Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity

Signals recorded from neurons with extracellular planar sensors have a wide range of waveforms and amplitudes. This variety is a result of different physical conditions affecting the ion currents through a cellular membrane. The transmembrane currents are often considered by macroscopic membrane models as essentially a homogeneous process. However, this assumption is doubtful, since ions move through ion channels, which are scattered within the membrane. Accounting for this fact, the present work proposes a theoretical model of heterogeneous membrane conductivity. The model is based on the hypothesis that both potential and charge are distributed inhomogeneously on the membrane surface, concentrated near channel pores, as the direct consequence of the inhomogeneous transmembrane current. A system of continuity equations having non-stationary and quasi-stationary forms expresses this fact mathematically. The present work performs mathematical analysis of the proposed equations, following by the synthesis of the equivalent electric element of a heterogeneous membrane current. This element is further used to construct a model of the cell-surface electric junction in a form of the equivalent electrical circuit. After that a study of how the heterogeneous membrane conductivity affects parameters of the extracellular electrical signal is performed. As the result it was found that variation of the passive characteristics of the cell-surface junction, conductivity of the cleft and the cleft height, could lead to different shapes of the extracellular signals

Inhibition of HERG1 K+ channel protein expression decreases cell proliferation of human small cell lung cancer cells

HERG (human ether-à-go-go-related gene) K+ currents fulfill important ionic functions in cardiac and other excitable cells. In addition, HERG channels influence cell growth and migration in various types of tumor cells. The mechanisms underlying these functions are still not resolved. Here, we investigated the role of HERG channels for cell growth in a cell line (SW2) derived from small cell lung cancer (SCLC), a malignant variant of lung cancer. The two HERG1 isoforms (HERG1a, HERG1b) as well as HERG2 and HERG3 are expressed in SW2 cells. Inhibition of HERG currents by acute or sustained application of E-4031, a specific ERG channel blocker, depolarized SW2 cells by 10–15 mV. This result indicated that HERG K+ conductance contributes considerably to the maintenance of the resting potential of about −45 mV. Blockage of HERG channels by E-4031 for up to 72 h did not affect cell proliferation. In contrast, siRNA-induced inhibition of HERG1 protein expression decreased cell proliferation by about 50%. Reduction of HERG1 protein expression was confirmed by Western blots. HERG current was almost absent in SW2 cells transfected with siRNA against HERG1. Qualitatively similar results were obtained in three other SCLC cell lines (OH1, OH3, H82), suggesting that the HERG1 channel protein is involved in SCLC cell growth, whereas the ion-conducting function of HERG1 seems not to be important for cell growth

Analysis of Intracellular State Based on Controlled 3D Nanostructures Mediated Surface Enhanced Raman Scattering

Near-infrared surface-enhanced Raman spectroscopy (SERS) is a powerful technique for analyzing the chemical composition within a single living cell at unprecedented resolution. However, current SERS methods employing uncontrollable colloidal metal particles or non-uniformly distributed metal particles on a substrate as SERS-active sites show relatively low reliability and reproducibility. Here, we report a highly-ordered SERS-active surface that is provided by a gold nano-dots array based on thermal evaporation of gold onto an ITO surface through a nanoporous alumina mask. This new combined technique showed a broader distribution of hot spots and a higher signal-to-noise ratio than current SERS techniques due to the highly reproducible and uniform geometrical structures over a large area. This SERS-active surface was applied as cell culture system to study living cells in situ within their culture environment without any external preparation processes. We applied this newly developed method to cell-based research to differentiate cell lines, cells at different cell cycle stages, and live/dead cells. The enhanced Raman signals achieved from each cell, which represent the changes in biochemical compositions, enabled differentiation of each state and the conditions of the cells. This SERS technique employing a tightly controlled nanostructure array can potentially be applied to single cell analysis, early cancer diagnosis and cell physiology research

Measurement of Contractile Stress Generated by Cultured Rat Muscle on Silicon Cantilevers for Toxin Detection and Muscle Performance Enhancement

Background: To date, biological components have been incorporated into MEMS devices to create cell-based sensors and assays, motors and actuators, and pumps. Bio-MEMS technologies present a unique opportunity to study fundamental biological processes at a level unrealized with previous methods. The capability to miniaturize analytical systems enables researchers to perform multiple experiments in parallel and with a high degree of control over experimental variables for high-content screening applications.Methodology/Principal Findings: We have demonstrated a biological microelectromechanical system (BioMEMS) based on silicon cantilevers and an AFM detection system for studying the physiology and kinetics of myotubes derived from embryonic rat skeletal muscle. It was shown that it is possible to interrogate and observe muscle behavior in real time, as well as selectively stimulate the contraction of myotubes with the device. Stress generation of the tissue was estimated using a modification of Stoney's equation. Calculated stress values were in excellent agreement with previously published results for cultured myotubes, but not adult skeletal muscle. Other parameters such as time to peak tension (TPT), the time to half relaxation (KRT) were compared to the literature. It was observed that the myotubes grown on the BioMEMS device, while generating stress magnitudes comparable to those previously published, exhibited slower TPT and KRT values. However, growth in an enhanced media increased these values. From these data it was concluded that the myotubes cultured on the cantilevers were of an embryonic phenotype. The system was also shown to be responsive to the application of a toxin, veratridine.Conclusions/Significance: The device demonstrated here will provide a useful foundation for studying various aspects of muscle physiology and behavior in a controlled high-throughput manner as well as be useful for biosensor and drug discovery applications

K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport

<p>Abstract</p> <p>Background</p> <p>Lung epithelial Na⁺channels (ENaC) are regulated by cell Ca²⁺signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K⁺channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K⁺channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport.</p> <p>Methods</p> <p>Verapamil-induced depression of heterologously expressed human αβγ ENaC in <it>Xenopus </it>oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and <it>in vivo </it>alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca²⁺signal in H441 cells was analyzed using Fluo 4AM.</p> <p>Results</p> <p>The rate of <it>in vivo </it>AFC was reduced significantly (40.6 ± 6.3% of control, <it>P </it>< 0.05, n = 12) in mice intratracheally administrated verapamil. K_Ca3.1(1-EBIO) and K_ATP(minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca²⁺signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca²⁺in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, K_V(pyrithione-Na), K _Ca3.1(1-EBIO), and K_ATP(minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na⁺and K⁺transport pathways.</p> <p>Conclusions</p> <p>Our observations demonstrate that K⁺channel openers are capable of rescuing reduced vectorial Na⁺transport across lung epithelial cells with impaired Ca²⁺signal.</p

Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Different Classes of Antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects