmias in rat ventricular myocytes. Sodium current (I Na), the L-type calcium current (I Ca,L), and the transient outward potassium current (I to) were evaluated using whole-cell patch-clamp recordings in rat ventricular myocytes following H/R injury. The H/R group significantly decreased sodium currents, while L-type calcium currents and transient outward potassium currents was significantly increased (all p<0.01). TMCC (200 and 400 μM) prevented abnormal sodium currents induced by H/R by inhibiting steady-state inactivation. It also counteracted abnormal L-type calcium currents induced by H/R by inhibiting steady-state activation and facilitating steady-state inactivation. In addition, it mitigated abnormal transient outward potassium currents induced by H/R by inhibiting steady-state activation. TMCC prevents H/R-induced arrhythmias in rat ventricular myocytes by modifying ion channel function.

Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide ppTG20 analogue

P7, an analogue of the cell-penetrating peptide (CPP) ppTG20, was derived by replacing Phe and Trp with Arg based on the structure–activity relationships of CPPs and antimicrobial peptides (AMPs). P7 showed antimicrobial activity against Salmonella typhimurium at 4μM and possessed broad antimicrobial activity greater than its parent peptide. P7 displayed good selectivity, with low haemolysis below its minimum inhibitory concentration range, but displayed cytotoxic activity against the HT29 and MDA-MB231 mammalian cell lines. Studies of calcein leakage from egg yolk l-α-phosphatidylcholine/egg yolk l-α-phosphatidyl-dl-glycerol (EYPC/EYPG) (bacterial membrane mimic) and EYPC/cholesterol (eukaryotic membrane mimic) vesicles also demonstrated that P7 exhibited high selectivity and caused pore formation in the bacterial membrane. Circular dichroism experiments suggested that the conformation of P7 underwent transitions from a random coil in sodium phosphate buffer to an α-helical conformation in bacterial model membranes. P7 induced influx of the membrane fluorescent probe 1-N-phenylnaphthylamine (NPN) and the nucleic acid fluorescent probe SYTOX® Green by increasing live S. typhimurium cell outer membrane and plasma membrane permeability, respectively. P7 also induced ion channel formation in the cell plasma membrane causing leakage of potassium ions. Flow cytometric analysis demonstrated that S. typhimurium cell membrane integrity was destroyed following incubation with P7. These results indicated that P7 exhibited good bacterial selectivity and exerted its antibacterial activity by a membrane damage mechanism. Furthermore, these results suggested that CPPs may represent a source of templates for AMP design.

Study on the Cr3+-Induced Ion Channel Based the Electron Transfer Rate Across Mimic Biomembrane Measured by Scanning Electrochemical Microscopy

Study on the Cr3+-Induced Ion Channel Based the Electron Transfer Rate Across Mimic Biomembrane Measured by Scanning Electrochemical Microscopy

Transport of Small Molecules across the Bacterial Translocation Channel SecYEG

Many proteins are translocated across the endoplasmic reticulum (ER) membrane or the bacterial plasma membrane through a conserved channel, formed by a heterotrimeric protein complex (called the Sec61p complex in eukaryotes and the SecYEG complex in bacteria and archaea). Cotranslational or postranslational translocation requires ribosome or SecA binding, respectively. The resting channel is impermeable to ions and water (Saparov et al., 2007). Here we have tested whether the channel remains a barrier to small molecules after a conformational transition occurred due to ligand binding. Therefore, we reconstituted the purified and fluorescently labeled translocation channel SecYEG into planar lipid bilayers. Positioning of the membrane on top of a laser scanning microscope with single molecule sensitivity allowed monitoring the reconstitution efficiency. Both the motor molecule SecA and ribosomes induced ion channel activity. The probability of the channel opening was derived from the number of open channels and the total number of reconstituted channels.Acknowledgments: The project was funded by the Austrian Science Fund.ReferenceS. M. Saparov, Karl Erlandson, Kurt Cannon, Julia Schaletzky, Sol Schulman, Tom A. Rapoport, and P. Pohl. Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol.Cell 26 (4):501-509, 2007.

Plasma Membrane-porating Domain in Poliovirus 2B Protein. A Short Peptide Mimics Viroporin Activity

Picornavirus 2B, a non-structural protein required for effective viral replication, has been implicated in cell membrane permeabilization during the late phases of infection. Here, we have approached the molecular mechanism of this process by assessing the pore-forming activity of an overlapping peptide library that spanned the complete 2B sequence. At non-cytopathic concentrations, only the P3 peptide, spanning 2B residues 35–55, effectively assembled hydrophilic pores that allowed diffusion of low molecular mass solutes across the cell plasma membrane (IC50≈4×10−7 M) and boundary liposome bilayers (starting at peptide to lipid molar ratios>1:104). Circular dichroism data were consistent with its capacity to fold as a helix in a membrane-like environment. Furthermore, addition of this peptide to a sealed plasma-membrane model, consisting of retinal rod outer segments patch-clamped in a whole-cell configuration, induced ion channel activity within seconds at concentrations as low as 10−8 M. Thus, we have established a “one-helix” 2B version that possesses the intrinsic pore-forming activity required to directly and effectively permeabilize the cell plasma membrane. We conclude that 2B viroporin can be classified as a genuine pore-forming toxin of viral origin, which is produced intracellularly at certain times post infection.

Improved perfusion conditions for patch–clamp recordings on human erythrocytes

Various configurations of the patch–clamp method are powerful tools for examining the transport of charged solutes across biological membranes. Originally developed for the study of relatively large cells which adhere to solid surfaces under in vitro culture, these methods have been increasingly applied to small cells or organelles in suspension. Under these conditions, a number of significant technical problems may arise as a result of the smaller geometry. Here, we examined these problems using human erythrocytes infected with the malaria parasite, Plasmodium falciparum, a system where experimental differences and the technical difficulty of erythrocyte patch–clamp have hindered universal agreement on the properties of the induced ion channels. We found that patch–clamp recordings on infected erythrocytes are especially susceptible to artifacts from mechanical perturbations due to solution flow around the cell. To minimize these artifacts, we designed a new perfusion chamber whose geometry allows controlled solution flow around the fragile erythrocyte. Not only were recordings acquired in this chamber significantly less susceptible to perfusion artifacts, but the chamber permitted rapid and reversible application of known inhibitors with negligible mechanical agitation. Electrophysiological recordings then faithfully reproduced several findings made with more traditional methods. The new perfusion chamber should also be useful for patch–clamp recordings on blood cells, protoplasts, and organelles.

Channel formation by CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii.

It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M(w) and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a beta-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum.

A recent study on malaria-infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host- or parasite-derived and their molecular nature have not been resolved. Here we report a comparison of a malaria-induced anion channel with an endogenous anion channel in Plasmodium falciparum-infected human RBCs. Ion channel activity was measured using the whole-cell, cell-attached and excised inside-out configurations of the patch-clamp method. Parasitised RBCs were cultured in vitro, using co-cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (~15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I(-) > Br(-) > Cl(-). In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (~80 pS, outward conductance) was spontaneously active. The host membrane of malaria-infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria-infected RBCs were shown to have a low open-state probability (P(o)) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole-cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up-regulated by the malaria parasite P. falciparum.

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora

The purified major outer membrane protein (37275 Da) from the psychrotrophic phytopathogen Erwinia carotovora MFCL0 was structurally characterised by MALDI-TOF mass spectrometry, N-terminal microsequencing and DNA sequence determinations, and secondary structure prediction analyses. The deduced amino acid sequence showed 76% and 72% of similarities with the Serratia marcescens and Escherichia coli OmpA proteins respectively. Dendrogram analysis allowed to point out that E. carotovora is close to the genus Serratia. After reconstitution into planar lipid bilayers, this major protein induced ion channels with a major conductance level of 630 pS in 1 M NaCl and a weak cationic selectivity. These functional and structural features allowed to identify this major outer membrane component of E. carotovora as an OmpA-like protein, i.e., a channel-forming protein which could be involved in the infection process of this phytopathogen agent.

Serotonin and norepinephrine transporters: possible relationship between oligomeric structure and channel modes of conduction.

In the past several years there has been significant progress made on the biophysics of neurotransmitter transporters, leading to the proposal of new models of substrate and ion permeation across membranes. Questions arising from these studies are as follows: How are substrate uptake and substrate-induced current related? Where and how does substrate-ion coupling occur? What is the functional significance of the coupled and uncoupled currents? Because of a long-standing interest and collaboration, and because of their importance for normal function and disease, the authors have focused on the properties of human norepinephrine and serotonin transporters, using other clones and mutations as specific needs arise. It has been know for decades that hNETs (human norepinephrine transporters) clear NE+ (norepinephrine) following its release in peripheral sympathetic and central noradrenergic synapses. Neuronal activity influences NE+ uptake, so one is also interested in the acute regulation of hNET. To study these problems, hNET-expressing cells have been developed that are suitable for patch clamp, radioligand uptake, biochemistry, and transiently expressed clones for structure-function analysis, and new protocols have been designed combining patch-clamp, microamperometry, Ca2+ imaging, and native catecholamine transporter preparations to study transporters in whole cells and isolated patches. Using these methods, Na-dependent, NE+-induced hNET currents that are blocked by cocaine and antidepressants, channel modes of NE+ conduction, voltage-dependent uptake coupled to NE+-induced ion channel activity, PKC (phosphokinase C) regulation of NE+ uptake, and transporter modulation by [Ca2+]i have all been discovered. There is also provocative new data on other transporters in this family, such as Li/Na mole fraction experiments in the Drosophila serotonin transporters and sided enkephalin block in proline transporters. These studies have led one to postulate the existence of a narrow pore within transporters through which the substrate (NE+ or serotonin, 5HT+) and other ions (principally Na+) pass. It is hypothesized that the pore resides in an oligomeric structure and that separate gene products of hNET or hSERT (human serotonin transporters) come together to form a channel.

Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28°C to 8°C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8°C and 28°C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65–75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8°C and 28°C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight β-strand OmpA N-terminal domain (OmpA 171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA 171, provides strong evidence of the existence of an eight-stranded β-barrel in the N-terminal domain of OprFs.

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli.

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.

MOMP (major outer membrane protein) of Campylobacter jejuni; a versatile pore-forming protein

The great majority of trimeric porins of Gram-negative bacteria cannot be dissociated into monomers without disrupting their folded conformation. The porin of Campylobacter jejuni, however, displays two folded structures, a classical oligomer and a monomer resistant to detergent denaturation. We probed the transition of trimer to monomer using light scattering experiments and examined the secondary structures of these two molecular states by infra-red spectroscopy. The channel-forming properties of both trimer and monomer were studied after incorporation into artificial lipid bilayers. In these conditions, the trimer induced ion channels with a conductance value of 1200 pS in 1 M NaCl. The pores showed marked cationic selectivity and sensitivity to low voltage. Analysis of the isolated monomer showed nearly the same single-channel conductance and the same selectivity and sensitivity to voltage. These results indicate that the folded monomer form of C. jejuni MOMP displays essentially the same pore-forming properties as the native trimer.

Properties of acetylcholine receptor ion channels in the acutely dissociated neurons of the marginal division in the rat striatum.

Cell-attached mode of patch clamp technique was employed to investigate the properties of acetylcholine (ACh)-induced ion channels in acutely dissociated neurons from the marginal division (MrD) of rat striatum. Two types of conductance states (25 pS and 60 pS) were recorded. The 25 pS channel (more than 80%) was the main type in the neurons of MrD and was described here. The amplitudes of inward currents increased with hyperpolorization and the reversing potential was about 0 mV. Both single short opening and long burst openings were observed in MrD neurons. Two-time constants of these two kinds of ion channels are 0.29 ms, 1.84 ms and 1.96 ms, 18.24 ms, respectively. Average close time can be fitted with two exponential functions, the two time constants are 1.7 ms and 54 ms. Probability of channel opening is about 0.012 and no voltage-dependence was found. The properties of reversing potential, voltage-independence and the form of agonist to the ion channels indicated that the recorded channel currents flow through AChR channels. The mAChR is involved in slow synaptic transmission and Ach can not induce the opening of mAChR ion channel. The binding site of ACh to AChR and the nAChR ion channel are the same protein, ACh can only activate nAChR ion channel directly. Therefore, the recorded ion channels in the present study are nAChR ion channels. The results suggest that nAChR ion channels exist in the neurons of MrD and the MrD probably is involved in learning and memory mechanism of the brain.

Cyclic voltammetry and a.c. impedance studies of Ca 2+-induced ion channels on Pt-BLM

Self-assembled phosphatidylcholine/cholesterol bilayer lipid membrane (BLM) was fabricated on Pt electrode, which had been cleaned by high power ultrasonic horn. The influence of Ca 2+ ions on the electron transfer of Fe(CN) 6 3−/4− couple on Pt supported BLM (Pt-BLM) was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). CV experiments showed directly that Ca 2+ ions could induce electron transfer on Pt-BLM. Three possibilities of the cause of redox reaction induced by Ca 2+ ions were discussed: (1) electronic tunneling across bilayer, (2) electronic tunneling across monolayer, (3) direct electron transfer between Fe(CN) 6 3−/4− ions and Pt electrode through Ca 2+ induced ion-channels. Results of calculations based on Marcus's theory indicate that electronic tunneling across bilayer and monolayer contributes little to the redox current. The theory of partially blocked electrode was employed to analyze the sigmoidal CV curves, and it was concluded that ion-channels generated by Ca 2+ ions were the main cause of electron transfer reaction on Pt-BLM. EIS measurement showed that Pt-BLM became porous after Ca 2+ ions were added to the electrolyte. This was consistent with the conclusion drawn from CV experiments. Since ion-channels play the role similar to micro defects, it is concluded from CV and EIS experiments that sigmoidal CV curves and depressed circular arcs are evidence for the existence of micro defects on unmodified solid-supported-BLM.