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Clozapine is an atypical neuroleptic used to manage treatment-resistant schizophrenia which is known to inhibit cardiac hERG/K V 11.1 potassium channels, a pharmacological property associated with increased risk of potentially fatal Torsades de Pointes (TdP) and sudden cardiac death (SCD). Yet, the long-standing clinical practice of clozapine does not show a consistent association with increased incidence of TdP, although SCD is considerably higher among schizophrenic patients than in the general population. Here, we have established the inhibitory profile of clozapine at the seven cardiac ion currents proposed by the ongoing comprehensive in vitro pro-arrhythmia (CiPA) initiative to better predict new drug cardio-safety risk. We found that clozapine inhibited all CiPA currents tested with the following rank order of potency: K V 11.1 > Na V 1.5 (late current) ≈ Ca V 1.2 ≈ Na V 1.5 (peak current) ≈ K V 7.1 > K V 4.3 > K ir 2.1 (outward current) . Half-maximal inhibitory concentrations (IC 50 ) at the repolarizing K V 11.1 and K V 7.1 channels, and at the depolarizing Ca V 1.2 and Na V 1.5 channels fell within a narrow half-log 3-10 µM concentration range, suggesting that mutual compensation could explain the satisfactory arrhythmogenic cardio-safety profile of clozapine. Although the IC 50 values determined herein using an automated patch-clamp (APC) technique are at the higher end of clozapine plasmatic concentrations at target therapeutic doses, this effective antipsychotic appears prone to distribute preferentially into the cardiac tissue, which supports the clinical relevance of our in vitro pharmacological findings., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Drinking fresh grapefruit juice is associated with a significant prolongation of the QT segment on the electrocardiogram (ECG) in healthy volunteers. Among the prominent polyphenols contained in citrus fruits and primarily in grapefruit, the flavonoid naringenin is known to be a blocker of the human ether-a-go-go related gene (hERG) potassium channel. Here we hypothesized that naringenin could interfere with other major ion channels shaping the cardiac ventricular action potential (AP). To test this hypothesis, we examined the effects of naringenin on the seven channels comprising the Comprehensive in vitro Pro-Arrhythmia (CiPA) ion channel panel for early arrhythmogenic risk assessment in drug discovery and development. We used automated population patch-clamp of human ion channels heterologously expressed in mammalian cells to evaluate half-maximal inhibitory concentrations (IC 50 ). Naringenin blocked all CiPA ion channels tested with IC 50 values in the 30-100 µM concentration-range. The rank-order of channel sensitivity was the following: hERG > K ir 2.1 > Na V 1.5 (late current) > Na V 1.5 (peak current) > K V 7.1 > K V 4.3 > Ca V 1.2. This multichannel inhibitory profile of naringenin suggests exercising caution when large amounts of grapefruit juice or other citrus juices enriched in this flavonoid polyphenol are drunk in conjunction with QT prolonging drugs or by carriers of congenital long-QT syndromes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human K ir 2.1 and hERG potassium channels in the 1 μM-100 μM range with a 2-4 fold enantiomeric separation. Na V 1.5 sodium currents and Ca V 1.2 calcium currents, as well as K V 4.3 and K V 7.1 potassium currents remained unaffected at up to 90 μM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 μM and 30 μM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background and Purpose: Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Na v 1.7 voltage-dependent Na + channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Na v channel targets., Experimental Approach: We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high-throughput automated patch-clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Na v -expressing cells in pull-down and cell distribution studies., Key Results: Two of the produced ProTx II analogues, Biot-ProTx II and ATTO488-ProTx II, best emulate the pharmacological properties of unlabelled ProTx II, whereas other analogues remain high affinity blockers of Na v 1.7. The biotinylated version of ProTx II efficiently works for the pull-down of several Na v isoforms tested in a concentration-dependent manner, whereas the fluorescent ATTO488-ProTx II specifically labels the Na v 1.7 channel over other Na v isoforms tested in various experimental conditions., Conclusions and Implications: The properties of these ProTx II analogues as tools for Na v channel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also be useful in the design of new drug screening strategies., (© 2021 The British Pharmacological Society.)

Cannabidiol (CBD) is a non-psychoactive component of Cannabis which has recently received regulatory consideration for the treatment of intractable forms of epilepsy such as the Dravet and the Lennox-Gastaut syndromes. The mechanisms of the antiepileptic effects of CBD are unclear, but several pre-clinical studies suggest the involvement of ion channels. Therefore, we have evaluated the effects of CBD on seven major cardiac currents shaping the human ventricular action potential and on Purkinje fibers isolated from rabbit hearts to assess the in vitro cardiac safety profile of CBD. We found that CBD inhibits with comparable micromolar potencies the peak and late components of the Na V 1.5 sodium current, the Ca V 1.2 mediated L-type calcium current, as well as all the repolarizing potassium currents examined except K ir 2.1. The most sensitive channels were K V 7.1 and the least sensitive were K V 11.1 (hERG), which underly the slow (I Ks ) and rapid (I Kr ) components, respectively, of the cardiac delayed-rectifier current. In the Purkinje fibers, CBD decreased the action potential (AP) duration more potently at half-maximal than at near complete repolarization, and slightly decreased the AP amplitude and its maximal upstroke velocity. CBD had no significant effects on the membrane resting potential except at the highest concentration tested under fast pacing rate. These data show that CBD impacts cardiac electrophysiology and suggest that caution should be exercised when prescribing CBD to carriers of cardiac channelopathies or in conjunction with other drugs known to affect heart rhythm or contractility., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Lysophosphatidic acid (LPA) is the natural ligand for two phylogenetically distinct families of receptors (LPA 1-3, LPA 4-6 ) whose pathways control a variety of physiologic and pathophysiological responses. Identifying the benefit of balanced activation/repression of LPA receptors has always been a challenge because of the high lability of LPA and the limited availability of selective and/or stable agonists. In this study, we document the discovery of small benzofuran ethanolamine derivatives (called CpX and CpY) behaving as LPA 1-3 agonists. Initially found as rabbit urethra contracting agents, their elusive receptors were identified from [ 35 S]GTP γ S-binding and β -arrestin2 recruitment investigations and then confirmed by [ 3 H]CpX binding studies (urethra, hLPA 1-2 membranes). Both compounds induced a calcium response in hLPA 1-3 cells within a range of 0.4-1.5-log lower potency as compared with LPA. The contractions of rabbit urethra strips induced by these compounds perfectly matched binding affinities with values reaching the two-digit nanomolar level. The antagonist, KI16425, dose-dependently antagonized CpX-induced contractions in agreement with its affinity profile (LPA 1 ≥LPA 3 >>LPA 2 ). The most potent agonist, CpY, doubled intraurethral pressure in anesthetized female rats at 3 µg/kg i.v. Alternatively, CpX was shown to inhibit human preadipocyte differentiation, a process totally reversed by KI16425. Together with original molecular docking data, these findings clearly established these molecules as potent agonists of LPA 1-3 and consolidated the pivotal role of LPA 1 in urethra/prostate contraction as well as in fat cell development. The discovery of these unique and less labile LPA 1-3 agonists would offer new avenues to investigate the roles of LPA receptors. SIGNIFICANCE STATEMENT: We report the identification of benzofuran ethanolamine derivatives behaving as potent selective nonlipid LPA 1-3 agonists and shown to alter urethra muscle contraction or preadipocyte differentiation. Unique at this level of potency, selectivity, and especially stability, compared with lysophosphatidic acid, they represent more appropriate tools for investigating the physiological roles of lysophosphatidic acid receptors and starting point for optimization of drug candidates for therapeutic applications., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Background and Purpose: The Na V 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target., Experimental Approach: We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models., Key Results: We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNa V 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNa V 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay., Conclusions and Implications: The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide., (© 2019 The British Pharmacological Society.)

Inwardly rectifying I K1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding K ir 2.1 channels, which primarily conduct ventricular I K1 , are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of K ir 2.1-mediated I K1 currents is a cardiosafety concern during new drug discovery and development. K ir 2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of K ir 2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human K ir 2.1 channels. We reproduced key electrophysiological and pharmacological features known for native I K1 , including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the K ir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of this assay authorizes medium throughput screening for cardiosafety purposes and could help to enrich the currently limited K ir 2.1 pharmacology.

[This corrects the article DOI: 10.3389/fphar.2018.01000.].

Although necessary for human survival, pain may sometimes become pathologic if long-lasting and associated with alterations in its signaling pathway. Opioid painkillers are officially used to treat moderate to severe, and even mild, pain. However, the consequent strong and not so rare complications that occur, including addiction and overdose, combined with pain management costs, remain an important societal and economic concern. In this context, animal venom toxins represent an original source of antinociceptive peptides that mainly target ion channels (such as ASICs as well as TRP, Ca V , K V and Na V channels) involved in pain transmission. The present review aims to highlight the Na V 1.7 channel subtype as an antinociceptive target for spider toxins in adult dorsal root ganglia neurons. It will detail (i) the characteristics of these primary sensory neurons, the first ones in contact with pain stimulus and conveying the nociceptive message, (ii) the electrophysiological properties of the different Na V channel subtypes expressed in these neurons, with a particular attention on the Na V 1.7 subtype, an antinociceptive target of choice that has been validated by human genetic evidence, and (iii) the features of spider venom toxins, shaped of inhibitory cysteine knot motif, that present high affinity for the Na V 1.7 subtype associated with evidenced analgesic efficacy in animal models.

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of Na V 1.7 subtype of voltage-gated sodium (Na V ) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and Na V 1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of Na V 1.6, compared to Na V 1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNa V 1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the Na V 1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.

Over the last six decades, voltage-gated sodium (Na v ) channels have attracted a great deal of scientific and pharmaceutical interest, driving fundamental advances in both biology and technology. The structure and physiological function of these channels have been extensively studied; clinical and genetic data have uncovered their implication in diseases such as epilepsy, arrhythmias, and pain, bringing them into focus as current and future drug targets. While different techniques have been established to record the activity of Na v channels, proper determination of their properties still presents serious challenges, depending upon the experimental conditions and the desired subtype of channel to be characterized. The aim of this unit is to review the characteristics of Na v channels, their properties, the cells in which they can be studied, and the currently available techniques. Topics covered include the determination of Na v -channel biophysical properties as well as the use of toxins to discriminate between subtypes using electrophysiological or optical methods. Perspectives on the development of high-throughput screening assays with their advantages and limitations are also discussed to allow a better understanding of the challenges encountered in voltage-gated sodium channel preclinical drug discovery. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Since the cloning of its first member in 1998, transient receptor potential (TRP) cation channels have become one of the most studied ion channel families in drug discovery. These channels, almost all calcium permeant, have been studied in many different (patho)-physiological and therapeutic areas as diverse as pain; neurodegenerative, cardiovascular, and inflammatory diseases; and cancer. At the same time, implementation of automated electrophysiology screening platforms has significantly increased the tractability of ion channels, mainly voltage gated, as drug targets. The work presented in this article shows the design and validation of TRP screening assays using the IonWorks Quattro platform (Molecular Devices, Sunnyvale, CA), allowing a significant increase in throughput to support drug discovery programs. This new player has a direct impact on resources and timelines by prioritizing potential candidates and reducing the number of molecules requiring final testing by manual patch-clamp, which is still today the gold standard technology for this challenging drug target class.

A new member of the two transmembrane domain potassium (K+) channel family was identified and isolated from a human brain cDNA library. The cDNA clone contains an open reading frame which encodes a 360 amino acid sequence with a characteristic P domain flanked by two hydrophobic regions representing the membrane spanning segments. The closest homologue of this gene product is the inwardly rectifying potassium channel subunit, Kir1.2 (identity approximately 42%). Northern blot analysis of human tissues with a selective cDNA probe for this new K+ subunit showed a single major transcript of 3.4 kb predominantly expressed at high levels in small intestine, with lower levels in stomach, kidney and brain. The main regions of expression in the central nervous system were medulla, hippocampus and corpus callosum. cRNA-injected oocytes and transiently transfected HEK293 cells expressed a K+ conductance which displays an inward rectification. This conductance is blocked by cesium and barium but is insensitive to tolbutamide and diazoxide even upon co-transfection of this novel subunit with the plasmid encoding the sulfonylurea receptor SUR1. Taken together, these results demonstrate that we have isolated and characterized a novel K+ channel subunit belonging to the inwardly rectifying K+ (Kir) channel family to which, upon homology classification, we have given the nomenclature Kir7.1.

A rise of intracellular calcium concentration triggered by the engagement of various membrane receptors is a key event in the control of cell growth. This increase involves both a release of calcium from intracellular stores and the opening of a transmembrane calcium conductive pathway. Using video imaging to measure intracellular calcium concentration in individual fura-2-loaded cells, we detected a defect in calcium influx in lymphocytes and fibroblasts collected from patients affected by a rare and new form of primary immunodeficiency. In these cells, pharmacological agents such as thapsigargin or ionomycin, and the physiological activator bradykinin, only induced transient increases in cytoplasmic calcium level, due to the emptying of internal stores, while in control cells, this initial step is followed by an additional and sustained transmembrane calcium influx. The fact that calcium influx is absent in patient's fibroblasts indicates that the related deficiency, which is clinically associated with a lack of proliferation of T lymphocytes, also affects cells of the non-hematopoietic lineages. This study emphasizes the adequacy of single cell imaging for determining whether some forms of pathologies are associated with a disregulation of ionic fluxes, and for identifying them accurately.