双分子层膜人工离子通道的合成

离子通道(ion channels)是由细胞膜上的一类特殊亲水性蛋白质构成的微孔道,它的主要功能就是传输离子跨膜,相当于细胞的通气孔。其结构与功能的异常往往引起上千种疾病,统称为离子通道病,这种疾病目前不能靠常规的仪器来检查,在确诊上有一定的难度。因此通过化学手段合成人工离子通道来模拟生物体内细胞膜上的离子通道的结构与功能,对于深入研究这些疾病并发现特异性治疗药物均具有十分重要的理论和实际意义。本论文就近三十年来人们设计合成的不同种类人工离子通道进行了综述,介绍了其研究进展并总结了各种人工离子通道的分子结构设计以及在膜上传输离子行为,展望了其在模拟天然离子通道功能的同时在生物医药以及生命科学等领域的应用前景。     

Characteristics of Single Ionic Channels Induced by Sulfonic Acid Derivatives of Dibenzo-18-Crown-6 in Bilayers

Sulfonic acid derivatives of dibenzo-18-crown-6 (DB18C6) have been shown to induce formation of ion channels permeable to monovalent cations in bilayer lipid membranes (BLM). Some electric characteristics of channels have been studied by means of the voltage clamp method. Channels displayed little interionic discrimination. The voltage dependence as well as the multiple state behaviors of the channels functioning was observed. The channels formation has been shown to be dependent on pH of bath solutions and on the presence of bivalent cations in them. The channels supposed to be formed from aggregates of complexes associates assembled in aqueous salt solutions.     

Rectification of the current in alpha-hemolysin pore depends on the cation type: the alkali series probed by MD simulations and experiments

A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel's end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.     

Observing a model ion channel gating action in model cell membranes in real time in situ: membrane potential change induced alamethicin orientation change

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split α/3(10)-helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer and was measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH = 6.7 the α-helix at the N-terminus and the 3(10)-helix at the C-terminus tilt at about 72° (θ(1)) and 50° (θ(2)) versus the surface normal, respectively. When pH increases to 11.9, θ(1) and θ(2) decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ(1) for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and the bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.     

AC conductance of transmembrane protein channels. The number of ionized residue mobile counterions at infinite dilution

Simultaneous measurements of the AC and DC conductances of alpha-hemolysin (alphaHL) ion channels and outer membrane protein F (OmpF) porins in dilute ionic solutions is described. AC conductance measurements were performed by applying a 10 mV rms AC voltage across a suspended planar bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the absence and presence of the protein and detecting the AC current response using phase-sensitive lock-in techniques. The conductances of individual alphaHL channels and OmpF porins were measured in symmetric KCl solutions containing between 5 and 1000 mM KCl. The AC and DC conductances of each protein were in agreement for all solution conditions, demonstrating the reliability of the AC method in single-channel recordings. Linear plots of conductance versus bulk KCl concentration for both proteins extrapolate to significant nonzero conductances (0.150 +/- 0.050 nS and 0.028 +/- 0.008 nS for OmpF and alphaHL, respectively) at infinite KCl dilution. The infinite dilution conductances are ascribed to mobile counterions of the ionizable residues within the protein lumens. A method of analyzing the plots of conductance vs KCl concentration is introduced that allows the determination of the concentration of mobile counterions associated with ionizable groups without knowledge of either the protein geometry or the ion mobilities. At neutral pH, an equivalent of 3 mobile counterions (K+ or Cl-) is estimated to contribute to the conductivity of the alphaHL channel.     

Ion channel formation from a calix[4]arene amide that binds HCl

The ion transport activity of calix[4]arene tetrabutylamide 1,3-alt 2 was studied in liposomes, planar lipid bilayers, and HEK-293 cells. These experiments, when considered together with (1)H NMR and X-ray crystallography data, indicate that calix[4]arene tetrabutylamide 2 (1) forms ion channels in bilayer membranes, (2) mediates ion transport across cell membranes at positive holding potential, (3) alters the pH inside liposomes experiencing a Cl(-) gradient, and (4) shows a significant Cl(-)/SO(4)(2)(-) transport selectivity. An analogue, calix[4]arene tetramethylamide 1, self-assembles in the presence of HCl to generate solid-state structures with chloride-filled and water-filled channels. Structureminus signactivity studies indicate that the hydrophobicity, amide substitution, and macrocyclic framework of the calixarene are essential for HCl binding and transport. Calix[4]arene tetrabutylamide 2 is a rare example of an anion-dependent, synthetic ion channel.     

Ion permeability through bilayer lipid membranes for biosensor development: control by chemical modification of interfacial regions between phase domains.

Based on the results of studies on cystic fibrosis, which implicated hydroxystearic acid (HSA) as a contributing factor in altered biomembrane function, solvent-free bilayer lipid membranes (BLMs) and monolayer films were prepared from a lipid mixture containing (by mass) 34% phosphatidylcholine, 19% dipalmitoylphosphatidyl serine, 47% cholesterol and variable amounts of 10- and 12-HSA (0-50%). Ion currents, resulting from K+ permeation through BLMs that were supported in 0.1 mol dm-3 KCl solutions buffered to pH 7.4, were monitored with use of a d.c. circuit. The structures of monolayer films at the air-water interface of a Langmuir-Blodgett trough were studied by pressure-area correlations and by further correlation with microscopic phase separation as revealed by fluorescence microscopy. In order to elucidate the role of the hydroxyl moieties in ion permeability, the transmembrane ion current was corrected for the effect of the negative surface charge of the carboxylic acid by replacement of the HSA component with stearic acid. The ion current was found to increase with the molar proportion of the HSAs. Two models for ion conduction through BLMs were considered: 'hopping' via hydrophilic sites within the hydrophobic zone of the BLMs, introduced by the hydroxyl moiety of 10- or 12-HSA; and transport through interfacial regions between phase domains that represent areas of low steric density and low structural order within monolayers. Although the two mechanisms are not distinct, the ion permeability results indicate a change in the response of ion current to HSA concentration at 35 mol-%, suggesting a change in the relative proportion of the mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence microscopy of the pressure-dependent structure of lipid bilayers suspended across conical nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.     

Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5-Ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5, 5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-1, 3-dioxane bromide (DTDB), was used toconstruct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochem-ical impedance spectroscopy (EIS), small-angle X-ray diffrac-tion (SAXD) and cyclic voltammetry were used to characterizethe s-BLM. Both EIS and SAXD data indicated that the syn-thetic lipid exists as a well-oriented bilayer in the membrane.     

Differences in ion-channel formation by ampullosporins B,C, D and semisynthetic desacetyltryptophanyl ampullosporin A

Peptaibol-type ampullosporins B (2) and D (4) are capable of forming ion-conducting pores in planar lipid bilayer membrane prepared from soybean phosphatidylcholine in a similar manner as it was shown for ampullosporin A (1). However, the isomeric ampullosporin C (3) was required in 20-fold higher concentration to afford a comparable effect. In contrast to 1, 2, 3 and 4, the desacetyltryptophanyl ampullosporin A (5) failed to form ion channels. The results suggest that the sequence of amino acids especially at positions 8-10, the nitrogen-terminal acetyl residues and tryptophane are major factors determining ion-channel formation within bilayer membranes. The differences in membrane activities were comparable to the observed biological activities.     

Acid-Containing Tyrosine-Derived Polycarbonates: Wettability and Surface Reactivity

Tyrosine-derived polycarbonates having carboxylic acid pendant groups were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). A pronounce decrease of receding angle as well as contact angle hysteresis as a function of acid composition strongly indicated that the acid groups are more accessible at the water/polymer interface after hydration. pH dependent contact angle confirmed an existence of carboxylic acid groups in the surface region. The receding angle transition appearing in the pH range of 4-6 was a consequence of hydrophilicity change due to interconverting from carboxylic acid (-COOH) to carboxylate ion (-COO⁻). The surface compositions of imidazole-labeled polymers as analyzed by XPS were consistent with the bulk stoichiometry of the polymers. Reactivity of acid groups towards chemical reaction at the surface was also investigated. The acid groups at the surface of polymers were capable of adsorbing a significant amount of calcium ion from simulated body fluid and being activated by a reaction with N-hydroxysuccinimide.     

Transmembrane ion channels constructed of cholic acid derivatives.

A new class of supramolecular transmembrane ion channels was prepared by linking two amphiphilic cholic acid methyl ethers through biscarbamate bonds to afford bis(7,12-dimethyl-24-carboxy-3-cholanyl)-N,N'-xylylene dicarbamate 2 and bis[7,12-dimethyl-24-(N,N,N-trimethylethanaminium-2-carboxylate)-3-cholanyl]-N,N'-xylylene dicarbamate dichloride 3. When incorporated into a planar bilayer membrane, both compounds showed stable (lasting 10 ms to 10 s) single ion channel currents. Only limited numbers of relatively small conductances were characterized for these channels (5-20 pS for 2 and 5-10 pS for 3, 10 and 17 pS for 2, and 9 pS for 3 in particular). Both channels were cation selective, and permeability ratios of potassium cation to chloride anion were 17 and 7.9 for 2 and 3, respectively, reflecting the difference in ionic species of the headgroup. Both channels 2 and 3 showed significant potassium selectivity over sodium by a factor of 3.1 and 3.2, respectively. No Li(+) currents were observed for 2, showing sharp discrimination between Na(+) or K(+).     

Alkylated glass partition allows formation of solvent-free lipid bilayer by Montal-Mueller technique

Formation of bilayer lipid membrane (BLM) by Montal-Mueller technique across a small aperture in a partition film traditionally requires coating of the aperture with a hydrophobic substance, often just an organic solvent. However, we demonstrate here that the most effective coating is not strictly hydrophobic but rather provides water/oil repellent properties. BLM were formed from diphytanoylphosphatidylcholine (DPhPC) on small 0.1-0.8 mm apertures made in specially prepared alkylated glass coverslips. The coverslips were either fluorosiliconized by 3,3,3-Trifluoropropyl-trimethoxysilane, which reduces adsorption of DPhPC in addition to creation of hydrophobic surface, or silanized, which promote adsorption of DPhPC. At fluorosiliconized surfaces stable BLM were formed. Specific capacitance of these BLM was 0.86 microF/cm(2)+/-5%, while their lateral tension was estimated as 4.3+/-0.4 mN/m. BLM were stable for hours under moderate voltage applied. At silanized surfaces stable BLM were formed only in acidic medium (3 相似文献   

Preparation and evaluation of an imidazole-coated capillary column for the electrophoretic separation of aromatic acids

An imidazole-coated capillary column for electrophoresis has been prepared by means of organosilanization. With mesityl oxide as neutral marker, the results indicated that the electroosmotic flow of the bonded phase displays a dramatic difference in pH dependence in comparison with that of the bare fused-silica column. The presence of positive charges on the coating surface and the anionic exchange property, due to the cationic property of the imidazole group at pH values below 6, allows the separation of geometric isomers that are very similar in ionic mobility. Separation parameters including buffer composition and concentration, pH, applied voltage, and the influence of other additives were investigated. By using acetate buffer (100 mM, pH 5.2) and an applied voltage of -15 kV with UV detection at 212 nm, the separation of 11 aromatic acids including mono-, di-, tri- and tetra-carboxylic acids could be achieved in less than 14 min. The average plate number was 3 x 10(5)/m. With acetate buffer (25 mM, pH 5.5) and an applied voltage of -25 kV, the addition of silver nitrate or beta-cyclodextrin significantly improved the resolution of some more highly charged carboxylic acids.     

Voltage control of droplet interface bilayer lipid membrane dimensions

A novel approach to control the area of anchor-free droplet interface bilayer (DIB) lipid membranes is presented. Unsupported DIB lipid membranes are formed at the interface of phospholipid-coated aqueous droplets dispensed in dodecane oil. Using electrodes inserted into the droplets, an external voltage is applied which modulates the effective DIB area. Electrical (capacitance or current) and optical (imaging of DIB lateral length) recordings were simultaneously performed. Alpha-hemolysin (αHL) single channel insertions into the DIB were recorded. Currents across the DIB were measured as a function of voltage and αHL concentration in the droplets. Nonlinear response is observed for current, DIB lateral length and area, and capacitance with respect to voltage. Voltage induced changes in interfacial tension modulated the DIB-oil contact angle and the membrane contact length, which provided control of membrane dimensions. Comparison of these results is made to the electrowetting effect, which is also governed by effect of voltage on the interfacial tension. This approach provides active control of the number of ion channels inserted into the DIB.     

充蜡石墨支撑的季铵离子/己二酸混合双层膜上钙离子诱发的离子通道行为

以充蜡石墨电极作为新型支撑体,成功制备了一种季铵离子为内层（包括四丁 基铵TBA,十六烷基三甲基铵CTrMA）,己二酸（HDA）为外层的新型自组装混合双 层膜,以循环伏安和电化学交流阻抗方法研究了膜的离子通道行为。该膜能够接受 Ca~(2+)的刺激作用而打开[Fe(CN)_6]~(3-/4-)电极氧化还原的离子通道,撤走该 刺激离子则通道关闭。提出了混合双层膜的结构和离子通道作用的模型,指出外层 膜HDA分子可能具有V型和W型两种结构。     

Local anesthetics facilitate ion transport across lipid planar bilayer membranes under an electric field: dependence on type of lipid bilayer

In order to elucidate the role of structural change of lipid membrane bilayer in the mode of action of local anesthetic, we studied the effects of local anesthetics, charged tetracaine and uncharged benzocaine, on ion permeability across various lipid planar bilayers (PC, mixed PC/PS (4/1, mol/mol); mixed PC/PE (1/1, mol/mol); mixed PC/SM (4/1, mol/mol)) under a constant applied voltage. The membrane conductances increased in the order of PC  PC/PS ≤ PC/SM  PC/PE. When the constant voltage of −100 or −70 mV was applied through the lipid bilayer membranes in the presence of positively charged tetracaine, the fluctuating current pulses with the large amplitude generated, but not appeared in the absence of tetracaine. The addition of uncharged benzocaine generated the fluctuating currents with the small amplitude. Both charged tetracaine and uncharged benzocaine facilitated electrophoretically the transport of small ions such as KCl in the buffer solution through the fluctuating pores in the lipid bilayer membranes formed by interaction with the local anesthetic under the negative applied membrane potential. The current pulses also contained actual transport of charged tetracaine together with the transport of the small ions. The amplitude and the duration time of the electrical current generated by adding the local anesthetics were dependent on the type of the lipid, the applied voltage and its voltage polarity.     

Ion diode logics for pH control

Electronic control over the generation, transport, and delivery of ions is useful in order to regulate reactions, functions, and processes in various chemical and biological systems. Different kinds of ion diodes and transistors that exhibit non-linear current versus voltage characteristics have been explored to generate chemical gradients and signals. Bipolar membranes (BMs) exhibit both ion current rectification and water splitting and are thus suitable as ion diodes for the regulation of pH. To date, fast switching ion diodes have been difficult to realize due to accumulation of ions inside the device structure at forward bias--charges that take a long time to deplete at reverse bias. Water splitting occurs at elevated reverse voltage bias and is a feature that renders high ion current rectification impossible. This makes integration of ion diodes in circuits difficult. Here, we report three different designs of micro-fabricated ion bipolar membrane diodes (IBMDs). The first two designs consist of single BM configurations, and are capable of either splitting water or providing high current rectification. In the third design, water-splitting BMs and a highly-rectifying BM are connected in series, thus suppressing accumulation of ions. The resulting IBMD shows less hysteresis, faster off-switching, and also a high ion current rectification ratio as compared to the single BM devices. Further, the IBMD was integrated in a diode-based AND gate, which is capable of controlling delivery of hydroxide ions into a receiving reservoir.     

A study of the mechanism of response of liquid ion exchanger calcium selective electrodes: Part III. Membrane behaviour under non-zero current conditions

The I-E response of the liquid membrane of the calcium selective electrode is studied under constant or linearly varying current and voltage. An increase in the membrane resistance, recorded when an electrical current crosses the membrane, is due to the outflow of Cl^? ions initially present in the membrane. When calcium ions are replaced by alkaline ions inside the membrane at constant current, the decrease of the membrane resistance due to an ion exchange is in agreement with the conductivity measurements (Part II). When the applied voltage is imposed besides the ion exchange one must take into account the interfacial overpotential to explain the important rectification effect observed. The interfacial transfer constant rate of alkaline ions seems greater than that of Ca²⁺ ion.     

Heterogeneous Electrospinning Nanofiber Membranes with pH-regulated Ion Gating for Tunable Osmotic Power Harvesting

Biological ion channels existing in organisms are critical for many biological processes. Inspired by biological ion channels, the heterogeneous electrospinning nanofiber membranes (HENM) with functional ion channels are constructed by electrospinning technology. The HENM successfully realizes ion-gating effects, which can be used for tunable energy conversions. Introduction of pyridine and carboxylic acid groups into the HENM plays an important role in generating unique and stable ion transport behaviors, in which gates become alternative states of open and close, responding to symmetric/asymmetric pH stimulations. Then we used the HENM to convert osmotic energy into electric energy which reach a maximum value up to 12.34 W m⁻² and the output power density of HENM-based system could be regulated by ion-gating effects. The properties of the HENM provide widespread potentials in application of smart nanofluidic devices, energy conversion, and water treatment.