Reconstitution Of Ion Channels Research Articles

Lipid nanodisc scaffold and size alter the structure of a pentameric ligand-gated ion channel

Lipid nanodiscs have become a standard tool for studying membrane proteins, including using single particle cryo-electron microscopy (cryo-EM). We find that reconstituting the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodiscs produces distinct structures by cryo-EM. The effect of the nanodisc on ELIC structure extends to the extracellular domain and agonist binding site. Additionally, molecular dynamic simulations indicate that nanodiscs of different size impact ELIC structure and that the nanodisc scaffold directly interacts with ELIC. These findings suggest that the nanodisc plays a crucial role in determining the structure of pLGICs, and that reconstitution of ion channels in larger nanodiscs may better approximate a lipid membrane environment.

Modulation of the Dipole Potential of Model Lipid Membranes with Phytochemicals: Molecular Mechanisms, Structure–Activity Relationships, and Implications in Reconstituted Ion Channels

Phytochemicals, such as flavonoids, stilbenoids, alkaloids, terpenoids, and related compounds, have a wide range of useful pharmacological properties which cannot be ascribed to binding to a single peptide or protein target alone. Due to the relatively high lipophilicity of phytochemicals, the lipid membrane is thought to mediate their effects via changes in the properties of the lipid matrix, in particular, by modulating the transmembrane distribution of the electrical potential and, consequently, the formation and functioning of the ion channels reconstituted in the lipid bilayers. Therefore, biophysical studies on the interactions between plant metabolites and model lipid membranes are still of interest. This review represents an attempt to provide a critical analysis of a variety of studies on altering membranes and ion channels with phytochemicals via disturbing the potential drop at the membrane-aqueous solution interface. Critical structural motifs and functioning groups in the molecules of plant polyphenols (alkaloids and saponins are identified) and the possible mechanisms of dipole potential modulation with phytochemicals are discussed.

Is the Membrane Lipid Matrix a Key Target for Action of Pharmacologically Active Plant Saponins?

This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.

Solubilization, purification, and functional reconstitution of human ROMK potassium channel in copolymer styrene-maleic acid (SMA) nanodiscs

Expression, purification, and functional reconstitution of mammalian ion channels are often challenging. Heterologous expression of mammalian channels in bacteria can be advantageous due to unrelated protein environment and the lack of risk of copurification of endogenous proteins, e.g., accessory channel subunits that can influence the channel activity. Also, direct recording of channel activity could be challenging due to their intracellular localization like in the case of mitochondrial channels. The activity of purified channels can be characterized at the single-molecule level by electrophysiological techniques, such as planar lipid bilayers (PLB). In this work, we describe a simple approach to accomplish PLB recording of the activity of single renal outer medullary potassium channels ROMK expressed in E. coli. We focused on the ROMK2 isoform that is present at low levels in the mitochondria and can be responsible for mitoKATP activity. We screened for the best construct to express the codon-optimized ROMK proteins with a 6xHis tag for protein purification. The strategy involved the use of optimal styrene-maleic acid (SMA) copolymer, which forms so-called polymer nanodiscs, to solubilize and purify ROMK-containing SMA lipid particles (SMALPs), which were amenable for fusion with PLB. Reconstituted ROMK channels exhibited ion selectivity, rectification, and pharmacological properties, which are in agreement with previous work on ROMK channels.

Ion channel reconstitution in lipid bilayers.

Incorporation of ion channels in planar lipid bilayers allows detecting and measuring ion channel activity in a well-controlled system. This technique provides critical information about ion channel kinetics, ion selectivity, gating mechanism, open probability, unitary conductance, subconductance states, voltage dependence, and burst opening events, particularly at the single molecule level. Planar lipid bilayers provide a unique controllable environment that enables maintaining specific regulatory components, including lipids, ligands, inhibitors, particular ions, and proteins, as well as the temperature that can modulate activity of many ion channels. Thus, this system provides explicit details about ion channel gating mechanism and enables identifying its particular regulatory molecules or components. This chapter will describe the planar lipid bilayer method using the example of a transient receptor potential (TRP) ion channel family member. The planar lipid bilayer electrophysiological approach has proven to be useful in studying intrinsic properties of TRP channels. This method is particularly valuable for our understanding of intrinsic temperature sensitivity of thermoreceptors such as TRP channels and direct effects of TRP channels agonists, antagonists, co-factors, and other modifiers.

The application of Poisson distribution statistics in ion channel reconstitution to determine oligomeric architecture.

During reconstitution, membrane proteins are randomly inserted into liposomes according to Poisson distribution statistics. When the protein to lipid ratios in the reconstitution mixture are varied systematically, the characteristics of this statistical capture permit inferences about the proteins themselves, such as the number of subunits that assemble into a single functional unit. This chapter describes the Poisson distribution as applied to the reconstitution of membrane proteins into proteoliposomes and focuses on an application whereby this statistical behavior is used to determine the number of ion channel subunits that assemble into a functional pore. Practical considerations for performing these experiments are emphasized. Harnessing Poisson dilution statistics provides a function-based method to determine ion channel oligomerization, complementing other biophysical, biochemical, or structural approaches.

1,3-Thiazine, 1,2,3,4-Dithiadiazole, and Thiohydrazide Derivatives Affect Lipid Bilayer Properties and Ion-Permeable Pores Induced by Antifungals.

Over the past decade, thiazines, thiadiazoles, and thiohydrazides have attracted increasing attention due to their sedative, antimicrobial, antiviral, antifungal, and antitumor activities. The clinical efficacy of such drugs, as well as the possibility of developing resistance to antimicrobials, will depend on addressing a number of fundamental problems, including the role of membrane lipids during their interaction with plasma membranes. The effects of the eight 1,3- thiazine-, 1,2,3,4- dithiadiazole-, and thiohydrazide-related compounds on the physical properties of model lipid membranes and the effects on reconstituted ion channels induced by the polyene macrolide antimycotic nystatin and antifungal cyclic lipopeptides syringomycin E and fengycin were observed. We found that among the tested agents, the fluorine-containing compound N′-(3,5-difluorophenyl)-benzenecarbothiohydrazide (C6) was the most effective at increasing the electric barrier for anion permeation into the hydrophobic region of the membrane and reducing the conductance of anion-permeable syringomycin pores. A decrease in the membrane boundary potential with C6 adsorption also facilitated the immersion of positively charged syringomycin molecules into the lipid bilayer and increases the pore-forming ability of the lipopeptide. Using differential scanning microcalorimetry, we showed that C6 led to disordering of membrane lipids, possibly by potentiating positive curvature stress. Therefore, we used C6 as an agonist of antifungals forming the pores that are sensitive to membrane curvature stress and lipid packing, i.e., nystatin and fengycin. The dramatic increase in transmembrane current induced by syringomycin E, nystatin, and fengycin upon C6 treatment suggests its potential in combination therapy for treating invasive fungal infections.

Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers.

Detergent-solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar "black lipid membranes" formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step-by-step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single-channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added. © 2018 by John Wiley & Sons, Inc.

Ion Channels Induced by Antimicrobial Agents in Model Lipid Membranes are Modulated by Plant Polyphenols Through Surrounding Lipid Media.

The potential therapeutic applications of plant polyphenols in various neurological, cardiovascular, metabolic and malignant disorders determine the relevance of studying the molecular mechanisms of their action on the cell membranes. Here, the quantitative changes in the physical parameters of model bilayer lipid membranes upon the adsorption of plant polyphenols were evaluated. It was shown that butein and naringenin significantly decreased the intrinsic dipole potential of cholesterol-free and cholesterol-enriched membranes. Cardamonin, 4'-hydroxychalcone, licochalcone A and liquiritigenin demonstrated the average efficiency, while resveratrol did not characterized by the ability to modulate the bilayer electrostatics. At the same time, the tested polyphenols affected melting of phospholipids with saturated acyl chains. The effects were attributed to the lipid disordering and a promotion of the positive curvature stress. According to DSC data and results of measurements of the threshold voltages that cause bilayer breakdown licochalcone A is the most effective agent. Furthermore, the role of the polyphenol induced changes in the electric and elastic properties of lipid host in the regulation of reconstituted ion channels was examined. The ability of the tested polyphenols to decrease the conductance of single ion channels produced by the antifungal cyclic lipopeptide syringomycin E was in agreement with their effects on the dipole potential of the lipid bilayers. The greatest effect of licochalcone A on the steady-state membrane conductance induced by the antifungal polyene macrolide antibiotic nystatin correlated with its greatest efficacy to induce the positive curvature stress. We also found that butein and naringenin bind specifically to a single pore formed by α-hemolysin from Staphylococcus aureus.

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers.

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F- channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.

Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization

Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that synaptosomes purified from rodent and human frozen brain (control and Alzheimer disease) can be studied both biochemically and functionally. This method opens the way to directly testing the effects of novel drugs on native ion channels.

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14–55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.

Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes.

The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.

Lipid reconstitution and recording of recombinant ion channels.

Ion channels are membrane proteins that facilitate passive ion movements down the electrochemical gradients across biological membranes. They regulate a wide range of important physiological functions, from neuronal excitation, muscle contraction, substance absorption in epithelial tissues, to innate immunity. In recent years, the membrane transport field has seen tremendous advances in membrane protein purification and structural determination. This paves the way for investigators to reconstitute ion channels into biochemically defined lipid systems free of other biological constituents and to study channel functions within a structural context. The goal of this chapter is to guide readers through the process of producing proteoliposomes, designing liposome assays for initial characterization of channel function, and performing electrical recordings for in-depth mechanistic investigation.

Solvent Free Bilayer Recordings using a Novel All-In-One Miniaturized Amplifier

We present a new minaturized all-in-one Amplifier suitable to perform high-quality electrophysiology measurements. The instrument has been evaluated on a platform where ion channel proteins are reconstituted in solvent-free bilayers and multiple corresponding experimental data sets are shown.Characterization of reconstituted ion channels is still challenging as low noise current measurements in the pA range have to be combined with a large bandwidth of tens of kilohertz. Traditional transimpedance amplifiers use resistive or capacitive feedback to generate an analog output which needs to be digitized for further processing and display, resulting in complex, bulky and expensive systems. The amplifier shown here uses a switched capacitor gain stage combined with a sigma-delta digitizer which directly produces a digital signal. The actual layout is completely done in one silicon die (ASIC). This novel approach allows the integration of a multiple gain stage, filters, signal conditioning circuitry, digitizer and USB interface into the size of a pack of cigarettes while noise performance (≈100fA rms @ 1KHz) and bandwidth (up to 100kHz) are comparable to state-of-the-art patch clamp amplifiers.Combined with an already established platform using planar glass chips for semi-automatic formation of solvent-free BLMs an integrated and easy-to-use system has been set up offering students and researches an easy entry to high level electrophysiology research.

Parallel Reconstitution of Bacterial Toxins, Porins and Ion Channels into Suspended Lipid Membrane Microarrays for High-Throughput Electrophysiology

High-throughput electrophysiological recordings in artificial membranes have a great potential for pharmaceutical screening and nanopore analytics. In addition to parallelization and automation of lipid bilayers formation, different protein reconstitution protocols are required depending on the concrete application.We report on functional reconstitutions of three classes of pore-forming proteins into suspended lipid bilayers on Micro Electrode Cavity Array (MECA). Three reconstitution strategies have been employed and optimized depending on the protein structure: self-insertion for pore-forming toxins, transfer from detergent micelles for porins and vesicle fusion for ion channels.By optimizing protein concentration and facilitating spontaneous insertion with short voltage pulses just below the electroporation threshold, a controlled insertion of alpha-hemolysin was achieved. Exactly one pore per bilayer was inserted in over 50% of bilayers in the array, which is substantially higher than the 37% predicted by the Poisson distribution.We investigated and optimized insertion of outer membrane porins OmpF, MspA and OccK1 into bilayer arrays via detergent dilution. Best strategy was to use mild nonionic detergents and sequentially dilute protein stock solution to minimize influence of the detergent on the membrane. Protein injection in the immediate vicinity of a bilayer array and thorough mixing of the solution were decisive factors for homogeneous distribution of functional porins.Reconstitution via detergent micelles was found to be problematic for ion channels, but proteovesicle fusion was successful. The tetrameric potassium channel KcsA was expressed in vitro with cotranslational insertion into lipid vesicles or nanodiscs. Transfer from nanodiscs into suspended bilayers was shown to be inefficient. Additionally, the presence of nanodiscs substantially destabilized the membranes. For proteovesicles best fusion rates were achieved upon addition of negatively charged phospholipids followed by extrusion of the recovered proteovesicles through a polycarbonate filter.

Nano-Bilayer Lipid Membranes Hosted on Biogenic Nanoporous Substrates

Lifetime and stability of lipid bilayer membranes are essential parameters in ion channel reconstitution experiments. Reports on nano-BLMs across porous silicon nitride and alumina membranes with nanometer-sized apertures show a dramatically enhanced bilayer lifetime. In addition, the particular porous structure of the membranes enables the formation of a nano-BLM array, where failure of one membrane will not affect the membranes suspended across the other nanopores. However, a tradeoff between membrane thickness and mechanical stability is necessary when manufacturing these devices, with a three-dimensional multi-layer structure, substantially raising the cost of top-down device fabrication. In our study, the use of biogenic nanopores as substrates to host nano-BLMs was explored. Diatoms are unicellular marine algae in porous silica shells, called frustules. These shells have a tiered structure, with pores of minimum diameter 40 nm. The hierarchical porous structure of these shells makes them exceptionally mechanically stable.Frustules obtained from the marine algae Coscinodiscus wailesii were supported on a silicon platform with a micron-sized through-wafer aperture that allows fluidic access to either side of the frustule.Nano-BLMs were formed across the frustule pores using bubble-collapse method and were monitored by measuring the seal resistance and capacitance. Untreated frustules are hydrophilic, preventing the organic solvent from wetting the surface, resulting in a low BLM formation probability. Our studies indicate a formation probability of 1 out of 10 attempts. To render the surface hydrophobic, frustules were functionalized usingsilane chemistry, significantly improving bilayer formation probability. To study the effect of pore size on the incorporation probability and functionality of membrane proteins, the self-insertion of outer membrane protein OmpF of E.Coli in the nano-BLMs was investigated using single channel recordings. Blocking of the channel in the presence of the antibiotic ampicillin indicates the full functionality of the channel protein.

Lipid bilayer arrays: Cyclically formed and measured

Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, and can potentially be applied in DNA sequencing. Droplet bilayers have significant technological potential for enabling many of these applications due to their compatibility with automation and array platforms. To further develop this potential, we have simplified the formation and electrical measurement of droplet bilayers using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and measurement over a 32-element array with ~80% yield and no operator input following fluid addition. Cycling these arrays resulted in the formation and measurement of 96 out of 120 possible bilayers in 80 minutes, a sustainable rate that could significantly increase with automation and greater parallelization. This turn-key, high-yield approach to making artificial lipid bilayers requires no training, making the capability of creating and measuring lipid bilayers and ion channels accessible to a much wider audience. In addition, this approach is low-cost, parallelizable, and automatable, allowing high-throughput studies of ion channels and pore proteins in lipid bilayers for sensing or screening applications.

Bilayer Measurement of Endoplasmic Reticulum Ca2+ Channels

Reconstitution of ion channels into planar lipid bilayers (also called black lipid membranes or BLM) is the most widely used method to conduct physiological studies of intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca(2+)) channels. The two main types of Ca(2+) release channels in the ER membrane are ryanodine receptors (RyanRs) and inositol(1,4,5)-trisphosphate receptors (InsP3Rs). Use of the BLM reconstitution technique enabled the initial description of the functional properties of InsP3R and RyanR at the single-channel level more than 20 years ago. Since then, BLM reconstitution methods have been used to study physiological modulation and to perform structure-function analysis of these channels, and to study pathological changes in the function of InsP3R and RyanR in various disease states. The BLM technique has also been useful for studies of other intracellular Ca(2+) channels, such as ER Ca(2+) leak presenilin channels and NAADP-gated lysosomal Ca(2+) channels encoded by TPC2. In this article, basic protocols used for BLM studies of ER Ca(2+) channels are introduced.

Reconstitution of Endoplasmic Reticulum InsP3 Receptors into Black Lipid Membranes

Intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca(2+)) channels, are most often studied through their reconstitution into planar lipid bilayers (also called black lipid membranes, or BLMs). General methods for making bilayers and for ion channel reconstitution into BLMs have been extensively detailed elsewhere; thus, here the focus is on specific details relevant for inositol(1,4,5)-trisphosphate receptor (InsP3R) recordings. These procedures describe how to perform single-channel recordings of native or recombinant InsP3Rs in BLMs. Similar procedures are used to study native or recombinant ryanodine receptors (RyanRs) in BLMs.