An efficient approach for the prediction of ion channels and their subfamilies

Ion channels are integral membrane proteins that are responsible for controlling the flow of ions across the cell. There are various biological functions that are performed by different types of ion channels. Therefore for new drug discovery it is necessary to develop a novel computational intelligence techniques based approach for the reliable prediction of ion channels families and their subfamilies. In this paper random forest based approach is proposed to predict ion channels families and their subfamilies by using sequence derived features. Here, seven feature vectors are used to represent the protein sample, including amino acid composition, dipeptide composition, correlation features, composition, transition and distribution and pseudo amino acid composition. The minimum redundancy and maximum relevance feature selection is used to find the optimal number of features for improving the prediction performance. The proposed method achieved an overall accuracy of 100%, 98.01%, 91.5%, 93.0%, 92.2%, 78.6%, 95.5%, 84.9%, MCC values of 1.00, 0.92, 0.88, 0.88, 0.90, 0.79, 0.91, 0.81 and ROC area values of 1.00, 0.99, 0.99, 0.99, 0.99, 0.95, 0.99 and 0.96 using 10-fold cross validation to predict the ion channels and non-ion channels, voltage gated ion channels and ligand gated ion channels, four subfamilies (calcium, potassium, sodium and chloride) of voltage gated ion channels, and four subfamilies of ligand gated ion channels and predict subfamilies of voltage gated calcium, potassium, sodium and chloride ion channels respectively.     

Natural products as tools for studies of ligand-gated ion channels

Ligand-gated ion channels, or ionotropic receptors, constitute a group of membrane-bound proteins that regulate the flux of ions across the cell membrane. In the brain, ligand-gated ion channels mediate fast neurotransmission. They are crucial for normal brain function and involved in many diseases in the brain. Historically, natural products have been used extensively in biomedical studies and ultimately as drugs or leads for drug design. In studies of ligand-gated ion channels, natural products have been essential for the understanding of their structure and function. In the following a short survey of natural products and their use in studies of ligand-gated ion channels is given.     

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。     

Negative‐ion/positive‐ion coincidence spectroscopy as a tool to identify anionic fragments: The case of core‐excited CHF3

We have studied the dissociation of the trifluoromethane molecule, CHF₃, into negative ionic fragments at the C 1s and F 1s edges. The measurements were performed by detecting coincidences between negative and positive ions. We observed five different negative ions: F^?, H^?, C^?, CF^?, and F₂^?. Their production was confirmed by the analysis of triple coincidence events (negative‐ion/positive‐ion/positive‐ion or NIPIPI coincidences) that were recorded with cleaner signals than those of the negative‐ion/positive‐ion coincidences. The intensities of the most intense NIPIPI coincidence channels were recorded as a function of photon energy across the C 1s and F 1s excitations and ionization thresholds. We also observed dissociation channels involving the formation of one negative ion and three positive ions. Our results demonstrate that negative‐ion/positive‐ion coincidence spectroscopy is a very sensitive method to observe anions, which at inner‐shell edges are up to three orders of magnitude less probable dissociation products than cations.     

Impact of novel screening technologies on ion channel drug discovery

Ion channels are a large superfamily of membrane proteins that pass ions across membranes. They are critical to diverse physiological functions in both excitable and nonexcitable cells and underlie many diseases. As a result, they are an important target class which is proven to be highly "druggable". However, for high throughput screening (HTS), ion channels are historically difficult as a target class due to their unique molecular properties and the limitations of assay technologies that are HTS-amendable. In this article, we describe the background of ion channels and current status and challenges for ion channel drug discovery, followed by an overview of both conventional and newly emerged ion channel screening technologies. The critical impact of such new technologies on current and future ion channel drug discovery is also discussed.     

Optimized precursor ion selection for labile ions in a linear ion trap mass spectrometer and its impact on quantification using selected reaction monitoring

The fragmentation of fragile ions during the application of an isolation waveform for precursor ion selection and the resulting loss of isolated ion intensity is well‐known in ion trap mass spectrometry (ITMS). To obtain adequate ion intensity in the selected reaction monitoring (SRM) of fragile precursor ions, a wider ion isolation width is required. However, the increased isolation width significantly diminishes the selectivity of the channels chosen for SRM, which is a serious problem for samples with complex matrices. The sensitive and selective quantification of many lipid molecules, including ceramides from real biological samples, using a linear ion trap mass spectrometer is also hindered by the same problem because of the ease of water loss from protonated ceramide ions. In this study, a method for the reliable quantification of ceramides using SRM with near unity precursor ion isolation has been developed for ITMS by utilizing alternative precursor ions generated by in‐source dissociation. The selected precursor ions allow the isolation of ions with unit mass width and the selective analysis of ceramides using SRM with negligible loss of sensitivity. The quantification of C18:0‐, C24:0‐ and C24:1‐ceramides using the present method shows excellent linearity over the concentration ranges from 6 to 100, 25 to 1000 and 25 to 1000 nM, respectively. The limits of detection of C18:0‐, C24:0‐ and C24:1‐ceramides were 0.25, 0.25 and 5 fmol, respectively. The developed method was successfully applied to quantify ceramides in fetal bovine serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Simulating ion channel activation mechanisms using swarms of trajectories

Atomic-level studies of protein activity represent a significant challenge as a result of the complexity of conformational changes occurring on wide-ranging timescales, often greatly exceeding that of even the longest simulations. A prime example is the elucidation of protein allosteric mechanisms, where localized perturbations transmit throughout a large macromolecule to generate a response signal. For example, the conversion of chemical to electrical signals during synaptic neurotransmission in the brain is achieved by specialized membrane proteins called pentameric ligand-gated ion channels. Here, the binding of a neurotransmitter results in a global conformational change to open an ion-conducting pore across the nerve cell membrane. X-ray crystallography has produced static structures of the open and closed states of the proton-gated GLIC pentameric ligand-gated ion channel protein, allowing for atomistic simulations that can uncover changes related to activation. We discuss a range of enhanced sampling approaches that could be used to explore activation mechanisms. In particular, we describe recent application of an atomistic string method, based on Roux's “swarms of trajectories” approach, to elucidate the sequence and interdependence of conformational changes during activation. We illustrate how this can be combined with transition analysis and Brownian dynamics to extract thermodynamic and kinetic information, leading to understanding of what controls ion channel function. © 2019 Wiley Periodicals, Inc.     

Techniques for recording reconstituted ion channels

This review describes and discusses techniques useful for monitoring the activity of protein ion channels in vitro. In the first section the biological importance and the classification of ion channels are outlined in order to justify the strong motivation for dealing with this important class of membrane proteins. The expression, reconstitution and integration of recombinant proteins into lipid bilayers are crucial steps to obtain consistent data when working with ion channels. In the second section recording techniques used in research are presented. Since this review focuses on analytical systems bearing reconstituted ion channels the industrial most important patch-clamp techniques of cells are only briefly mentioned. In section three, artificial systems developed in the last decades are described while the emerging technologies using nanostructured supports or microfluidic systems are presented in section four. Finally, the remaining challenges of membrane protein analysis and its potential applications are briefly outlined.     

Voltammetric study on ion transport across a bilayer lipid membrane in the presence of a hydrophobic ion or an ionophore

This review describes voltammetric studies on ion transport from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) containing a hydrophobic ion, valinomycin (Val) or gramicidin A (GA). In particular, the ion transport mechanisms are discussed in terms of the distribution of a pair of ions between aqueous and BLM phases. By addition of a small amount of hydrophobic ion into W1 and/or W2 containing a hydrophilic salt as a supporting electrolyte, the hydrophobic ion was distributed into the BLM with the counter ion to maintain electroneutrality within the BLM phase. It was found that the counter ion was transferred between W1 and W2 across the BLM by applying a membrane potential. Facilitated transport of alkali ions across a BLM containing Val as an ion carrier compound, could be interpreted by considering not only the formation of the alkali metal ion–Val complex but also the distribution of both the objective cation and the counter ion. In the case of addition of GA as a channel-forming compound into the BLM, the facilitated transport of alkali ions across the BLM depended on the ionic species of the counter ions. It was discovered that the influence of the counter ion on the facilitated transport of alkali ions across the BLM could be explained in terms of the hydrophobicity and the ionic radius of the counter ion.     

Artificial ion channel formed by cucurbit[n]uril derivatives with a carbonyl group fringed portal reminiscent of the selectivity filter of K+ channels

Novel artificial ion channels (1 and 2) based on CB[n] (n = 6 and 5, respectively) synthetic receptors with carbonyl-fringed portals (diameter 3.9 and 2.4 A, respectively) can transport proton and alkali metal ions across a lipid membrane with ion selectivity. Fluorometric experiments using large unilamellar vesicles showed that 1 mediates proton transport across the membranes, which can be blocked by a neurotransmitter, acetylcholine, reminiscent of the blocking of the K+ channels by polyamines. The alkali metal ion transport activity of 1 follows the order of Li+ > Cs+ approximately Rb+ > K+ > Na+, which is opposite to the binding affinity of CB[6] toward alkali metal ions. On the other hand, the transport activity of 2 follows the order of Li+ > Na+, which is also opposite to the binding affinity of 2 toward these metal ions, but virtually no transport was observed for K+, Rb+, and Cs+. It is presumably because the carbonyl-fringed portal size of 2 (diameter 2.4 A) is smaller than the diameters of these alkali metal ions. To determine the transport mechanism, voltage-clamp experiments on planar bilayer lipid membranes were carried out. The experiments showed that a single-channel current of 1 for Cs+ transport is approximately 5 pA, which corresponds to an ion flux of approximately 3 x 107 ions/s. These results are consistent with an ion channel mechanism. Not only the structural resemblance to the selectivity filter of K+ channels but also the remarkable ion selectivity makes this model system unique.     

Metal–Organic Framework Membrane Nanopores as Biomimetic Photoresponsive Ion Channels and Photodriven Ion Pumps

Biological ion channels and ion pumps with sub-nanometer sizes modulate ion transport in response to external stimuli. Realizing such functions with sub-nanometer solid-state nanopores has been an important topic with wide practical applications. Herein, we demonstrate a biomimetic photoresponsive ion channel and photodriven ion pump using a porphyrin-based metal–organic framework membrane with pore sizes comparable to hydrated ions. We show that the molecular-size pores enable precise and robust optoelectronic ion transport modulation in a broad range of concentrations, unparalleled with conventional solid-state nanopores. Upon decoration with platinum nanoparticles to form a Schottky barrier photodiode, photovoltage across the membrane is generated with “uphill” ion transport from low concentration to high concentration. These results may spark applications in energy conversion, ion sieving, and artificial photosynthesis.     

Amperometric tape ion sensors for cadmium(II) ion analysis

This paper describes a novel tape platform ion sensing methodology specific to the detection of cadmium(II) ions in aqueous solution based on assisted ion transfer reactions across a polarized water | organic gel micro-interface. The tape ion sensors were constructed to incorporate the micro-water | polyvinylchloride-2-nitrophenylethyl ether (PVC-NPOE) gel interfaces referred to as ionodes. The sensors have overall thicknesses less than 300 μm, allowing their packaging in a disposable tape format. The detection methodology is based on the selective assisted transfer of the cadmium ion in aqueous phase by ETH 1062 present in the PVC-NPOE gel layer and was first investigated using cyclic voltammetry. Quantitative analysis of cadmium(II) ions in aqueous solution using the tape sensors was then conducted under stop-flow conditions. Detection limits as low as 20 ppb (178 nM) for Cd(II) ions in very small volumes as low as a single 20 μl droplet without any sample preconcentration was achieved in an analysis time of approximately 20 s, which could be easily employed for the direct measurement of Cd(II) ion levels in various field applications. The tape ion sensor can also be used in a flow-cell geometry to preconcentrate Cd(II) ions from aqueous samples and further improve the detection limit.     

A Voltage-Responsive Synthetic Cl−-Channel Regulated by pH

Transmembrane protein channels are an important inspiration for the design of artificial ion channels. Their dipolar structure helps overcome the high energy barrier to selectively translocate water and ions sharing one pathway, across the cell membrane. Herein, we report that the amino-imidazole (Imu) amphiphiles self-assemble via multiple H-bonding to form stable artificial Cl⁻-channels within lipid bilayers. The alignment of water/Cl⁻ wires influences the conduction of ions, envisioned to diffuse along the hydrophilic pathways; at acidic pH, Cl⁻/H⁺ symport conducts along a partly protonated channel, while at basic pH, higher Cl⁻/OH⁻ antiport translocate through a neutral channel configuration, which can be greatly activated by applying strong electric field. This voltage/pH regulated channel system represents an unexplored alternative for ion-pumping along artificial ion-channels, parallel to that of biology.     

Selective complexation and transport of europium ions at the interface of vesicles

The aim of the present work was to design functionalized lipidic membranes that can selectively interact with lanthanide ions at the interface and to exploit the interaction between membranes induced by this molecular-recognition process with a view to building up self-assembled vesicles or controlling the permeability of the membrane to lanthanide ions. Amphiphilic molecules bearing a beta-diketone unit as head group were synthesized and incorporated into phospholipidic vesicles. Binding of Eu(III) ions to the amphiphilic ligand can lead to formation of a complex involving ligands of the same vesicle membrane (intravesicular complex) or of two different vesicles (intervesicular complex). The effect of Eu(III) ions on vesicle behavior was studied by complementary techniques such as fluorimetry, light scattering, and electron microscopy. The formation of an intravesicular luminescent Eu/beta-diketone ligand (1/2) complex was demonstrated. The linear increase in the binding constant with increasing concentration of ligands in the membrane revealed a cooperative effect of the ligands distributed in the vesicle membrane. The luminescence of this complex can be exploited to monitor the kinetics of complexation at the interface of the vesicles, as well as ion transport across the membrane. By encapsulation of 2,6-dipicolinic acid (DPA) as a competing ligand which forms a luminescent Eu/DPA complex, the kinetics of ion transport across the membrane could be followed. These functional vesicles were shown to be an efficient system for the selective transport of Eu(III) ions across a membrane with assistance by beta-diketone ligands.     

Metal–Organic Framework Membrane Nanopores as Biomimetic Photoresponsive Ion Channels and Photodriven Ion Pumps

Biological ion channels and ion pumps with sub‐nanometer sizes modulate ion transport in response to external stimuli. Realizing such functions with sub‐nanometer solid‐state nanopores has been an important topic with wide practical applications. Herein, we demonstrate a biomimetic photoresponsive ion channel and photodriven ion pump using a porphyrin‐based metal–organic framework membrane with pore sizes comparable to hydrated ions. We show that the molecular‐size pores enable precise and robust optoelectronic ion transport modulation in a broad range of concentrations, unparalleled with conventional solid‐state nanopores. Upon decoration with platinum nanoparticles to form a Schottky barrier photodiode, photovoltage across the membrane is generated with “uphill” ion transport from low concentration to high concentration. These results may spark applications in energy conversion, ion sieving, and artificial photosynthesis.     

Mechanical actuation of ion channels using a piezoelectric planar patch clamp system

High-throughput screening of ion channels is now possible with the advent of the planar patch clamp system. This system drastically increases the number of ion channels that can be studied, as multiple ion channel experiments can now be conducted in parallel. However, due to tedious, usually pressure-driven mechanotransduction techniques, there has been a slow integration of this technology into the field of mechanosensitive ion channels. By implementing a piezoelectric quartz substrate into a planar patch clamp system, we show that the patch clamp substrate itself can be used to mechanically actuate ion channels. The piezoelectric substrate transduces an external, applied electric field into a mechanical tension, so precise actuation of the membrane can be accomplished. By applying this electric field only to the outer edges of the substrate, no ulterior electric field is created in the vicinity of the membrane during actuation. Further, with resonant frequencies ranging from 1 kHz to 200 MHz, quartz substrates can be used to apply a wide range of time-varying tensions to cell membranes. This will allow for new and instructive investigations into the dynamic mechanotransductive properties of ion channels.     

An improved pseudotargeted metabolomics approach using multiple ion monitoring with time-staggered ion lists based on ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Pseudotargeted metabolomics is a novel strategy integrating the advantages of both untargeted and targeted methods. The conventional pseudotargeted metabolomics required two MS instruments, i.e., ultra-high performance liquid chromatography/quadrupole-time- of-flight mass spectrometry (UHPLC/Q-TOF MS) and UHPLC/triple quadrupole mass spectrometry (UHPLC/QQQ-MS), which makes method transformation inevitable. Furthermore, the picking of ion pairs from thousands of candidates and the swapping of the data between two instruments are the most labor-intensive steps, which greatly limit its application in metabolomic analysis. In the present study, we proposed an improved pseudotargeted metabolomics method that could be achieved on an UHPLC/Q-TOF/MS instrument operated in the multiple ion monitoring (MIM) mode with time-staggered ion lists (tsMIM). Full scan-based untargeted analysis was applied to extract the target ions. After peak alignment and ion fusion, a stepwise ion picking procedure was used to generate the ion lists for subsequent single MIM and tsMIM. The UHPLC/Q-TOF tsMIM MS-based pseudotargeted approach exhibited better repeatability and a wider linear range than the UHPLC/Q-TOF MS-based untargeted metabolomics method. Compared to the single MIM mode, the tsMIM significantly increased the coverage of the metabolites detected. The newly developed method was successfully applied to discover plasma biomarkers for alcohol-induced liver injury in mice, which indicated its practicability and great potential in future metabolomics studies.     

Synthesis and ion transport activity of oligoesters containing an environment-sensitive fluorophore

A series of oligoesters based on a rigid triphenyl-diyne core is described. The molecules were readily synthesized from key intermediates, and retained good solubility properties. One of the compounds displayed modest ion transport activity in vesicles, was capable of forming highly conducting single channels in planar bilayers and exhibited an irregular non-linear current-voltage response. All the reported molecules had minimal aqueous fluorescence while being highly fluorescent in less-polar media including lipid vesicles; their partitioning into the membrane could be monitored by a significant blue-shift and increase in fluorescence intensity, as well as a decreased extent of quenching in vesicles over that in water. The combined data indicated that the compounds are highly aggregated in aqueous solution, which limits their membrane partitioning and ion transport activity, in agreement with mechanisms proposed for other 'simple' oligoester channels.