Effect of gating currents of ion channels on the collective spiking activity of coupled Hodgkin-Huxley neurons

Based on the coupled stochastic Hodgkin-Huxley neurons, we numerically studied the effect of gating currents of ion channels, as well as coupling and the number of neurons, on the collective spiking rate and regularity in the coupled system. It was found, for a given coupling strength and with a relatively large number of neurons, when gating currents are applied, the collective spiking regularity decreases; meanwhile, the collective spiking rate increases, indicating that gating currents can aggravate the desynchronization of the spikings of all neurons. However, gating currents caused hardly any effect in the spiking of any individual neuron of the coupled system. This result, different from the reduction of the spiking rate by gating currents in a single neuron, provides a new insight into the effect of gating currents on the global information processing and signal transduction in real neural systems. Supported by the Science Foundation of Ludong University (Grant Nos. 23140301, L20072805)     

Delay-induced coherence bi-resonance-like behavior in stochastic Hodgkin-Huxley neuron networks

In this paper, we study how information transmission delays affect the spiking behavior of electrically coupled stochastic Hodgkin-Huxley (HH) neurons on Newman-Watts networks. It is found that the spiking behavior becomes the most regular at an optimal time delay, indicating the occurrence of delay-induced coherence resonance-like (CR-like) behavior. Interestingly, there are different CR-like types, depending on the membrane patch size of the neuron. For a smaller patch size, only single CR-like behavior occurs; while for a larger patch size, coherence bi-resonance-like (CBR) behavior appears. These findings show that the delay-induced CR-like behavior is closely related to the channel noise strength, and the coupled neurons may exhibit different spiking behaviors under the interplay of the channel noise and time delay. Therefore, the channel noise should be taken into account in the study of time delay-related spiking activity in stochastic HH neurons. This work provides new insight into the role of channel noise and information transmission delays in realistic neural systems.     

Synthetic ion channel activity documented by electrophysiological methods in living cells

Hydraphiles are synthetic ion channels that use crown ethers as entry portals and that span phospholipid bilayer membranes. Proton and sodium cation transport by these compounds has been demonstrated in liposomes and planar bilayers. In the present work, whole cell patch clamp experiments show that hydraphiles integrate into the membranes of human embryonic kidney (HEK 293) cells and significantly increase membrane conductance. The altered membrane permeability is reversible, and the cells under study remain vital during the experiment. Control compounds that are too short (C(8)-benzyl channel) to span the bilayer or are inactive owing to a deficiency in the central relay do not induce similar conductance increases. Control experiments confirm that the inactive channel analogues do not show nonspecific effects such as activation of native channels. These studies show that the combination of structural features that have been designed into the hydraphiles afford true, albeit simple, channel function in live cells.     

Voltage activation and hysteresis of the non-selective voltage-dependent channel in the intact human red cell

Suspension of intact human red cells in media with low chloride and sodium concentrations (isotonic sucrose substitution) results in strongly inside positive membrane potentials, which activate the voltage-dependent non-selective cation (NSVDC) channel. By systematic variation of the initial Nernst potentials for chloride (degree of ion substitution) as well as the chloride conductance (block by NS1652), and by exploiting the interplay between the Ca(2+)-permeable NSVDC channel, the Ca(2+)-activated K+ channel (the Gárdos channel) and the Ca(2+)-pump, a graded activation of the NSVDC channel was achieved. Under these conditions, it was shown that the NSVDC channels exist in two states of activation depending on the initial conditions for the activation. The hysteretic behaviour, which in patch clamp experiments has been found for the individual channel unit, is thus retained at the cellular level and can be demonstrated with red cells in suspension.     

Incidence of partial charges on ion selectivity in potassium channels

Potassium channels are membrane proteins known to select potassium over sodium ions at a high diffusion rate. We conducted ab initio calculations on a filter model of KcsA of about 300 atoms at the Hartree-Fock level of theory. Partial charges were derived from the quantum mechanically determined electrostatic potential either with Merz-Kollman or Hinsen-Roux schemes. Large polarization and/or charge transfer occur on potassium ions located in the filter, while the charges on sodium ions remain closer to unity. As a result, a weaker binding is obtained for K(+) ions. Using a simplified version of a permeation model based on the concerted-motion mechanism for ion translocation within the single-file ion channel [P. H. Nelson, J. Chem. Phys. 117, 11396 (2002)], we discuss how differences in polarization effects in the adducts with K(+) and Na(+) can play a role as for ionic selectivity and conductance.     

Effect of Protein Dimerization on Ion Conductivity of Gramicidin A Channel Studied Using Polarizable Force Field

In studies of ion channel systems, due to the huge computational cost of polarizable force fields, classical force fields remain the most widely used for a long time. In this work, we used the AMOEBA polarizable atomic multipole force field in enhanced sampling simulations of single-channel gramicidin A (gA) and double-channel gA systems and investigated its reliability in characterizing ion-transport properties of the gA ion channel under dimerization. The influence of gA dimerization on the permeation of potassium and sodium ions through the channel was described in terms of conductance, diffusion coefficient, and free energy profile. Results from the polarizable force field simulations show that the conductance of potassium and sodium ions passing through the single- and double-channel agrees well with experimental values. Further data analysis reveals that the molecular mechanism of protein dimerization affects the ion-transport properties of gA channels, i.e., protein dimerization accelerates the permeation of potassium and sodium ions passing through the double-channel by adjusting the environment around gA protein (the distribution of phospholipid head groups, ions outside the channel, and bulk water), rather than directly adjusting the conformation of gA protein.     

Enhancement of spike coherence by the departure from Gaussian noise in a Hodgkin-Huxley neuron

Experimental study has shown that non-Gaussian noise exists in sensory systems like neurons. The departure from Gaussian behavior is a characteristic parameter of non-Gaussian noise. In this paper, we have numerically studied the effect of a particular kind of non-Gaussian colored noise (NGN), especially its departure q from Gaussian noise (q = 1), on the spiking activity in a deterministic Hodgkin-Huxley (HH) neuron driven by sub-threshold periodic stimulus. Simulation results show that the departure q can affect the spiking activity induced by noise intensity D. For smaller q values, the minimum in the variation coefficient (CV) as a function of noise intensity (D) becomes smaller, showing that D-induced stochastic resonance (SR) becomes strengthened. Meanwhile, depending on the value of D, q can either enhance or reduce the spiking regularity. Interestingly, CV changes non-monotonously with varying q and passes through a minimum at an intermediate q, representing the presence of “departure-induced SR”. This result shows that appropriate departures of the NGN can enhance the spike coherence in the HH neuron. Since the departure of the NGN determines the probability distribution and hence may denote the type of the noise, “departure-induced SR” shows that different types of noise can enhance the spike coherence, and hence may improve the timing precision of sub-threshold signal encoding in the HH neuron. Supported by the Science Foundation of Ludong University (L20072805)     

Spatial distribution of ion channel activity in biological membranes: the role of noise

A new approach is proposed to model a collective ion channel dynamics. We have assumed that ion channels create a two-component spatio-temporal interaction field. Every channel at its current spatial location in membrane contributes permanently to this field with its state (open or closed) and coupling strength to other channels. This field is described by a reaction-diffusion equation, the transition of ion channel from closed to open state (and vice versa) is described by a master equation, and migration of channels in membrane is described by a set of Langevin equations coupled by the interaction field. Within this model, we have investigated critical conditions for spatial distribution of ion channel activity.     

Synthesis and evaluation of novel α-aminoamides with substituted benzene scaffold for the treatment of neuropathic pain

Small molecule sodium ion channel blockers with a pharmacophore of α-aminoamide have exhibited anti-allodynia effects on neuropathic pain. A library of new α-aminoamide derivatives containing a scaffold of substituted benzene were designed and synthesized. These compounds were evaluated in mice formalin model and they exhibited significant analgesic activities. However, the anti-allodynia mechanism of these compounds remains unclear; some of the target compounds can only moderately inhibit the sodium ion channel, Nav1.7, in a whole-cell patch clamp assay. These results suggest that introduction of the moiety of substituted benzene to α-aminoamide derivatives can improve their bioactivity and further study is warranted.     

基于分子模拟方法预测PIP2在双孔钾通道TREK-1上结合位点的研究

磷脂酰肌醇4,5-二磷酸酯(PIP₂)是一类分布在质膜内层的信号磷脂分子, 对钾、 钠和氯等离子通道和转运蛋白等多种跨膜蛋白具有调节作用. TREK-1是一类重要的背景钾通道, 受温度、 机械拉伸及胞内pH等多种因素调节, PIP₂在特定浓度范围内可激活TREK-1通道, 在内面向外膜片钳记录TREK-1通道电流中使用PIP₂抗结剂(如多聚赖氨酸)可导致TREK-1通道关闭. 利用分子对接和全原子分子动力学模拟探索了PIP₂与双孔钾通道TREK-1的相互作用. 分子对接计算结果表明, PIP₂在TREK-1通道上有两个可能的结合位点. 进一步的分子动力学模拟和均力势(PMF)计算结果表明, 其中位于螺旋M4和螺旋M1的位点可能是PIP₂激活TREK-1的优先结合位点. 模拟展示了PIP₂与TREK-1结合的可能构象. PIP₂的肌醇头部磷酸根与位于M1和M4上的碱性残基K45, K304和R311形成稳定盐桥; M1螺旋上的一系列疏水残基对稳定PIP₂的脂肪长链具有关键作用.     

Epithelial Sodium Channel Inhibition by Amiloride Addressed with THz Spectroscopy and Molecular Modeling

THz spectroscopy is important for the study of ion channels because it directly addresses the low frequency collective motions relevant for their function. Here we used THz spectroscopy to investigate the inhibition of the epithelial sodium channel (ENaC) by its specific blocker, amiloride. Experiments were performed on A6 cells’ suspensions, which are cells overexpressing ENaC derived from Xenopus laevis kidney. THz spectra were investigated with or without amiloride. When ENaC was inhibited by amiloride, a substantial increase in THz absorption was noticed. Molecular modeling methods were used to explain the observed spectroscopic differences. THz spectra were simulated using the structural models of ENaC and ENaC—amiloride complexes built here. The agreement between the experiment and the simulations allowed us to validate the structural models and to describe the amiloride dynamics inside the channel pore. The amiloride binding site validated using THz spectroscopy agrees with previous mutagenesis studies. Altogether, our results show that THz spectroscopy can be successfully used to discriminate between native and inhibited ENaC channels and to characterize the dynamics of channels in the presence of their specific antagonist.     

高氯酸镱对大鼠背根神经元细胞毒性及膜上钾通道的影响

研究了高氯酸镱(Yb(ClO4)3)诱导大鼠背根神经(DRG)元凋亡、引起胞内钙离子浓度变化以及对膜上钾离子通道的影响.急性分离大鼠DRG细胞,用不同浓度的Yb(ClO4)3处理DRG细胞24和96h,采用流式细胞仪和激光共聚焦法,检测细胞的凋亡和细胞内钙离子荧光强度的变化.利用全细胞膜片钳法,记录Yb(ClO4)3对细胞膜上不同钾通道电流的影响.结果表明,10,100,1000μmol/L Yb(ClO4)3处理DRG神经元24h,细胞基本不表现凋亡;处理96h,细胞出现明显的凋亡(P0.05～0.01),尤其是1000μmol/L Yb(ClO4)3,凋亡率达到了(55.23±3.76)%(P0.01).经Yb(ClO4)3孵育的DRG神经元胞内的Ca2+的荧光强度显著增大;Yb(ClO4)3抑制背根神经节纤维和神经元突起的生长.Yb(ClO4)3抑制DRG神经元膜上的钾电流,胞内和胞外的Yb(ClO4)3作用钾通道的部位不同.细胞外液中的Yb(ClO4)3不同程度地阻断了瞬间外向钾电流IA,对延迟整流钾电流几乎没影响;往电极内液中加入同样浓度的Yb(ClO4)3对IA影响很小,却特异性地阻断了延迟整流钾电流IK.10μmol/L Yb(ClO4)3使IA的激活和失活过程都显著右移,延长了瞬间外向钾电流达到峰值的时间和快速失活时间常数,增加神经元的兴奋性.     

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca²⁺-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca²⁺-regulated potassium (mitoBK_Ca) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BK_Ca channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary β3-β4 subunits of BK_Ca channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately β3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBK_Ca channel in a bronchial epithelial cell line were described.     

Ion channel drug potency assay with an artificial bilayer chip

The potency of pharmaceutical compounds acting on ion channels can be determined through measurements of ion channel conductance as a function of compound concentration. We have developed an artificial lipid bilayer chip for simple, fast, and high-yield measurement of ion channel conductance with simultaneous solution perfusion. Here we show the application of this chip to the measurement of the mammalian cold and menthol receptor TRPM8. Ensemble measurements of TRPM8 as a function of concentration of menthol and 2-aminoethoxydiphenyl borate (2-APB) enabled efficient determination of menthol's EC(50) (111.8 μM ± 2.4 μM) and 2-APB's IC(50) (4.9 μM ± 0.2 μM) in agreement with published values. This validation, coupled with the compatibility of this platform with automation and parallelization, indicates significant potential for large-scale pharmaceutical ion channel screening.     

Micro‐ and Nano‐Technologies for Lipid Bilayer‐Based Ion‐Channel Functional Assays

Ion channel proteins provide gated pores that allow ions to passively flow across cell membranes. Owing to their crucial roles in regulating transmembrane ion flow, ion channel proteins have attracted the attention of pharmaceutical investigators as drug targets for use in the studies of both therapeutics and side effects. In this review, we discuss the current technologies that are used in the formation of ion channel‐integrated bilayer lipid membranes (BLMs) in microfabricated devices as a potential platform for next‐generation drug screening systems. Advances in BLM fabrication methodology have allowed the preparation of BLMs in sophisticated formats, such as microfluidic, automated, and/or array systems, which can be combined with channel current recordings. A much more critical step is the integration of the target channels into BLMs. Current technologies for the functional reconstitution of ion channel proteins are presented and discussed. Finally, the remaining issues of the BLM‐based methods for recording ion channel activities and their potential applications as drug screening systems are discussed.     

Spider venoms: a rich source of acylpolyamines and peptides as new leads for CNS drugs

Advances in NMR and mass spectrometry as well as in peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous products from spider venoms, previously explored due to technical limitations. The chemical composition of spider venoms is diverse, ranging from low molecular weight organic compounds such as acylpolyamines to complex peptides. First, acylpolyamines (< 1000 Da) have an aromatic moiety linked to a hydrophilic lateral chain. They were characterized for the first time in spider venoms and are ligand-gated ion channel antagonists, which block mainly postsynaptic glutamate receptors in invertebrate and vertebrate nervous systems. Acylpolyamines represent the vast majority of organic components from the spider venom. Acylpolyamine analogues have proven to suppress hippocampal epileptic discharges. Moreover, acylpolyamines could suppress excitatory postsynaptic currents inducing Ca+ accumulation in neurons leading to protection against a brain ischemic insult. Second, short spider peptides (< 6000 Da) modulate ionic currents in Ca2+, Na+, or K+ voltage-gated ion channels. Such peptides may contain from three to four disulfide bridges. Some spider peptides act specifically to discriminate among Ca2+, Na+, or K+ ion channel subtypes. Their selective affinities for ion channel subfamilies are functional for mapping excitable cells. Furthermore, several of these peptides have proven to hyperpolarize peripheral neurons, which are associated with supplying sensation to the skin and skeletal muscles. Some spider N-type calcium ion channel blockers may be important for the treatment of chronic pain. A special group of spider peptides are the amphipathic and positively charged peptides. Their secondary structure is alpha-helical and they insert into the lipid cell membrane of eukaryotic or prokaryotic cells leading to the formation of pores and subsequently depolarizing the cell membrane. Acylpolyamines and peptides from spider venoms represent an interesting source of molecules for the design of novel pharmaceutical drugs.     

Potential analytical applications of gated artificial ion channels

Biosensors based on natural ion channels combine a biological recognition mechanism with a physical transduction technique in a very selective and sensitive manner. This kind of molecular sensor will contribute to drug screening and environmental screening. Key information about channel gating, ion transport, and molecular mechanism is provided by the patch-clamp technique, commonly used for electrophysiological analysis. Here we report the synthesis of light-gated artificial ion channels, necessary constituents for construction of biosensors based on natural ion channels. The artificial gated ion channels described here are based on calix[4]resorcinarene. Opening and closing of the artificial ion channel is achieved by azo groups, which work like a lid. Azo groups alter their conformation on irradiation with light, and are chemically quite stable. Addition of a gate function will enhance the potential of synthetic channels to be used in sensors as molecular switches.Dedicated to the memory of Wilhelm Fresenius     

Ion channel screening

Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the G protein coupled and nuclear hormone receptor families. A historical limitation on ion channel targeted drug discovery in the form of the extremely low throughput nature of the gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, has been overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. These have taken advantage of various approaches to monitor ion flux or membrane potential using radioactive, non-radioactive, spectroscopic and fluorescence measurements and have significantly impacted both high-throughput screening and lead optimization efforts. In addition, major advances have been made in the development of automated electrophysiological platforms to increase capacity for cell-based screening using formats aimed at recapitulating the gold standard assay. This review addresses the options available for cell-based screening of ion channels with examples of their utility and presents case studies on the successful implementation of high-throughput screening campaigns for a ligand-gated ion channel using a fluorescent calcium indicator, and a voltage-gated ion channel using a fluorescent membrane potential sensitive dye.     

Study of the interaction of unaggregated and aggregated amyloid β protein (10-21) with outward potassium channel

Metal ion-induced aggregation of Aβinto insoluble plaques is a central factor in Alzheimer's disease. Zn2+ is the only physiologically available transition metal ion responsible for aggregating Aβ at pH 7.4. To make it clear that the neurotoxicity of Zn2+-induced aggregation of Aβ on neurons is the key to understand Aβ mechanism of action further. In this paper, we choose Aβ (10-21) as the model fragment to research hippocampal CA1 pyramidal neurons. For the first time, we adopt the combination of spectral analysis with patch-clamp technique for the preliminary study of the mutual relations of Zn2+, Aβ and ion channel from the cell level. The following expounds upon the effects and mode of action of two forms (unaggregated and aggregated) of Aβ (10-21) on hippocampus outward potassium channel three processes (activation, inactivation and reactivation). It also shows the molecular mechanics of AD from the channel level. These results are significant for the further study of Aβ nosogenesis and the development of new types of target drugs for the treatment of AD.     

High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device

Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.