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Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10–100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1–10 µM) blunted the signaling response of nicotine. The pesticides (at 3–30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.

Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3β4, and α4β2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2–3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4β2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03168-z.

Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

A high-throughput screening campaign has identified 1,4-diazepane compounds which are potent Cannabinoid receptor 2 agonists with excellent selectivity against the Cannabinoid receptor 1. This class of compounds suffered from low metabolic stability. Following various strategies, compounds with a good stability in liver microsomes and rat PK profile have been identified.

The histamine H3 receptor (H3R) plays a regulatory role in the presynaptic release of histamine and several other neurotransmitters, and thus, it is an attractive target for central nervous system indications including cognitive disorders, narcolepsy, attention-deficit hyperactivity disorder, and pain. The development of H3R antagonists was complicated by the similarities between the pharmacophores of H3R and human Ether-a-go-go related gene (hERG) channel blockers, a fact that probably prevented promising compounds from being progressed into the clinic. Using a three-dimensional in silico modeling approach complemented with automated and manual patch clamping, we were able to separate these two pharmacophores and to develop highly potent H3R antagonists with reduced risk of hERG liabilities from initial hit series with low selectivity identified in a high-throughput screening campaign.

High-information screening formats, using more physiologically relevant cellular models and readout approaches, are slowly replacing traditional, target-orientated approaches in drug discovery programs. With improved access to primary cells, as well as label-free, non-intrusive methods of compound interrogation (such as automated electrophysiology), high-thoughput screening facilities have to adapt to more complex assay scenarios. The implementation of novel cellular systems, readout technologies and data management in a drug discovery company are essential to improve the current falling productivity evident in recent years throughout the pharmaceutical industry.

Following its involvement in life-threatening cardiac arrhythmias, the catchword 'hERG' has become infamous in the drug discovery community. The blockade of the ion channel coded by the human ether-á-go-go-related gene (hERG) has been correlated to a prolongation of the QT interval in the ECG, which again is correlated to a potential risk of a life-threatening polymorphic ventricular tachycardia - torsades de pointes (TdP). Therefore, in vitro investigations for blockade of this ion channel have become a standard, starting early in most drug discovery projects and often accompanying the whole project; at some stage, scientists in many medicinal chemistry programs have to deal with hERG channel liabilities. Data for the compound effects on hERG channel activity are generally part of the safety pharmacology risk assessment in regulatory submissions and, at this stage, are ideally conducted in compliance with good laboratory practice. With the withdrawal of clobutinol from the market, owing to its perceived risk of introducing TdP, the importance of the hERG channel has very recently been reconfirmed. Despite being of such importance for drug discovery, the relevance and impact of hERG data are sometimes misinterpreted, as there are drugs that block the hERG-coded ion channel but do not cause TdP, and drugs that cause TdP but do not block the hERG channel. This review aims to provide an overview of TdP, including the cardiac action potential and the ion channels involved in it, as well as on the relevance and interpretation of in vitro hERG channel data and their impact for drug discovery projects. Finally, novel cardiac safety test systems beyond in vitro hERG channel screening are discussed.

A functional cell-based assay was developed using a generic proprietary assay protocol, based on a membrane-potential sensitive dye, for the identification of small-molecule antagonists against the Kv1.3 potassium ion channel. A high-throughput screen (HTS) was subsequently performed with 20,000 compounds from the Evotec library, preselected using known smallmolecule antagonists for both sodium and potassium ion channels. Following data analysis, the hit rate was measured at 1.72%, and subsequent dose-response analysis of selected hits showed a high hit confirmation rate yielding approximately 50 compounds with an apparent IC50 value lower than 10 µM. Subsequent electrophysiological characterization of selected hits confirmed the initial activity and potency of the identified hits on the Kv1.3 target and also selectivity toward Kv1.3 through measurements on HERG as well as Kv1.3-expressing cell lines. Follow-up structure-activity relationship analysis revealed a variety of different clusters distributed throughout the library as well as several singlicates. In comparison to known Kv1.3 blockers, new chemical entities and scaffolds showing potency and selectivity against the Kv1.3 ion channel were detected. In addition, a screening strategy for ion channel drug discovery HTS, medicinal chemistry, and electrophysiology is presented. (Journal of Biomolecular Screening 2006:57-64)

Atomic force microscopy (AFM) was used to measure the forces stabilizing human aquaporin-1 (hAQP1), a tetrameric transmembrane protein that forms highly specific water channels. To this end, the AFM tip was attached to the C-terminus of hAQP1 and secondary structure elements were extracted from the membrane while the single-molecule force-extension curve was being recorded. Force peaks, reflecting the unfolding of secondary structure elements, could be interpreted in depth using the atomic model of hAQP1. Different classes of force-extension curves indicated the existence of alternative unfolding pathways for individual proteins. In addition, transmembrane helices at the periphery of the hAQP1 tetramer exhibited smaller extraction forces than helices at the interface between hAQP1 monomers. These results represent the first direct assessment of intermolecular forces defining the oligomeric state of a membrane protein.

Summary The atomic force microscope (AFM) is an exquisitely delicate probe measuring the height of a specimen at discrete sampling points in a fixed two-dimensional (2D) raster. The resulting topograph is a 2D digital image, with each pixel representing a distinct height measurement. The height of an object is determined as the average of the maximum heights measured above the supporting surface. We show that such object heights derived from a variety of organic samples depend critically on the sampling or pixel size of the 2D raster. It is concluded that to obtain accurate specimen heights, the pixel size must be small enough to resolve submolecular structures and thus ensure representative sampling of the height variation on the surface.

Blockade of the cardiac ion channel coded by human ether-a-gogo-related gene (hERG) can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. Automated electrophysiological patch clamp allows assessment of hERG channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. However, a number of potential sources of errors in setting up hERG channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, Copenhagen, Denmark) technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.

Here we discuss the experimental approaches that have allowed high resolution atomic force microscopy (AFM) imaging, and review results that show AFM to be of great interest for biologists, AFM allows single proteins to be imaged under physiologically relevant conditions. The exceptional signal-to-noise ratio and resolution of AFM topographs enables the oligomerization state and characteristic substructures of individual proteins to be resolved. Several examples demonstrate the capabilities of AFM to directly observe single proteins, and their conformational changes, to study protein-protein interactions and to follow the assembly of membrane proteins. We consider the AFM techniques that have allowed high resolution imaging, and review results that show AFM to be a powerful method to analyze biological processes at the level of single molecules.

Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.

Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulness of high-resolution TMAFM images. Topographs exhibited a lateral resolution between 1.1 and 1. 5 nm and a vertical resolution of approximately 0.1 nm. For all protein surfaces, TMAFM and CMAFM topographs were in excellent agreement. TMAFM was capable of imaging the fragile polypeptide loop connecting the transmembrane alpha-helices E and F of bacteriorhodopsin in its native extended conformation. The standard deviation (SD) of averages calculated from TMAFM topographs exhibited an enhanced minimum (between 0.1 and 0.9 nm) that can be assigned to the higher noise of the raw data. However, the SD difference, indicating the flexibility of protein subunits, exhibited an excellent agreement between the two imaging modes. This demonstrates that the recently invented imaging-mode TMAFM has the ability to faithfully record high-resolution images and has sufficient sensitivity to contour individual peptide loops without detectable deformations.

Ion channels are integral membrane proteins that regulate the flow of ions across the plasma membrane and the membranes of intracellular organelles of both excitable and non-excitable cells. Ion channels are vital to a wide variety of biological processes and are prominent components of the nervous system and cardiovascular system, as well as controlling many metabolic functions. Furthermore, ion channels are known to be involved in many disease states and as such have become popular therapeutic targets. For many years now manual patch-clamping has been regarded as one of the best approaches for assaying ion channel function, through direct measurement of ion flow across these membrane proteins. Over the last decade there have been many remarkable breakthroughs in the development of technologies enabling the study of ion channels. One of these breakthroughs is the development of automated planar patch-clamp technology. Automated platforms have demonstrated the ability to generate high-quality data with high throughput capabilities, at great efficiency and reliability. Additional features such as simultaneous intracellular and extracellular perfusion of the cell membrane, current clamp operation, fast compound application, an increasing rate of parallelization, and more recently temperature control have been introduced. Furthermore, in addition to the well-established studies of over-expressed ion channel proteins in cell lines, new generations of planar patch-clamp systems have enabled successful studies of native and primary mammalian cells. This technology is becoming increasingly popular and extensively used both within areas of drug discovery as well as academic research. Many platforms have been developed including NPC-16 Patchliner(®) and SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich), CytoPatch™ (Cytocentrics AG, Rostock), PatchXpress(®) 7000A, IonWorks(®) Quattro and IonWorks Barracuda™, (Molecular Devices, LLC); Dynaflow(®) HT (Cellectricon AB, Mölndal), QPatch HT (Sophion A/S, Copenhagen), IonFlux HT (Fluxion Bioscience Inc, USA), which have demonstrated the capability to generate recordings similar in quality to that of conventional patch clamping. Here we describe features of Nanion's NPC-16 Patchliner(®) and processes and protocols suited for this particularly flexible and successful high-throughput automated platform, which is based on planar patch-clamp technology. However, many of the protocols and notes given in this chapter can be applied to other automated patch-clamp platforms, similarly.

Ion channels are integral membrane proteins that regulate the flow of ions across the plasma membrane and the membranes of intracellular organelles of both excitable and non-excitable cells. Ion channels are vital to a wide variety of biological processes and are prominent components of the nervous system and cardiovascular system, as well as controlling many metabolic functions. Furthermore, ion channels are known to be involved in many disease states and as such have become popular therapeutic targets. For many years now manual patch-clamping has been regarded as one of the best approaches for assaying ion channel function, through direct measurement of ion flow across these membrane proteins. Over the last decade there have been many remarkable breakthroughs in the development of technologies enabling the study of ion channels. One of these breakthroughs is the development of automated planar patch-clamp technology. Automated platforms have demonstrated the ability to generate high-quality data with high throughput capabilities, at great efficiency and reliability. Additional features such as simultaneous intracellular and extracellular perfusion of the cell membrane, current clamp operation, fast compound application, an increasing rate of parallelization, and more recently temperature control have been introduced. Furthermore, in addition to the well-established studies of over-expressed ion channel proteins in cell lines, new generations of planar patch-clamp systems have enabled successful studies of native and primary mammalian cells. This technology is becoming increasingly popular and extensively used both within areas of drug discovery as well as academic research. Many platforms have been developed including NPC-16 Patchliner(®) and SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich), CytoPatch™ (Cytocentrics AG, Rostock), PatchXpress(®) 7000A, IonWorks(®) Quattro and IonWorks Barracuda™, (Molecular Devices, LLC); Dynaflow(®) HT (Cellectricon AB, Molndal), QPatch HT (Sophion A/S, Copenhagen), IonFlux HT (Fluxion Bioscience Inc, USA), which have demonstrated the capability to generate recordings similar in quality to that of conventional patch clamping. Here we describe features of Nanion's NPC-16 Patchliner(®) and processes and protocols suited for this particularly flexible and successful high-throughput automated platform, which is based on planar patch-clamp technology. However, many of the protocols and notes given in this chapter can be applied to other automated patch-clamp platforms, similarly.

The field of automated patch-clamp electrophysiology has emerged from the tension between the pharmaceutical industry’s need for high-throughput compound screening versus its need to be conservative due to regulatory requirements. On the one hand, hERG channel screening was increasingly requested for new chemical entities, as the correlation between blockade of the ion channel coded by hERG and Torsades de Pointes cardiac arrhythmia gained increasing attention. On the other hand, manual patch-clamping, typically quoted as the gold-standard for understanding ion channel function and modulation, was far too slow (and, consequently, too expensive) for keeping pace with the numbers of compounds submitted for hERG channel investigations from pharmaceutical R&D departments. In consequence it became more common for some pharmaceutical companies to outsource safety pharmacological investigations, with a focus on hERG channel interactions. This outsourcing has allowed those pharmaceutical companies to build up operational flexibility and greater independence from internal resources, and allowed them to obtain access to the latest technological developments that emerged in automated patch-clamp electrophysiology – much of which arose in specialized biotech companies. Assays for nearly all major cardiac ion channels are now available by automated patch-clamping using heterologous expression systems, and recently, automated action potential recordings from stem-cell derived cardiomyocytes have been demonstrated. Today, most of the large pharmaceutical companies have acquired automated electrophysiology robots and have established various automated cardiac ion channel safety screening assays on these, in addition to outsourcing parts of their needs for safety screening.

A common over-the-counter (OTC) non-opioid antitussive drug, clobutinol, was recently withdrawn from the market due to its potential to induce cardiac arrhythmias by a blockade of the potassium channel coded by the human ether-a-go-go-related gene (hERG). In this study, we investigated the effects of a number of antitussive compounds on the hERG ion channel current using patch-clamp electrophysiology, and compared the effects to that of clobutinol. The compounds clobutinol, pentoxyverine, dextromethorphan, and codeine inhibited the outward current in hERG transfected cells with half-maximal inhibition concentrations (IC50) of 1.9 microM, 3.0 microM, 5.1 microM, and 97 microM, respectively. For theobromine, no significant effect on the hERG current at a concentration up to 100 microM was detected. Safety margins between the effects of the drugs on the hERG ion channel current and their calculated maximal free therapeutic plasma concentration were calculated. These results were compared to assess potential risks of the compounds to induce torsade de pointes-type arrhythmias.

Steroids are known to exert direct and indirect effects on cardiovascular functions, and women have been found to be more susceptible to QT prolongation than men. Although many clinical studies have been performed, the effects of steroids on cardiac repolarization are not yet fully understood. We examined the effects of 17-beta-estradiol (estradiol) on the major cardiac currents that are correlated to clinical observations of arrhythmias. Effects on the two major currents responsible for repolarization of the cardiac action potential (mediated by the human ether-a-gogo related gene (HERG) product), and by the potassium channel Q1 (KCNQ1) co-expressed with the potassium channel accessory subunit E1 (KCNE1) were examined, as well as effects on the sodium inward current (mediated by the sodium channel 5A (SCN5A) and generating the rapid upstroke of the action potential). A concentration-dependent effect of estradiol on the KCNQ1/KCNE1-mediated potassium current was observed. The half-maximal inhibition concentration (IC(50)) of estradiol on the KCNQ1/KCNE1 ion channel was calculated to 1.13+/-0.23 microM. The HERG-mediated potassium and the SCN5A-mediated sodium currents, however, were only slightly reduced by estradiol at concentrations of up to 30 microM. This suggests that alterations of the cardiac action potentials by steroids may be mediated by interaction with the KCNQ1/KCNE1 ion channel.

The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane α-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the α-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.

In tapping-mode atomic force microscopy, the phase shift between excitation and response of the cantilever is used as a material-dependent signal complementary to topography. The localization of information in the phase signal is demonstrated with 1.4-nm lateral resolution on purple membrane of Halobacterium salinarum in buffer solution. In a first-order approximation, the phase signal is found to correlate with modulations of the tip oscillation amplitude, induced by topography. Extending the analysis to contributions of the tip-sample interaction area as a second-order approximation, a method is proposed to extract information about the interaction from the phase signal for surfaces with a roughness in the order of the tip radius.

Atomic force microscopy (AFM) allows the observation of surface structures of purple membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transmembrane α-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.

A review, with 27 refs., on the application of at. force microscopy to examine the structure of proteins.