Modulation Of Ionic Currents Research Articles

Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.

Electrical Detection of Protein Biomarkers Using Nanoneedle Biosensors

Abstract:Here we present the development of an array of electrical nano-biosensors in a microfluidic channel, called Nanoneedle biosensors. Then we present the proof of concept study for protein detection. A Nanoneedle biosensor is a real-time, label-free, direct electrical detection platform, which is capable of high sensitivity detection, measuring the change in ionic current and impedance modulation, due to the presence or reaction of biomolecules such as proteins or nucleic acids. We show that the sensors which have been fabricated and characterized for the protein detection. We have functionalized Nanoneedle biosensors with receptors specific to a target protein using physical adsorption for immobilization. We have used biotinylated bovine serum albumin as the receptor and sterptavidin as the target analyte. The detection of streptavidin binding to the receptor protein is also presented.

A Bipolar Vacuum Microelectronic Device

This report provides the first demonstration of a vacuum microelectronic device that utilizes both positive and negative charge states (i.e., a bipolar vacuum microelectronic device), thus enabling the possibility of device designs that are not previously possible in traditional vacuum microelectronics. In the same way that complimentary metal-oxide-semiconductor (complimentary n-channel MOS and p-channel MOS) were required in solid-state electronics before digital logic applications could be addressed, vacuum microelectronic devices benefit from a second charge state to realize many applications. This advance could enable integrated circuits for radiation-intensive environments (nuclear power facilities and space-based communications such as satellites) and high-temperature applications (engines and materials processing). A microelectromechanical systems platform was used to construct pentode structures with integrated carbon nanotube field emitters for electron emission and bias electrodes for separate electron- and ion-current modulation. Ions were generated via electron impact in an argon ambient, and devices were tested in both voltage sweep and pulsed modes. Current that is greater than 2 mA/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> was modulated at the anode between ion and electron collection, demonstrating that this novel platform has the potential to foster a new class of bipolar vacuum microelectronics.

Cardiac Resynchronization and Electrophysiological Reverse-Remodeling in the Failing Heart

The electrophysiological hallmark of cells and tissues isolated from failing hearts is prolongation of action potential duration (APD) and conduction slowing. In human studies and a number of animal models of heart failure, functional downregulation of K+ currents and alterations in depolarizing Na+ and Ca2+ currents and transporters are demonstrated. Alterations in intercellular ion channels and matrix contribute to heterogeneity of APD and conduction slowing. The changes in cellular and tissue function are regionally heterogenous particularly in the heart failure with dyssynchronous LV contraction (DHF). Furthermore, β-adrenergic signaling and modulation of ionic currents is blunted in heart failure. Cardiac resynchronization therapy (CRT) partially reversed the DHF-induced downregulation of K+ current and improved Na+ channel gating. CRT significantly improved Ca2+ homeostasis especially in myocytes from late-activated, lateral wall, and restores the DHF-induced blunted β-adrenergic receptor responsiveness. CRT abbreviated DHF-induced prolongation of APD in the lateral wall myocytes and reduced the LV regional gradient of APD, and suppressed development of early afterdepolarizations. In conclusion, CRT partially restores the DHF-induced ion channel remodeling, abnormal Ca2+ homeostasis, blunted β-adrenergic response and regional heterogeneity of APD, thus may suppress ventricular arrhythmias and contribute to the mortality benefit of CRT as well as improve mechanical performance of the heart.

Proposal and experimental validation of the electrophoretic Coulter method for analyzing microparticles and biological cells

Zeta potential measurement is one of the simplest methods for analyzing the electrical surface properties of microparticles and biological cells in a solution. The authors propose a new methodology for simultaneous measurement of the number, size and zeta potential of different specimens in a microchannel, referred to as the electrophoretic Coulter (EC) method. First, a microchannel is fabricated using soft lithography, a small amount of a specimen is injected into it, and ion current modulation through the microchannel is measured while a DC electric field is applied. The results are then compared with those obtained using the conventional methods involving dynamic light scattering (DLS) method and microscopy. The results of the EC method show good agreement with those of the conventional approaches. Accordingly, the technique enables high-throughput analysis of different specimens including nano materials and biomolecules using a micro/nanochannel, thereby significantly contributing to the field of bio-nano fusion research.

Electrophysiological Remodeling in Heart Failure Dyssynchrony vs. Resynchronization

The electrophysiological hallmark of cells and tissues isolated from failing hearts is prolongation of action potential duration (APD) and conduction slowing. In human studies and a number of animal models of heart failure, functional downregulation of K currents and alterations in depolarizing Na and Ca currents and transporters are demonstrated. Alterations in intercellular ion channels and matrix contribute to heterogeneity of APD and conduction slowing. The changes in cellular and tissue function are regionally heterogenous particularly in the heart failure with dyssynchronous LV contraction (DHF). Furthermore, β‐adrenergic signaling and modulation of ionic currents is blunted in heart failure.Cardiac resynchronization therapy (CRT) partially reversed the DHF‐induced downregulation of K current and improved Na channel gating. CRT significantly improved Ca homeostasis especially in myocytes from late‐activated, lateral wall, and restores the DHF‐induced blunted β‐adrenergic receptor responsiveness. CRT abbreviated DHF‐induced prolongation of APD in the lateral wall myocytes and reduced the LV regional gradient of APD, and suppressed development of early afterdepolarizations.In conclusion, CRT partially restores DHF‐induced ion channel remodeling, abnormal Ca homeostasis, blunted β‐adrenergic response and regional heterogeneity of APD, thus may suppress ventricular arrhythmias and contribute to the mortality benefit of CRT as well as improve mechanical performance of the heart.

Electrophysiological Remodeling in Heart Failure Dyssynchrony vs. Resynchronization

The electrophysiological hallmark of cells and tissues isolated from failing hearts is prolongation of action potential duration (APD) and conduction slowing. In human studies and a number of animal models of heart failure, functional downregulation of K currents and alterations in depolarizing Na and Ca currents and transporters are demonstrated. Alterations in intercellular ion channels and matrix contribute to heterogeneity of APD and conduction slowing. The changes in cellular and tissue function are regionally heterogenous particularly in the heart failure with dyssynchronous LV contraction (DHF). Furthermore, β-adrenergic signaling and modulation of ionic currents is blunted in heart failure. Cardiac resynchronization therapy (CRT) partially reversed the DHF-induced downregulation of K current and improved Na channel gating. CRT significantly improved Ca homeostasis especially in myocytes from late-activated, lateral wall, and restores the DHF-induced blunted β-adrenergic receptor responsiveness. CRT abbreviated DHF-induced prolongation of APD in the lateral wall myocytes and reduced the LV regional gradient of APD, and suppressed development of early afterdepolarizations. In conclusion, CRT partially restores DHF-induced ion channel remodeling, abnormal Ca homeostasis, blunted β-adrenergic response and regional heterogeneity of APD, thus may suppress ventricular arrhythmias and contribute to the mortality benefit of CRT as well as improve mechanical performance of the heart.

Electrical remodeling in the failing heart

We focus on the molecular and cellular basis of excitability, conduction and electrical remodeling in heart failure with dyssynchronous left ventricular contraction (DHF) and its restoration by cardiac resynchronization therapy (CRT) using a canine tachy-pacing heart failure model. The electrophysiological hallmark of cells and tissues isolated from failing hearts is prolongation of action potential duration (APD) and conduction slowing. In human studies and a number of animal models of heart failure, functional downregulation of K currents and alterations in depolarizing Na and Ca currents and transporters are demonstrated. Alterations in intercellular ion channels and extracellular matrix contribute to heterogeneity of APD and conduction slowing. The changes in cellular and tissue function are regionally heterogeneous, particularly in the DHF. Furthermore, beta-adrenergic signaling and modulation of ionic currents is blunted in heart failure. CRT partially reverses the DHF-induced downregulation of K current and improves Na channel gating. CRT significantly improves Ca homeostasis, especially in lateral myocytes, and restores the DHF-induced blunted beta-adrenergic receptor responsiveness. CRT abbreviates DHF-induced prolongation of APD in the lateral myocytes, reduces the left ventricular regional gradient of APD and suppresses development of early afterdepolarizations. CRT partially restores DHF-induced electrophysiological remodeling, abnormal Ca homeostasis, blunted beta-adrenergic responsiveness, and regional heterogeneity of APD, and thus may suppress ventricular arrhythmias and contribute to the mortality benefit of CRT as well as improving mechanical performance of the heart.

Control of bursting activity by modulation of ionic currents

Our study is focused on modulation of dynamics of single leech heart interneurons (HNs). We consider two models of HNs representing these neurons under two different pharmacological treatments: (1) blocking of Ca2+ currents and inhibitory coupling with the Ca2+-containing saline and partial blocking of K+ currents; (2) decoupling HNs with bicuculline. In (1), an HN demonstrates slow plateau-like oscillations [1,2]. In (2), an HN demonstrates endogenously bursting activity [3]. We analyze how the interburst interval and burst duration could be controlled by manipulating hyperpolarization-activated current, Ih, and persistent Na+ current, IP, namely by variation of their conductances and the half-activation voltages, V1/2. For example, burst duration increases greatly from 1.7 s to 8.9 s as Vh,1/2 increased from -30 mV to 4 mV. The interburst interval grows from 0.6 s to 125 s as the Vh,1/2 decreases from 4 [mV] to -56 [mV] in accordance with a saddlenode bifurcation. In (2), we similarly show that the variation of Vh,1/2 could be a target for modulation of the bursting. In both cases, we show co-existence of bursting and silence. Interestingly, the co-existence is sensitive to gh (and to maximal conductance of fast Ca2+ current, gCaF too in (2)) and is not sensitive to the maximal conductances of other currents. In (1), if gh is increased from 4 nS to 8 nS, the bistability is then observed in an almost five-fold larger range of the leak conductance values, gleak. In (2), if either gh is changed from 4 nS to 8 nS or gCaF is changed from 5 nS to 0 nS, the bistability is observed in an almost two-fold larger range of gleak. If the bistability is an indication of a dysfunctional dynamics, this observation describes a new, potentially pathological role of overexpression of Ih and ICaF.

Lattice-Boltzmann Simulations of Ionic Current Modulation by DNA Translocation.

We present a numerical study of the effect of DNA translocation on the ionic current through a nanopore. We use a coarse-grained model to solve the electrokinetic equations at the Poisson-Boltzmann level for the microions, coupled to a lattice-Boltzmann equation for the solvent hydrodynamics. In most cases, translocation leads to a reduction in the ionic current. However, at low salt concentrations (large screening lengths) we find ionic current enhancement due to translocation. In an unstructured pore, translocation of the helical charge distribution of the DNA has no effect on the ionic current. However, if a localized charge probe is placed on the wall of the nanopore, we observe ionic current modulations that, though weak, should be experimentally observable.

Effects of brain-derived neurotrophic factor (BDNF) on activity mediated by NMDA receptors in rat spinal cord cultures

Brain-derived neurotrophic factor (BDNF) is involved in the differentiation and the survival of neurons. It has also been shown to be associated with the regrowth of neurons of damaged spinal cord and the modulation of ionic currents by acting on sodium channels and NMDA receptors through tyrosine kinase B (TrkB) receptors. We investigated the effects of BDNF on rhythm generation induced by disinhibition in dissociated cultures from embryonic rat spinal cord (E14), with extracellular multisite recordings (MultiElectrode Arrays, MEAs) or intracellular patch-clamp recordings. Exogenous BDNF had only minor effects on the bursting by increasing the activity during the burst. This increase of activity is suggested to be mediated by a potentiation of the postsynaptic NMDA receptors because it has been found that BDNF potentiates the NMDA-evoked depolarization in cultures incubated with BDNF for 10 min. Possible direct effects of BDNF on sodium channels were also investigated by local application of BDNF to the soma of patched neurons but no depolarization was observed. Long-term application of BDNF strongly decreased the activity during the burst and also the number of active electrodes, possibly due to a decrease in network density.

Fast modulation of heat-activated ionic current by proinflammatory interleukin 6 in rat sensory neurons

The pro-inflammatory cytokine interleukin-6 (IL-6) together with its soluble receptor (sIL-6R) induces and maintains thermal hyperalgesia. It facilitates the heat-induced release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Here we report that exposure of nociceptive neurons to the IL-6-sIL-6R complex or the gp130-stimulating designer IL-6-sIL-6R fusion protein Hyper-IL-6 (HIL-6) resulted in a potentiation of heat-activated inward currents (I(heat)) and a shift of activation thresholds towards lower temperatures without affecting intracellular calcium levels. The Janus tyrosine kinase inhibitor AG490, the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide 1 (BIM1), as well as rottlerin, a selective blocker of the PKCdelta isoform, but not the cyclooxygenase inhibitor indomethacin, effectively reduced the effect. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed expression of mRNA for the signal-transducing beta subunit of the receptor gp130 in neuronal somata, rather than satellite cells in rat dorsal root ganglia. Together, the results suggest that IL-6-sIL-6R acts directly on sensory neurons. It increases their susceptibility to noxious heat via the gp130/Jak/PKCdelta signalling pathway.

Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is abundantly present in the serum and mediates multiple biological responses. With the aim of extending our knowledge on the role played by S1P in the regulation of cytoskeletal reorganization, native as well as C2C12 myoblasts stably transfected with green fluorescent protein (GFP)-tagged alpha- and beta-actin constructs were stimulated with S1P (1 microM) and observed under confocal and multiphoton microscopes. The addition of S1P induced the appearance of actin stress fibres and focal adhesions through Rho- and phospholipase D (PLD)-mediated pathways. The cytoskeletal response was dependent on the extracellular action of S1P through its specific surface receptors, since the intracellular delivery of the sphingolipid by microinjection was unable to modify the actin cytoskeletal assembly. Interestingly, it was revealed by whole-cell patch-clamp that S1P-induced stress fibre formation was associated with increased ion currents and conductance through stretch-activated channels (SACs), thereby suggesting a possible regulatory role for organized actin in channel sensitivity. Experiments aimed at stretching the plasma membrane of C2C12 cells, using the cantilever of an atomic force microscope, indicated that there was a Ca2+ influx through putative SACs. In conclusion, the present data suggest novel mechanisms of S1P signalling involving actin cytoskeletal reorganization and Ca2+ elevation through SACs that might influence myoblastic functions.

Collisional ion dynamics in capacitively coupled rf discharges

Based on a one-dimensional fluid model, the collision-dominated ion dynamics in capacitively coupled rf discharges are studied theoretically as well as numerically in the low rf frequency regime ω⩽VB∕l (here ω is the rf frequency, VB is the Bohm velocity, and l is the plasma length). It is found that the ion current entering the sheath varies with time as we have shown in the collisionless sheath [N. Xiang and F. L. Waelbroeck, J. Appl. Phys. 93, 5034 (2003)]. The mechanisms for the ion current modulation in the collision-dominated and collisionless sheaths, however, are different. It is also shown that the sheath dynamics and ion energy distribution are affected significantly by this time-varying ion current.

Molecular heterogeneity of calcium channel beta-subunits in canine and human heart: evidence for differential subcellular localization.

Multiple Ca2+ channel beta-subunit (Ca(v)beta) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Ca(v)beta subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Ca(v)beta genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Ca(v)beta isoforms. A total of 18 different Ca(v)beta isoforms were detected in both canine and human ventricles including splice variants from all four Ca(v)beta genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Ca(v)beta subunit genes are expressed at the protein level, and the Ca(v)beta subunits show differential subcellular localization with Ca(v)beta1b, Ca(v)beta2, and Ca(v)beta3 predominantly localized to the T-tubule sarcolemma, whereas Ca(v)beta1a and Ca(v)beta4 are more prevalent in the surface sarcolemma. Coexpression of the novel Ca(v)beta2c subunits (Ca(v)beta(2cN1), Ca(v)beta(2cN2), Ca(v)beta(2cN4)) with the pore-forming alpha1C (Ca(v)1.2) and Ca(v)alpha2delta subunits in HEK 293 cells resulted in a marked increase in ionic current and Ca(v)beta2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Ca(v)beta subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.

Influence of permeant ions on voltage sensor function in the Kv2.1 potassium channel.

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K+ occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K+]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K+] modulate the rate of channel activation. Elevation of symmetrical [K+] or [Rb+] from 0 to 3 mM doubled the rate of on-gating charge movement (Qon), measured at 0 mV. Cs+ produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03–3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Qon. However, for each of these permeant ions, the speeding of Qon occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K+-dependent reorientation of the outer vestibule, also abolished the K+-dependence of Qon. Together, the data indicate that the K+-dependent reorientation in the outer vestibule was responsible for the change in Qon. Moreover, similar [K+]-dependence and effects of mutagenesis indicate that the K+-dependent change in rate of Qon can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K+ reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.

Microtubules mobility affects the modulation of L-type ICa by muscarinic and β-adrenergic agonists in guinea-pig cardiac myocytes

To investigate the interaction of cytoskeleton with the receptor modulation of ionic currents, we studied the effect of muscarinic and β-adrenergic stimulation in adult guinea-pig ventricular cardiac myocytes treated with paclitaxel and colchicine, two drugs that respectively stabilize or destabilize microtubules. We observed that the stabilization of microtubules with paclitaxel (1 μM for 1–4 h) did not markedly affect either the kinetics of I Ca, or the stimulatory effect of isoproterenol (Iso, 1 μM); however paclitaxel significantly blunted the response to carbachol (CCh, 1 μM). In agreement with the electrophysiological measurements, Iso induced a similar enhancement of intracellular cAMP levels in both control and paclitaxel-treated cells, while the response to CCh 1 μM was significantly reduced in paclitaxel-treated cells. The reduction of muscarinic response induced by paclitaxel was also evident in atrial cells, in which the stimulation of I KACh by CCh 1 μM was reduced to about 10%. Compared to the muscarinic response, paclitaxel did not have significant effect on the purinergic (adenosine 1–10 μM) modulation of I Ca. In contrast to paclitaxel, in colchicine-treated cells, I Ca was not enhanced by β-adrenergic stimulation, but instead reduced by CCh, even in the absence of previous stimulation. In conclusion, our data suggest that microtubule stabilization significantly affects the muscarinic modulation of I Ca, by interacting with the receptor or the G-protein rather than on the intracellular signaling cascade.

Flavonoid modulation of ionic currents mediated by GABA A and GABA C receptors

The modulation of ionotropic γ-aminobutyric acid (GABA) receptors (GABA-gated Cl − channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by α 1β 1γ 2s GABA A and ρ 1 GABA C receptors expressed in Xenopus laevis oocytes in the micromolar range. α 1β 1γ 2s GABA A and ρ 1 GABA C receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by α 4β 2 neuronal nicotinic acetylcholine, serotonin 5-HT 3A and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized α 1β 1γ 2s GABA A receptors. Effects of apigenin and quercetin on α 1β 1γ 2s GABA A receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation.

Ion flux from vacuum arc cathode spots in the absence and presence of a magnetic field

Because plasma production at vacuum cathode spots is approximately proportional to the arc current, arc current modulation can be used to generate ion current modulation that can be detected far from the spot using a negatively biased ion collector. The drift time to the ion detector can used to determine kinetic ion energies. A very wide range of cathode materials have been used. It has been found that the kinetic ion energy is higher at the beginning of each discharge and approximately constant after 150 μs. The kinetic energy is correlated with the arc voltage and the cohesive energy of the cathode material. The ion erosion rate is in inverse relation to the cohesive energy, enhancing the effect that the power input per plasma particle correlates with the cohesive energy of the cathode material. The influence of three magnetic field configurations on the kinetic energy has been investigated. Generally, a magnetic field increases the plasma impedance, arc burning voltage, and kinetic ion energy. However, if the plasma is produced in a region of low field strength and streaming into a region of higher field strength, the velocity may decrease due to the magnetic mirror effect. A magnetic field can increase the plasma temperature but may reduce the density gradients by preventing free expansion into the vacuum. Therefore, depending on the configuration, a magnetic field may increase or decrease the kinetic energy of ions.

The modulation of ionic currents in excitable tissues by n-3 polyunsaturated fatty acids.

The modulation of ionic currents in excitable tissues by n-3 polyunsaturated fatty acids.