Value of a secretomic approach for distinguishing patients with COVID-19 viral pneumonia among patients with respiratory distress admitted to intensive care unit.

In intensive care units, COVID-19 viral pneumonia patients (VPP) present symptoms similar to those of other patients with Nonviral infection (NV-ICU). To better manage VPP, it is therefore interesting to better understand the molecular pathophysiology of viral pneumonia and to search for biomarkers that may clarify the diagnosis. The secretome being a set of proteins secreted by cells in response to stimuli represents an opportunity to discover new biomarkers. The objective of this study is to identify the secretomic signatures of VPP with those of NV-ICU. Plasma samples and clinical data from NV-ICU (n = 104), VPP (n = 30) or healthy donors (HD, n = 20) were collected at Nantes Hospital (France) upon admission. Samples were enriched for the low-abundant proteins and analyzed using nontarget mass spectrometry. Specifically deregulated proteins (DEP) in VPP versus NV-ICU were selected. Combinations of 2 to 4 DEPs were established. The differences in secretome profiles of the VPP and NV-ICU groups were highlighted. Forty-one DEPs were specifically identified in VPP compared to NV-ICU. We describe five of the best combinations of 3 proteins (complement component C9, Ficolin-3, Galectin-3-binding protein, Fibrinogen alpha, gamma and beta chain, Proteoglycan 4, Coagulation factor IX and Cdc42 effector protein 4) that show a characteristic receptor function curve with an area under the curve of 95.0%. This study identifies five combinations of candidate biomarkers in VPP compared to NV-ICU that may help distinguish the underlying causal molecular alterations.

Nav1.2 and BK channel interaction shapes the action potential in the axon initial segment.

In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm ) reaches the threshold for activation of the voltage-gated Na+ channels (VGNCs) Nav 1.2 and Nav 1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na+ , Vm and Ca2+ imaging in combination with partial block of Nav 1.2 by the peptide G1 G4 -huwentoxin-IV, we demonstrate an exclusive role of Nav 1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Nav 1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca2+ -activated K+ channels (CAKCs). Indeed, Ca2+ influx via Nav 1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9-anhydrotetrodotoxin, a moderately selective inhibitor of Nav 1.6, we concluded that the Ca2+ influx shaping the early phase of the AP is exclusive of Nav 1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav 1.2 reported here is important for understanding the physiology and pathology of neuronal excitability. KEY POINTS: We optically analysed the action potential generated in the axon initial segment of mouse layer-5 neocortical pyramidal neurons and its associated Na+ and Ca2+ currents using ultrafast imaging techniques. We found that partial selective block of the voltage-gated Na+ channel Nav 1.2, produced by a recently developed peptide, widens the shape of the action potential in the distal part of the axon initial segment. We demonstrate that this effect is due to a reduction of the Ca2+ influx through Nav 1.2 that activates BK Ca2+ -activated K+ channels. To validate our conclusions, we generated a neuron model that reproduces the ensemble of our experimental results. The present results indicate a specific role of Nav 1.2 in the axon initial segment for shaping of the action potential during its generation.

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells.

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the ß-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Screening an In-House Isoquinoline Alkaloids Library for New Blockers of Voltage-Gated Na+ Channels Using Voltage Sensor Fluorescent Probes: Hits and Biases.

Voltage-gated Na+ (NaV) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel NaV channel ligands. With the objective of discovering new blockers of NaV channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of NaV channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC50. These five alkaloids were then assayed using the Na+ fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNaV1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na+ currents elicited by the hNaV1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na+ currents elicited by the hNav1.2 and hNav1.6 channels, with IC50 values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block NaV channels, with promising therapeutical applications.

Modelling sudden cardiac death risks factors in patients with coronavirus disease of 2019: the hydroxychloroquine and azithromycin case.

AIMS: Coronavirus disease of 2019 (COVID-19) has rapidly become a worldwide pandemic. Many clinical trials have been initiated to fight the disease. Among those, hydroxychloroquine and azithromycin had initially been suggested to improve clinical outcomes. Despite any demonstrated beneficial effects, they are still in use in some countries but have been reported to prolong the QT interval and induce life-threatening arrhythmia. Since a significant proportion of the world population may be treated with such COVID-19 therapies, evaluation of the arrhythmogenic risk of any candidate drug is needed. METHODS AND RESULTS: Using the O'Hara-Rudy computer model of human ventricular wedge, we evaluate the arrhythmogenic potential of clinical factors that can further alter repolarization in COVID-19 patients in addition to hydroxychloroquine (HCQ) and azithromycin (AZM) such as tachycardia, hypokalaemia, and subclinical to mild long QT syndrome. Hydroxychloroquine and AZM drugs have little impact on QT duration and do not induce any substrate prone to arrhythmia in COVID-19 patients with normal cardiac repolarization reserve. Nevertheless, in every tested condition in which this reserve is reduced, the model predicts larger electrocardiogram impairments, as with dofetilide. In subclinical conditions, the model suggests that mexiletine limits the deleterious effects of AZM and HCQ. CONCLUSION: By studying the HCQ and AZM co-administration case, we show that the easy-to-use O'Hara-Rudy model can be applied to assess the QT-prolongation potential of off-label drugs, beyond HCQ and AZM, in different conditions representative of COVID-19 patients and to evaluate the potential impact of additional drug used to limit the arrhythmogenic risk.

Maurocalcin and its analog MCaE12A facilitate Ca2+ mobilization in cardiomyocytes.

Ryanodine receptors are responsible for the massive release of calcium from the sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the effect of MCa and its analog MCaE12A on isolated cardiac ryanodine receptor (RyR2), and showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ transients, calcium sparks and contraction on cardiomyocytes isolated from adult rats or differentiated from human-induced pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes and promotes the abnormal opening of RyR2. We also investigated the effect of MCaE12A on the pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A improves pacemaker activity of sinus node cells obtained from mice lacking L-type Cav1.3 channel, or following selective pharmacologic inhibition of calcium influx via Cav1.3. Our results identify MCaE12A as a high-affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca2+ homeostasis and dynamic of cardiac cells.

An O-GlcNAcylomic Approach Reveals ACLY as a Potential Target in Sepsis in the Young Rat.

Sepsis in the young population, which is particularly at risk, is rarely studied. O-GlcNAcylation is a post-translational modification involved in cell survival, stress response and metabolic regulation. O-GlcNAc stimulation is beneficial in adult septic rats. This modification is physiologically higher in the young rat, potentially limiting the therapeutic potential of O-GlcNAc stimulation in young septic rats. The aim is to evaluate whether O-GlcNAc stimulation can improve sepsis outcome in young rats. Endotoxemic challenge was induced in 28-day-old rats by lipopolysaccharide injection (E. Coli O111:B4, 20 mg·kg-1) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharide injection, rats were randomly assigned to no therapy, fluidotherapy (NaCl 0.9%, 10 mL·kg-1) ± NButGT (10 mg·kg-1) to increase O-GlcNAcylation levels. Physiological parameters and plasmatic markers were evaluated 2h later. Finally, untargeted mass spectrometry was performed to map cardiac O-GlcNAcylated proteins. Lipopolysaccharide injection induced shock with a decrease in mean arterial pressure and alteration of biological parameters (p < 0.05). NButGT, contrary to fluidotherapy, was associated with an improvement of arterial pressure (p < 0.05). ATP citrate lyase was identified among the O-GlcNAcylated proteins. In conclusion, O-GlcNAc stimulation improves outcomes in young septic rats. Interestingly, identified O-GlcNAcylated proteins are mainly involved in cellular metabolism.

Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Functional Impact of BeKm-1, a High-Affinity hERG Blocker, on Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells.

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

Aptamer Efficacies for In Vitro and In Vivo Modulation of αC-Conotoxin PrXA Pharmacology.

The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.

Arrhythmias precede cardiomyopathy and remodeling of Ca2+ handling proteins in a novel model of long QT syndrome.

AIM: Deletion of QKP1507-1509 amino-acids in SCN5A gene product, the voltage-gated Na+ channel Nav1.5, has been associated with a large phenotypic spectrum of type 3 long QT syndrome, conduction disorder, dilated cardiomyopathy and high incidence of sudden death. The aim of this study was to develop and characterize a novel model of type 3 long QT syndrome to study the consequences of the QKP1507-1509 deletion. METHODS AND RESULTS: We generated a knock-in mouse presenting the delQKP1510-1512 mutation (Scn5a+/ΔQKP) equivalent to human deletion. Scn5a+/ΔQKP mice showed prolonged QT interval, conduction defects and ventricular arrhythmias at the age of 2 weeks, and, subsequently, structural defects and premature mortality. The mutation increased Na+ window current and generated a late Na+ current. Ventricular action potentials from Scn5a+/ΔQKP mice were prolonged. At the age of 4 weeks, Scn5a+/ΔQKP mice exhibited a remodeling leading to [Ca2+]i transients with higher amplitude and slower kinetics, combined with enhanced SR Ca2+ load. SERCA2 expression was not altered. However, total phospholamban expression was higher whereas the amount of Ca2+-calmodulin-dependent kinase II (CaMKII)-dependent T17-phosphorylated form was lower, in hearts from 4-week-old mice only. This was associated with a lower activity of CaMKII and lower calmodulin expression. In addition, Scn5a+/ΔQKP cardiomyocytes showed larger Ca2+ waves, correlated with the presence of afterdepolarizations during action potential recording. Ranolazine partially prevented action potential and QT interval prolongation in 4-week-old Scn5a+/ΔQKP mice and suppressed arrhythmias. CONCLUSION: The Scn5a+/ΔQKP mouse model recapitulates the clinical phenotype of mutation carriers and provides new and unexpected insights into the pathological development of the disease in patients carrying the QKP1507-1509 deletion.

Bioinformatic analysis of a plakophilin-2-dependent transcription network: implications for the mechanisms of arrhythmogenic right ventricular cardiomyopathy in humans and in boxer dogs.

AIMS: Previous studies in murine hearts and in cell systems have shown that modifications in the expression or sequence integrity of the desmosomal molecule plakophilin-2 (PKP2) can alter the downstream expression of transcripts necessary for the electrical and mechanical function of the heart. These findings have provided support to mechanistic hypotheses that seek to explain arrhythmogenic right ventricular cardiomyopathy (ARVC) in humans. However, the relation between PKP2 expression and the transcriptome of the human heart remains poorly explored. Furthermore, while a number of studies have documented the clinical similarity between familial ARVC in humans and inheritable ARVC in boxer dogs, there is a puzzling lack of convergence as to the possible genetic causes of disease in one species vs. the other. METHODS AND RESULTS: We implemented bioinformatics analysis tools to explore the relation between the PKP2-dependent murine and human transcriptomes. Our data suggest that genes involved in intracellular calcium regulation, and others involved in intercellular adhesion, form part of a co-ordinated gene network. We further identify PROX1 and PPARA (coding for the proteins Prox1 and PPAR-alpha, respectively) as transcription factors within the same network. CONCLUSION: On the basis our analysis, we hypothesize that the molecular cascades initiated by the seemingly unrelated genetic mutations in humans and in boxers actually converge downstream into a common pathway. This can explain the similarities in the clinical manifestation of ARVC in humans and in the boxer dogs.

Analysis of the effect of the scorpion toxin AaH-II on action potential generation in the axon initial segment.

The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na+ channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Nav1.2 and Nav1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na+ imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na+ influx in the AIS. Finally, using ultrafast Ca2+ imaging, we observed that 7 nM AaH-II produces a spurious slow Ca2+ influx via Ca2+-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.

SARS-CoV-2 E and 3a Proteins Are Inducers of Pannexin Currents.

Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses induced by these proteins. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly acquired round shapes that detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this by using flow cytometry. In adhering cells expressing E or 3a protein, the whole-cell currents were not different from those of the control, suggesting that E and 3a proteins are not plasma membrane viroporins. In contrast, recording the currents on detached cells uncovered outwardly rectifying currents much larger than those observed in the control. We illustrate for the first time that carbenoxolone and probenecid block these outwardly rectifying currents; thus, these currents are most probably conducted by pannexin channels that are activated by cell morphology changes and also potentially by cell death. The truncation of C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent these outwardly rectifying currents. This suggests distinct pathways for the induction of these cellular events by the two proteins. We conclude that SARS-CoV-2 E and 3a proteins are not viroporins expressed at the plasma membrane.

Structure-function relationship of new peptides activating human Nav1.1.

Nav1.1 is an important pharmacological target as this voltage-gated sodium channel is involved in neurological and cardiac syndromes. Channel activators are actively sought to try to compensate for haploinsufficiency in several of these pathologies. Herein we used a natural source of new peptide compounds active on ion channels and screened for drugs capable to inhibit channel inactivation as a way to compensate for decreased channel function. We discovered that JzTx-34 is highly active on Nav1.1 and subsequently performed a full structure-activity relationship investigation to identify its pharmacophore. These experiments will help interpret the mechanism of action of this and formerly identified peptides as well as the future identification of new peptides. We also reveal structural determinants that make natural ICK peptides active against Nav1.1 challenging to synthesize. Altogether, the knowledge gained by this study will help facilitate the discovery and development of new compounds active on this critical ion channel target.

Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin.

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

In vivo spatiotemporal control of voltage-gated ion channels by using photoactivatable peptidic toxins.

Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.

A functional network of highly pure enteric neurons in a dish.

The enteric nervous system (ENS) is the intrinsic nervous system that innervates the entire digestive tract and regulates major digestive functions. Recent evidence has shown that functions of the ENS critically rely on enteric neuronal connectivity; however, experimental models to decipher the underlying mechanisms are limited. Compared to the central nervous system, for which pure neuronal cultures have been developed for decades and are recognized as a reference in the field of neuroscience, an equivalent model for enteric neurons is lacking. In this study, we developed a novel model of highly pure rat embryonic enteric neurons with dense and functional synaptic networks. The methodology is simple and relatively fast. We characterized enteric neurons using immunohistochemical, morphological, and electrophysiological approaches. In particular, we demonstrated the applicability of this culture model to multi-electrode array technology as a new approach for monitoring enteric neuronal network activity. This in vitro model of highly pure enteric neurons represents a valuable new tool for better understanding the mechanisms involved in the establishment and maintenance of enteric neuron synaptic connectivity and functional networks.

Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp.

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.