A 0-Memory Model for Single Ion Channel

This paper discusses a 0-memory model for a single ion channel. The renewal rates of the open-class and the close-class are proposed to deseribe kinetic properties of a single ion channel. Further more, a procedure to estimate the parameters in the model is suggested and illustrated with examples in pharmacology.     

Hill Coefficients of a Polymodal Monod-Wyman-Changeux Model for Ion Channel Gating

Allosteric transitions of ion channels can be driven by multiple sources of free energies. One class of model for describing such transitions is the multistimulus Monod-Wyman-Changeux model, in which each stimulus interacts with a specific sensor on the protein and activation of the sensor is allosterically coupled to conformational changes of the protein. In general, when a protein is stressed by multiple stimuli, one stimulus can influence the response to another, which can result in both a shift of the midpoint of the dose-response curve and a change of the slope of the curve. Here I show that, for a Monod-Wyman-Changeux model with independent sensors, the different dose-response curves of open probability for one stimulus have the same slope at the same agonist concentration. In the other words, the slope of the dose-response curve for one stimulus is an intrinsic property of the sensors for that stimulus; it is independent of other stimuli or their sensor properties. As the dose-response curve for many receptors can be fit to a Boltzmann or Hill equation, this property provides a practical, usable test for applicability of such models.     

Design of an Efficient Inhibitor for the Influenza A Virus M2 Ion Channel

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H+ proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.     

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.     

多囊蛋白2离子通道功能及在常染色体显性多囊肾发病中的作用机制

多囊蛋白2 (polycystin-2,PC2,或称TRPP2,PKD2)是一种瞬时受体电位通道(transient receptor potential channel,TRP),在维持细胞正常的Ca²⁺信号传导中起着关键作用,也是最常见的单基因常染色体显性遗传多囊肾病(transient receptor potential channel,ADPKD)的潜在病因之一。PC2可自身组装为同源四聚体离子通道或与其他蛋白质形成异源受体-离子通道复合物,参与调节机械感觉、细胞极性、细胞增殖和凋亡等多种生理功能,导致囊性细胞从正常的吸收、静止状态转变为病理性分泌、增殖状态。本文阐述了PC2蛋白相关结构域以及通道特性在维持细胞内Ca²⁺信号传导中的关键作用,并总结了PC2在细胞膜、纤毛、内质网以及线粒体等特定亚细胞定位形成多囊蛋白复合物,参与多种细胞分化、增殖、存活和凋亡相关信号通路,为确定特异性的有效的ADPKD干预治疗途径和靶点药物提供新的思考方向。     

Ion and pH Sensitivity of a TMBIM Ca2+ Channel

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A General Channel Model Accounts for Channel,Carrier, Countertransport and Cotransport Kinetics

In this work we propose a unifying model of mediated membrane transport, based upon the idea that the integral membrane proteins involved in these processes operate via complex channel mechanisms. In the first part, we briefly review literature about the structural aspects of membrane transporters. We conclude that there is a substantial amount of evidence suggesting that most membrane proteins performing transport are embodied with channel-like structures that may constitute the translocation paths. This includes cases where the phenomenological transport kinetics do not correspond to the classical channel behavior. In the second part of this article we introduce the general channel model of mediated transport and employ it to derive specific examples, like simple one- or two-ligand channels, water-ligand channels, simple carriers, co- and counter-transport systems and more complex water-ligand carriers. We show that, for the most part, these particular cases can be obtained by the application of the techniques of diagram reduction to the full model. The necessary conditions for diagram reduction reflect physical properties of the protein and its surroundings.     

A Stored Charge Model for the Sodium Channel

A new model is proposed to account for the apparent conductance changes of the sodium, or early, channel in nerve fiber membranes. In this model it is assumed that the channels are gated at the interior side of the membrane and are resistively limited at the exterior side by sodium selective barriers of high resistance to ion flow. Under resting conditions the closed channels accumulate a store of sodium ions, dependent on the exterior sodium concentration. With the application of a depolarizing clamp the interior gates open allowing the stored ions to discharge into the interior low sodium concentration solution. In this model the initial rise in the early current results from the opening of more and more gates in response to the depolarizing clamp. The subsequent fall in the early current results from the “capacitative” discharge of the opened channels, limited by the high resistive barrier at the exterior end. Upon repolarization, the gates reclose and sodium ions reaccumulate in the channels from the high concentration external solution, but at a slow rate determined by the resistive barrier. Preliminary tests of this model, using a number of simplifying assumptions, show that it has the ability to account, at least semiquantitatively, for the major characteristics of the experimental clamp results.     

Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT₃) or anion selective (e.g., GlyR, GABA_A and GluCl) membrane channels. Among others, 5-HT₃ is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT₃ is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT₃A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT₃A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT₃A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT₃A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested.     

Ion Permeation and Block of P2X2 Purinoceptors: Single Channel Recordings

P2X₂ purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG⁺, Tris⁺, TMA⁺, and TEA⁺. The selectivity sequence for currents carried by alkali metal ions is: K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG⁺, Tris⁺, TMA⁺ and TEA⁺ were virtually impermeant. The divalent ions (Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn²⁺ > Mg²⁺ > Ca²⁺ > Ba²⁺. Received: 3 May 1999/Revised: 23 August 1999     

Launching Channels: A New Milestone in the Ion Channel Field

Ion channels underlie a plethora of physiological functions not only in the animal kingdom, but also in plants and microorganisms such as bacteria. Even though we have only known of the existence of channels for about four decades, a PubMed search for channels yields over 120,000 papers, with 40,000 of those appearing in the past five years alone. Even before ion channels had been formally discovered, their existence was hypothesized by Hodgkin and Huxley who were awarded the Nobel Prize in Physiology/Medicine for their work on electrical activity in axons. Subsequent Nobel awards for ion channel electrophysiology techniques (Bert Sakmann and Erwin Neher for Medicine in 1991) and ion channel structure and physiology (Rod MacKinnon and Peter Agre for Chemistry in 2003) underscored the contemporary importance of ion channel research. It is noteworthy that single channel recording is one of the most sensitive techniques in biology – allowing researchers to study the function of a single molecule in its native environment in real time.     

Kernel Estimates for One- and Two-Dimensional Ion Channel Dwell-Time Densities

In this paper, we compare nonparametric kernel estimates with smoothed histograms as methods for displaying logarithmically transformed dwell-time distributions. Kernel density plots provide a simpler means for producing estimates of the probability density function (pdf) and they have the advantage of being smoothed in a well-specified, carefully controlled manner. Smoothing is essential for multidimensional plots because, with realistic amounts of data, the number of counts per bin is small. Examples are presented for a 2-dimensional pdf and its associated dependency-difference plot that display the correlations between successive dwell times.     

电导检测低压离子色谱法分析甘氨酸

采用低压离子色谱电导检测氨基酸分析方法,对甘氨酸含量进行了测定,实验结果表明采用该方法测定甘氨酸,标准偏差为4.72%(测定次数n=7),回收率分别为94.75%和97.30%,,表明用该方法分析样品中的甘氨酸含量准确度高,精密度好。     

Kinetic Analysis of Ca2+/K+ Selectivity of an Ion Channel by Single-Binding-Site Models

Current-voltage relationships of a cation channel in the tonoplast of Beta vulgaris, as recorded in solutions with different activities of Ca²⁺ and K⁺ (from Johannes & Sanders 1995, J. Membrane Biol. 146:211–224), have been reevaluated for Ca²⁺/K⁺ selectivity. Since conversion of reversal voltages to permeability ratios by constant field equations is expected to fail because different ions do not move independently through a channel, the data have been analyzed with kinetic channel models instead. Since recent structural information on K⁺ channels show one short and predominant constriction, selectivity models with only one binding site are assumed here to reflect this region kinetically. The rigid-pore model with a main binding site between two energy barriers (nine free parameters) had intrinsic problems to describe the observed current-saturation at large (negative) voltages. The alternative, dynamic-pore model uses a selectivity filter in which the binding site alternates its orientation (empty, or occupied by either Ca²⁺ or K⁺) between the cytoplasmic side and the luminal side within a fraction of the electrical distance and in a rate-limiting fashion. Fits with this model describe the data well. The fits yield about a 10% electrical distance of the selectivity filter, located about 5% more cytoplasmic than the electrical center. For K⁺ translocation, reorientation of the unoccupied binding site (with a preference of about 6:5 to face the lumenal side) is rate limiting. For Ca²⁺, the results show high affinity to the binding site and low translocation rates (<1% of the K⁺ translocation rate). With the fitted model Ca²⁺ entry through the open channel has been calculated for physiological conditions. The model predicts a unitary open channel current of about 100 fA which is insensitive to cytoplasmic Ca²⁺ concentrations (between 0.1 and 1 μm) and which shows little sensitivity to the voltage across the tonoplast. Received: 19 February 1997/Revised: 19 May 1997     

Exhaled Breath Analysis for Lung Cancer Detection Using Ion Mobility Spectrometry

Background

Conventional methods for lung cancer detection including computed tomography (CT) and bronchoscopy are expensive and invasive. Thus, there is still a need for an optimal lung cancer detection technique.

Methods

The exhaled breath of 50 patients with lung cancer histologically proven by bronchoscopic biopsy samples (32 adenocarcinomas, 10 squamous cell carcinomas, 8 small cell carcinomas), were analyzed using ion mobility spectrometry (IMS) and compared with 39 healthy volunteers. As a secondary assessment, we compared adenocarcinoma patients with and without epidermal growth factor receptor (EGFR) mutation.

Results

A decision tree algorithm could separate patients with lung cancer including adenocarcinoma, squamous cell carcinoma and small cell carcinoma. One hundred-fifteen separated volatile organic compound (VOC) peaks were analyzed. Peak-2 noted as n-Dodecane using the IMS database was able to separate values with a sensitivity of 70.0% and a specificity of 89.7%. Incorporating a decision tree algorithm starting with n-Dodecane, a sensitivity of 76% and specificity of 100% was achieved. Comparing VOC peaks between adenocarcinoma and healthy subjects, n-Dodecane was able to separate values with a sensitivity of 81.3% and a specificity of 89.7%. Fourteen patients positive for EGFR mutation displayed a significantly higher n-Dodecane than for the 14 patients negative for EGFR (p<0.01), with a sensitivity of 85.7% and a specificity of 78.6%.

Conclusion

In this prospective study, VOC peak patterns using a decision tree algorithm were useful in the detection of lung cancer. Moreover, n-Dodecane analysis from adenocarcinoma patients might be useful to discriminate the EGFR mutation.     

The Proapoptotic Influenza A Virus Protein PB1-F2 Forms a Nonselective Ion Channel

Background

PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization.

Methodology/Principal Findings

Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAV_PR8), from A/Brevig Mission/1/1918(H1N1) (IAV_SF2) or the H5N1 consensus sequence (IAV_BF2) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns.

Conclusion/Significance

The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development.     

Improved Skin Test for Detection of T-2 Toxin

A modified rat skin test based on dermatitic properties of trichothecenes is described which is quick, convenient, and sensitive to 0.05 mug of T-2 toxin.