Screening of ion channel receptor agonists using capillary electrophoresis-patch clamp detection with resensitized detector cells

Efficient techniques for identifying endogenous and synthetic ligands of ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.     

Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.     

Ligand binding to nicotinic acetylcholine receptor investigated by surface plasmon resonance

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A K(D) = 3.5 × 10(-)(6) M for the detergent-solubilized receptor and a K(D) = 1.4 × 10(-)(5) M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a K(D) = 4.5 × 10(-)(5) M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligand-receptor interactions in case of small ligand molecules by mass-sensitive techniques.     

Direct chromatographic determination of dissociation rate constants of ligand-receptor complexes: assessment of the interaction of noncompetitive inhibitors with an immobilized nicotinic acetylcholine receptor-based liquid chromatography stationary phase

A liquid chromatographic stationary phase containing immobilized membranes from a cell line expressing the alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) has been used to assess dissociation rate constants (kd) of 12 noncompetitive inhibitor-nAChR complexes. The pharmacological effects of the noncompetitive inhibitors, expressed as percent recovery of activity at 7 min and 4 h postexposure to the inhibitor, were also determined. The results demonstrate that the kd values correlated with the pharmacological effect and that this approach can be used to identify molecular structures associated with differences in kd values. The method can be adapted for use with membrane-bound receptors, ion channels, and transporters and represents a direct and facile technique for the assessment of dissociation rate constants (kd) of ligand-receptor complexes.     

Entrapment of highly active membrane-bound receptors in macroporous Sol-Gel derived silica

The immobilization of membrane-associated proteins remains a challenging task. Herein, we report on the entrapment of two classes of membrane-bound receptors into sol-gel derived silica. Both nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel, and dopamine D(2Short) receptor (D2R), a G-protein coupled receptor, were entrapped into a series of sol-gel derived nanocomposite materials. In cases where the silica had a bimodal pore size distribution wherein both mesopores and macropores were present, the two receptors showed 40-80% of solution activity over periods of at least 1 month. Furthermore, the dissociation constants of entrapped nAChR and D2R for binding to known agonists and antagonists were very close to the values obtained for free receptors in solution. These results indicate that membrane-bound receptors entrapped into bimodal meso/macroporous silica should provide a useful platform for the development of bioanalytical devices such as bioaffinity columns or microarrays, which could aid in diagnosis and high-throughput drug screening.     

Microtiter plate binding assay for cholinergic compounds utilizing the nicotinic acetylcholine receptor.

A receptor-based binding assay for the determination of cholinergic compounds of the nicotinic acetylcholine receptor has been developed. By conducting the assay in a 96-well microtiter plate, the method is suitable for large-scale screening in drug development. Solid-phase extraction of the enzyme label significantly simplifies the assay protocol compared to earlier methods. The assay is based on immobilization of biotin-BSA on the microtiter which takes up avidin-labeled peroxidase due to avidin-biotin interaction. To perform the assay, a ligand (the analyte) and a biotin alpha-bungarotoxin conjugate (alpha Bgt-biotin) sequentially bind to a vesicle bound nicotinic acetylcholine receptor. This is done either in a test tube, assay I, or in a biotinylated microtiter well, assay II. Avidin-HRP is then added to this mixture; free alpha Bgt-biotin conjugate and immobilized biotin-BSA compete for the avidin sites. After the assay solution has been aspirated off, bound enzyme activity is determined which is directly related to the amount of alpha Bgt-biotin added. Dose-response curves of cholinergic compounds and Scatchard plots were generated to evaluate the apparent binding constants. Kinetic studies were conducted for the purpose of optimization. The final assay can be performed in under 4 h with a minimum of sample handling.     

Nicotine-like discriminative stimulus effects of acetylcholinesterase inhibitors and a muscarinic receptor agonist in Rhesus monkeys

Acetylcholinesterase (AChE) inhibitors and positive allosteric nicotinic acetylcholine receptor (nAChR) modulators are potential pharmacotherapies for nicotine dependence. Because some smoking cessation aids (e.g. varenicline) appear to work by mimicking the effects of nicotine, we used drug discrimination to examine whether AChE inhibitors and nAChR allosteric modulators mimic the effects of nicotine. Rhesus monkeys discriminated 1.78?mg/kg of nicotine s.c. under an FR5 schedule of stimulus-shock termination. Nicotine and the AChE inhibitors donepezil and galantamine dose-dependently increased responding on the nicotine-appropriate lever with ED₅₀ values of 0.35, 0.22, and 0.77?mg/kg, respectively. Donepezil (0.56?mg/kg) produced nicotine-like effects for at least 6?h, whereas the duration of action of galantamine (1.78?mg/kg) was less than 3?h. The positive allosteric nAChR modulator PNU-120596 (up to 10?mg/kg) and midazolam (up to 1.0?mg/kg) produced no more than 22% nicotine-lever responding. Oxotremorine, a muscarinic acetylcholine receptor agonist that was used to explore the extent to which muscarinic receptor agonism might contribute to the effects of AChE inhibitors, produced 94% nicotine-lever responding (ED₅₀ value 0.013?mg/kg). The muscarinic antagonist atropine significantly antagonized the effects of both oxotremorine and nicotine; however, the dose of atropine antagonizing oxotremorine was smaller than the dose required to antagonize nicotine. Collectively, these results suggest that AChE inhibitors can mimic the effects of nicotine by indirectly stimulating both nicotinic and muscarinic receptors. Inasmuch as some smoking cessation aids work by exerting nicotine-like effects, the current results are consistent with the potential use of AChE inhibitors as novel smoking cessation aids.     

Ion channel model reduction using manifold boundaries

Mathematical models of voltage-gated ion channels are used in basic research, industrial and clinical settings. These models range in complexity, but typically contain numerous variables representing the proportion of channels in a given state, and parameters describing the voltage-dependent rates of transition between states. An open problem is selecting the appropriate degree of complexity and structure for an ion channel model given data availability. Here, we simplify a model of the cardiac human Ether-à-go-go related gene (hERG) potassium ion channel, which carries cardiac I_Kr, using the manifold boundary approximation method (MBAM). The MBAM approximates high-dimensional model-output manifolds by reduced models describing their boundaries, resulting in models with fewer parameters (and often variables). We produced a series of models of reducing complexity starting from an established five-state hERG model with 15 parameters. Models with up to three fewer states and eight fewer parameters were shown to retain much of the predictive capability of the full model and were validated using experimental hERG1a data collected in HEK293 cells at 37°C. The method provides a way to simplify complex models of ion channels that improves parameter identifiability and will aid in future model development.     

Application of natural receptors in sensors and assays

Biosensors are analytical devices that use a biological or biologically derived material immobilized at a physicochemical transducer to measure one or more analytes. Although there are a large number of reviews on biosensors in general, there has been little systematic information presented on the application of natural receptors in sensor technology. This perspective discusses broadly the fundamental properties of natural receptors, which make them an attractive option for use as biorecognition elements in sensor technology. It analyses the current situation by reference to typical examples, such as the application of nicotinic acetylcholine receptor and G protein-linked receptors in affinity sensors and analyses the problems that need to be resolved prior to any commercialization of such devices.     

Reconstitution of nicotinic acetylcholine receptors into gel-protected lipid membranes

We report the functional reconstitution of nicotinic acetylcholine receptors into gel-protected bilayer lipid membranes using two different methods. In the first case, reconstitution was achieved by direct membrane formation from an emulsion of glycerol monooleate, hexane, and a membrane receptor extract. In the second case, incorporation was achieved via the fusion of vesicles from a preparation of membrane-bound receptors into preformed membranes after diffusion through the protective front gel layer. Measurement of the dc conductivity of the membranes in the presence of either acetylcholine or alpha-bungarotoxin was used to test for the functional activity of incorporated receptors.     

A biosensing system based on extracellular potential recording of ligand-gated ion channel function overexpressed in insect cells

We have used outer cell potential measurement to record agonist-dependent cellular responses in cells engineered to express ligand-gated ion channels and grown on a microelectrode surface. Application of glutamate, a natural agonist, induced a complex and robust potentiometric response in cells expressing homomeric GluR-D glutamate receptor, but not in nonexpressing control cells. The response consisted of an initial decrease in outer potential followed by a transient increase and was not obtained for other amino acids devoid of agonist activity at glutamate receptors. Furthermore, the pharmacological agonist of the GluR-D receptor, kainate, also produced the potentiometric response whereas 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive antagonist, was not active in itself but attenuated the responses to glutamate. The time course of the measured changes was slow, which may be partially due to the ligand being applied by free diffusion but may also reflect a contribution by secondary changes in the behavior of the cells. This novel approach should be applicable to other ligand-gated ion channels and holds promise as a cell-based biosensor for high-throughput drug screening and other applications.     

Effective Gene Delivery into Human Stem Cells with a Cell‐Targeting Peptide‐Modified Bioreducible Polymer

Stem cells are poorly permissive to non‐viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell‐binding ligand with a polymer that releases nucleic acids in a cytoplasm‐responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9‐derived hESC. Conjugating RVG to a redox‐sensitive biodegradable dendrimer‐type arginine‐grafted polymer (PAM‐ABP) enabled nanoparticle formation with plasmid DNA without altering the environment‐sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG‐PAM‐ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. ～60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG‐PAM‐ABP is thus a novel bioreducible, biocompatible, non‐toxic, synthetic gene delivery system for nAchR‐expressing stem cells. Our data also demonstrates that a cell‐binding ligand like RVG can cooperate with a gene delivery system like PAM‐ABP to enable transfection of poorly‐permissive cells.     

A Bioinspired Platform for Effective Delivery of Protein Therapeutics to the Central Nervous System

Central nervous system (CNS) diseases are the leading cause of morbidity and mortality; their treatment, however, remains constrained by the blood–brain barrier (BBB) that impedes the access of most therapeutics to the brain. A CNS delivery platform for protein therapeutics, which is achieved by encapsulating the proteins within nanocapsules that contain choline and acetylcholine analogues, is reported herein. Mediated by nicotinic acetylcholine receptors and choline transporters, such nanocapsules can effectively penetrate the BBB and deliver the therapeutics to the CNS, as demonstrated in mice and non‐human primates. This universal platform, in general, enables the delivery of any protein therapeutics of interest to the brain, opening a new avenue for the treatment of CNS diseases.     

Effects of poly-(lactide-co-glycolide) nanoparticles on electrophysiological properties of enteroendocrine cells

PLGA nanoparticles are widely used to deliver pharmacological compounds and genes to a variety of cell types. Despite the fact that many of these cells types depend critically on ion channel activity to function normally, there have been no studies on the effect of nanoparticles on the ion channel activity. To this end, we have investigated the effect of nanoparticles on cholecystokinin (CCK)-releasing enteroendocrine cell (EEC) line STC-1. It has been shown that regulation of CCK release from STC-1 cells in response to food depends on the normal electrogenic properties of these cells, including the activity of voltage-gated calcium and potassium channels. Due to the importance of voltage-gated ion channels in the normal physiological responses of STC-1 cells, we performed electrophysiological (patch clamp) experiments to assess the effects of PLGA nanoparticles on the voltage-gated calcium and potassium channels. Whole-cell patch clamp recordings on STC-1 cells containing 100 nm nanoparticles show no macroscopic differences in calcium and potassium channel activity. Additional experiments determined that the activation, inactivation, and use-dependent inactivation of these voltage-gated ion channels did not have any significant effect of nanoparticles on these basic biophysical properties. Lastly, we have examined the effects of PLGA nanoparticles on stimulus-induced rise in intracellular calcium concentration in STC-1 cells, which is necessary for release of CCK. Our data demonstrate that the use of PLGA nanoparticles did not alter the electrophysiological properties of STC-1 cells and supports the use of PLGA nanoparticles as an attractive option for delivering pharmaceuticals/genes to cells of the digestive system that might eventually prove useful for reducing appetite/food intake and in treatment of various gastrointestinal illnesses.     

A 3D Magnetic Hyaluronic Acid Hydrogel for Magnetomechanical Neuromodulation of Primary Dorsal Root Ganglion Neurons

Neuromodulation tools are useful to decipher and modulate neural circuitries implicated in functions and diseases. Existing electrical and chemical tools cannot offer specific neural modulation while optogenetics has limitations for deep tissue interfaces, which might be overcome by miniaturized optoelectronic devices in the future. Here, a 3D magnetic hyaluronic hydrogel is described that offers noninvasive neuromodulation via magnetomechanical stimulation of primary dorsal root ganglion (DRG) neurons. The hydrogel shares similar biochemical and biophysical properties as the extracellular matrix of spinal cord, facilitating healthy growth of functional neurites and expression of excitatory and inhibitory ion channels. By testing with different neurotoxins, and micropillar substrate deflections with electrophysical recordings, it is found that acute magnetomechanical stimulation induces calcium influx in DRG neurons primarily via endogenous, mechanosensitive TRPV4 and PIEZO2 channels. Next, capitalizing on the receptor adaptation characteristic of DRG neurons, chronic magnetomechanical stimulation is performed and found that it reduces the expression of PIEZO2 channels, which can be useful for modulating pain where mechanosensitive channels are typically overexpressed. A general strategy is thus offered for neuroscientists and material scientists to fabricate 3D magnetic biomaterials tailored to different types of excitable cells for remote magnetomechanical modulation.     

Action potential properties are gravity dependent

The functional properties of neuronal tissue critically depend on cellular composition and intercellular comunication. A basic principle of such communication found in various types of neurons is the generation of action potentials (APs). These APs depend on the presence of voltage gated ion channels and propagate along cellular processes (e.g. axons) towards target neurons or other cells. It has already been shown that the properties of ion channels depend on gravity. To discover whether the properties of APs also depend on gravity, we examined the propagation of APs in earthworms (invertebrates) and isolated nerve fibres (i.e. bundles of axons) from earthworms under conditions of micro- and macro-gravity. In a second set of experiments we could verify our results on rat axons (vertebrates). Our experiments carried out during two parabolic flight campaigns revealed that microgravity slows AP propagation velocity and macrogravity accelerates the transmission of action potentials. The relevance for live-science related questions is considerable, taking into account that altered gravity conditions might affect AP velocity in man during space flight missions.     

High-throughput mass spectrometer using atmospheric pressure ionization and a cylindrical ion trap array

The analytical performance of an atmospheric pressure sampling, multiple-channel, high-throughput mass spectrometer was investigated using samples of a variety of types. The instrument, based on an array of cylindrical ion traps, was built with four independent channels and here is operated using two fully multiplexed channels (sources, ion optics, ion traps, detectors) capable of analyzing different samples simultaneously. Both channels of the instrument were incorporated within the same vacuum system and operated using a common set of control electronics. A multichannel electrospray ionization source was assembled and used to introduce samples including solutions of organic compounds, peptides, and proteins simultaneously into the instrument in a high-throughput fashion. Cross-talk between the channels of the instrument occurred in the detection system and could be minimized to 1-2% using shielding between detector channels. In this initial implementation of the instrumentation, an upper mass/charge limit of approximately 1300 Th was observed (+13 charge state of myoglobin) and unit mass/charge resolution was achieved to approximately 800 Th. The rather limited dynamic range (2-3 orders of magnitude for low-concentration analytes) is due to cross-talk contributions from more concentrated species introduced into a different channel. Analysis of mixtures of alkylamines and peptides is demonstrated, but analysis of mixtures with a wide spread in mass/charge ratios was not possible due to mass discrimination in the ion optics. Further refinement of the vacuum system and ion optics will allow the addition of more channels of parallel mass analysis and facilitate applications in fields such as proteomics and metabolomics.     

Hollow-Particles Quasi-Solid-State Electrolytes with Biomimetic Ion Channels for High-Performance Lithium-Metal Batteries

Solid-state electrolytes (SSEs) are the core material of solid-state lithium metal batteries (SLMBs), which are being researched urgently owing to their high energy and safety. Both high ionic conductivity and excellent cycling stability remain the primary goal of solid-state electrolytes. Herein, inspired by K⁺/Na⁺ ion channels in cell membrane of eukaryotes, a novel hollow UiO-66 with biomimetic ion channels based on quasi-solid-state electrolytes (QSSEs) is designed. The hollow UiO-66 spheres containing biomimetic ion channels can spontaneously combine anions and incorporate more lithium ions, creating improved ionic conductivity (1.15 × 10⁻³ S cm⁻¹) and lithium-ion transference number (0.70) at room temperature. The long-term cycling of symmetric batteries and COMSOL simulations demonstrate that this biomimetic strategy enables uniform ion flux to suppress Li dendrites. Furthermore, the Li metal full cells paired with LiFePO₄ cathode exhibit excellent cycling stability and rate performance. Consequently, the strategy of designing biomimetic QSSEs opens up a new path for developing high-performance electrolytes for SLMBs.     

Potassium-selective atomic force microscopy on ion-releasing substrates and living cells

A new method for simultaneous mapping of cell topography and ion fluxes was developed. A highly sensitive ion sensor system was generated by coating atomic force microscopy tips with a PVC layer containing valinomycin, an ionophore for potassium. The activity of specific ions was traced on artificial ion-releasing PVC substrates. A boundary potential was generated owing to the selective exchange of a specific ion between coated tip and ion-releasing substrate. The boundary potential was detectable as a force induced by ion-selective electrostatic interactions. The selectivity coefficient of valinomycin for potassium against sodium (K(K,Na)f) was -2.5 +/- 0.5. Potassium efflux was measured on living MDCK-F1 cells expressing BK(Ca) channels. We could demonstrate localized areas of high potassium concentrations at the cell surface. The potassium efflux could be reversibly inhibited by thapsigargin, which is known to inhibit the efflux of potassium from BK(Ca) channels by suppression of calcium ATPase.