**Diana Chapeton-Montes 1, Holger Brüggemann 2, Michel R. Popoff 1,\***


**Abstract:** Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) share similar structures and molecular modes of action, albeit BoNTs target motor-neuron endings leading to a flaccid paralysis (botulism) and TeNT interacts with central inhibitory interneurons causing spastic paralysis (tetanus). BoNTs and TeNT are produced by anaerobic and sporulating bacteria of the *Clostridium* genus, *C. botulinum* and *C. tetani*, respectively. The synthesis of BoNTs and TeNT is a highly regulated process. Among the diverse *C. botulinum* types, toxinogenesis was mainly investigated in *C. botulinum* A. In both *C. botulinum* A and *C. tetani*, a gene coding for an alternative sigma factor lies upstream of the neurotoxin gene and is required for the expression of the toxin gene. *C. botulinum* and *C. tetani* contain numerous two-component systems (TCSs) (39 in *C. botulinum* A and 30 in *C. tetani*), which are major regulatory systems in response to environmental conditions in prokaryotes. Albeit most TCS genes are homologous in both species, *C. botulinum* A and *C. tetani* use distinct sets of TCSs in the regulation of toxin synthesis. Only one TCS, which is homologous in *C. botulinum* A and *C. tetani*, has a similar function of negative regulation of toxin synthesis in both species. In contrast to *C. botulinum* A, inorganic phosphate and carbonate are environmental factors controlling toxin synthesis in *C. tetani.* In addition, a non-coding RNA downstream the *tent* gene negatively regulates TeNT synthesis. No homologous RNA sequence was found in *C. botulinum* A. Thereby, *C. botulinum* and *C. tetani* are two environmental bacteria that use distinct regulatory pathways to synthesize potent neurotoxins.

**Keywords:** *Clostridium botulinum*; *Clostridium tetani*; botulinum neurotoxin; tetanus neurotoxin; toxin synthesis regulation

#### **References**


*5.8. In Vivo Spatiotemporal Control of Voltage-Gated Ion Channels by Engineered Photoactivatable Peptidic Toxins*

**Jérôme Montnach 1,2, Laila Ananda Blömer 2,3, Ludivine Lopez 1,2,4, Luiza Filipis 2,3, Hervé Meudal 5, Aude Lafoux 6, Sébastien Nicolas 1,2, Duong Chu 7, Cécile Caumes 4, Rémy Béroud 4, Chris Jopling 8, Frank Bosmans 9, Corinne Huchet 6, Céline Landon 5, Marco Canepari 2,3, Michel De Waard 1,2,4,\***

<sup>1</sup> L'institut du thorax, INSERM, CNRS, UNIV NANTES, F-44007 Nantes, France.

<sup>2</sup> Laboratory of Excellence Ion Channels, Science & Therapeutics, F-06560 Valbonne, France. <sup>3</sup> Laboratoire Interdisciplinaire de Physique, Université Grenoble Alpes, CNRS UMR 5588, 38402 Saint Martin-d'Hères cedex, France.

<sup>4</sup> Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France.

<sup>5</sup> Center for Molecular Biophysics, CNRS, rue Charles Sadron, CS 80054, Orléans 45071, France.

<sup>6</sup> Therassay Platform, IRS2-Université de Nantes, Nantes, France.

<sup>7</sup> Queen's University Faculty of Medicine, Kingston, ON, Canada.

<sup>8</sup> Institut de Génomique Fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier, France.

<sup>9</sup> Department of Basic and Applied Medical Sciences, Ghent University, Ghent, Belgium. \* Correspondence: michel.dewaard@univ-nantes.fr

**Abstract:** Photoactivatable drugs targeting ligand-gated ion channels opened 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. We developed caged HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels. We first validated physico-chemical parameters of photolysis, and by using a high-throughput patch-clamp system (SyncroPatch364, Nanion), we validated the activity and photosensitivity of caged HwTxIV-Nvoc in vitro in HEK293 cells. We further pursued our investigations in ex vivo brain slices and in vivo on mice neuromuscular junctions and zebrafish models. We found that caged HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. We developed multiple photoactivatable toxins and therefore demonstrated the broad applicability of the toxin-photoactivation technology as a tool for experiments but also as a relevant clinical approach in disease management.

**Keywords:** ion channel; photopharmacology; toxin

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

**Stephan De Waard 1,2,#, Jérôme Montnach 1,#, Barbara Ribeiro 1, Sébastien Nicolas 1, Virginie Forest 1, Flavien Charpentier 1, Matteo Elia Mangoni 2,3, Nathalie Gaborit 1, Michel Ronjat 1,2, Gildas Loussouarn 1, Patricia Lemarchand 1, Michel De Waard 1,2,4,\***

<sup>1</sup> L'institut du thorax, INSERM, CNRS, Université de Nantes, F-44007 Nantes, France.

<sup>2</sup> LabEx Ion Channels, Science & Therapeutics, F-06560 Valbonne, France.

<sup>3</sup> Institut de Génomique Fonctionnelle, CNRS, INSERM, Université de Montpellier, F34094 Montpellier, France.

<sup>4</sup> Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France.

\* Correspondence: michel.dewaard@univ-nantes.fr

**Abstract:** 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 with 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. By 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 an easily modifiable peptide, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

**Keywords:** BeKm-1; hERG; hiPS-cardiomyocyte; LQTS

*5.10. An Agonist of the CXCR4 Receptor Is Therapeutic for the Neuroparalysis Induced by Bungarus Snake Envenoming*

**Marco Stazi 1,\*, Federico Fabris 1, Kae Yi Tan 2, Aram Megighian 1, Alessandro Rubini 1, Andrea Mattarei 3, Samuele Negro 1, Giorgia D'Este 1, Florigio Lista 4, Ornella Rossetto 1, Choo Hock Tan 5, Cesare Montecucco 1,6**

<sup>1</sup> Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, Padova 35131, Italy.

<sup>2</sup> Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.

<sup>3</sup> Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova 35131, Italy.

<sup>4</sup> Center of Medical and Veterinary Research of the Ministry of Defense, Policlinico Militare, Via Santo Stefano Rotondo 4, Rome 00184, Italy.

<sup>5</sup> Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.

<sup>6</sup> CNR Institute of Neuroscience, Padova 35131, Italy.

\* Correspondence: marco.stazi@studenti.unipd.it

**Abstract:** Snake envenoming is a major but neglected human tropical disease. Among venomous snakes, kraits (snakes of the *Bungarus* genus) are medically important venomous species that cause reversible peripheral neuroparalysis. Hospitalization and use of antivenoms derived from an animal immunized with *Bungarus* venoms are the primary therapies that prevent death from early-onset respiratory paralysis. There is a general consensus that additional and non-expensive treatments, which can be delivered even long after the snakebite, are needed. Traumatic or toxic degeneration of peripheral motor neurons, with ensuing neuroparalysis, is characterized by the activation of a pro-regenerative intercellular signaling program. A major player is the intercellular signaling axis consisting of the chemokine CXCL12a, produced by perisynaptic Schwann cells and acting on the CXCR4 receptor expressed on the damaged neuronal axons. The CXCR4 agonist NUCC-390 was recently found to promote axonal growth. Here, we tested its efficacy on the neuroparalysis induced by the venoms of three major krait species, i.e., *Bungarus caeruleus*, *B. multicinctus* and *B. candidus* that are prevalent in Asia. These venoms cause a complete degeneration of motor axon terminals. Functional recovery of the neuromuscular junction was assessed by electrophysiological recordings and by imaging. We report that NUCC-390 administration to venom-injected mice greatly accelerates the recovery from paralysis. These data candidate NUCC-390 to be tested as novel therapeutics to reduce death by respiratory deficits and to improve the recovery of normal neuromuscular physiology, thus reducing the human and hospital costs of envenoming. NUCC-390 can be administered any time after the snakebite and has great potential of becoming a non-expensive addition to the currently available antivenom treatments whose efficacy is limited to a short period after snakebite. This drug is expected to decrease the long and expensive post-snakebite mechanical ventilation phase.

**Keywords:** *Bungarus* venom; CXCR4 agonist; neuromuscular junction

*5.11. Neutralization of Crotamine by Polyclonal Antibodies against Two Rattlesnake Venoms and a Novel Recombinant Fusion Protein*

**Roberto Ponce-López 1, Alejandro Olvera-Rodríguez 1,\*, Miguel Borja-Jiménez 2, Edgar Neri-Castro 1, Leticia Olvera-Rodríguez 1, Alejandro Alagón <sup>1</sup>**

<sup>1</sup> Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.

<sup>2</sup> Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango, Gómez Palacio, Durango, México.

\* Correspondence: alejandro.olvera@ibt.unam.mx

**Abstract:** Current pit-viper antivenoms for human use in Mexico have shown a low level of protection against one neurotoxin found in some rattlesnakes (*Crotalus* spp.), crotamine. This toxin has a low molecular weight (~5 kDa) and is well known for its spastic paralysis symptom provoked in mice with no evidence of neutralization. Recently, it was reported that crotamine is the major toxin found in some rattlesnake venoms such as *C. molossus nigrescens* (~50%) and *C. oreganus helleri* (62%). On the other hand, sphingomyelinase D (SMD) is a highly immunogenic enzyme (MW: ~30 kDa) found in *Loxosceles* spp. spider venom. In this study, we aimed to neutralize the crotamine-induced main symptom of paralysis by employing, as immunogens, two rattlesnake venoms and a novel recombinant fusion protein made from crotamine and SMD, used as a carrier. **Methods**—Crotamine cDNA was synthetized from venom gland mRNA of a *C. m. nigrescens* individual from Mexico, while one plasmid containing *L. reclusa* SMD was available in the lab. We cloned the sphingomyelinase D and crotamine in tandem into the expression vector pQE30 to transform competent Origami *Escherichia coli* cells for the production of the protein. By using the recombinant protein and the whole venoms of *C. o. helleri* and *C. m. nigrescens*, we performed separate immunization protocols in rabbits to measure the antibody recognition to crotamine (ELISA and Western blot) and the neutralization capacity in mice against the main toxin-induced paralysis symptom. **Results**—The fusion protein was obtained at ~10 mg/L of bacterial culture with the expected 37.5 kDa molecular mass as analyzed by SDS-PAGE and Western-blot. The recombinant protein and the two whole crotamineenriched venoms generated antibodies with cross-reactivity against crotamine from up to seven species. The three experimental antivenoms were able to neutralize the paralysis symptom provoked by crotamine in the mice model. **Discussion/Conclusion**—We showed, for the first time, the neutralization of the crotamine-induced spastic paralysis symptom by three experimental antivenoms. The recombinant SMD-Crotamine fusion protein as well as crotamine-enriched venoms could be good candidate immunogens for the improvement of Mexican antivenoms.

**Keywords:** antivenom; crotamine; rattlesnake; sphingomyelinase-D

**Acknowledgments:** Fordecyt 303045.

*5.12. Anti-Cancer Effect of the Moroccan cobra (Naja haje) Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture*

**Ayoub Lafnoune 1,2,\*, Su-Yeon Lee 3, Jin-Yeong Heo 4, Salma Chakir 1,5, Khadija Daoudi 1,2, Bouchra Darkaoui 1,2, Aziz Hmyene 5, Rachida Cadi 2, Khadija Mounaji 2, David Shum 4, Haeng-Ran Seo 3, Naoual Oukkache <sup>1</sup>**

<sup>1</sup> Laboratoire des Venins et Toxines, Département de Recherche, Institut Pasteur du Maroc, 1 place Louis Pasteur, Casablanca 20360, Morocco.

<sup>2</sup> Laboratoire Physiopathologie, Génétique Moléculaire & Biotechnologie, Faculté des Sciences Ain-Chock, Hassan II University of Casablanca, B.P 5366 Maarif, Casablanca 20000, Morocco.

<sup>3</sup> Cancer Biology Research Laboratory, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488 Rep. of Korea.

<sup>4</sup> Screening Discovery Platform, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488 Rep. of Korea.

<sup>5</sup> Laboratoire de Biochimie Environnement et Agroalimentaire, Faculté des Sciences et Techniques de Mohammedia, Mohammedia, Morocco.

\* Correspondence: ayoublafnoune@gmail.com

**Abstract:** Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer-related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine, is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra *Naja haje* venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of *N. haje* venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of *N. haje* venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cell proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

**Keywords:** anticancer molecule; hepatocellular carcinoma; multicellular tumor spheroid; *Naja haje*; venom

#### **6. Poster Presentations (When More than One Author, the Underlined Name Is That of the Presenter)**

*6.1. Circadian Variations in Inflammatory Response and Oxidative Stress during Experimental Scorpion Envenomation*

#### **Fares Daachi, Sonia Adi-Bessalem \*, Amal Megdad-Lamraoui, Fatima Laraba-Djebari**

University of Science and Technology Houari Boumediene (USTHB), Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP 32 El-Alia, Bab Ezzouar, Algiers, Algeria.

\* Correspondence: soniabessalem@hotmail.com

**Abstract:** The mammalian circadian clock orchestrates diverse physiological processes by synchronizing with the nervous and cardiovascular systems, immune response, and metabolic homeostasis. Little is known about the circadian rhythm in the immune response in vivo settings during envenomation—few studies have demonstrated that a circadian pattern might exist. The aim of this study is to investigate whether circadian rhythm affects the inflammation and oxidative stress in envenomed model. In this study, the systemic inflammatory response is induced by the administration of a sublethal dose of *Androctonus australis hector* (Aah) venom to mice during light (1 HALO) and dark (18 HALO) periods. The hypothalamic–pituitary–adrenal (HPA) axis activity was evaluated by measurement of adrenocorticotropic (ACTH) and corticosterone plasma hormones levels as well as by analysis of CD68 immunohistochemistry staining. The inflammation was assessed by the measurement of the serum level of the proinflammatory cytokines, IL-17 and IL-6, evaluation of neutrophil infiltration and oxidative/nitrosative stress markers (Nitrites, MDA, H2O2, CAT and GSH). Glucose level and activities of AST and ALT as well as histopathological analysis of hepatic tissue, were also performed in the two groups of

animals (1 HALO and 18 HALO). Obtained results showed a high increase of serum ACTH and corticosterone levels as well as positive immunostaining of CD68 cells during the dark period indicating activation of the HPA axis associated with local inflammation. The results also showed day-night variations, with significantly high levels of nitrite, hydrogen peroxide, myeloperoxidase and lipid peroxidation during the daytime compared with the nighttime. Significant variations in catalase activity and GSH levels are also observed with the highest evening values. Furthermore, an increase in IL-6 levels was observed during the active phase, while no differences were observed in the IL-17 levels between day and night times. These results indicate also that Aah venom induced a time-dependent increase of metabolic parameters during the dark phase and severe hepatocellular injury. In conclusion, the daily variation of the HPA axis activation and inflammatory response appears to be closely related to the circadian moment of envenomation. Future studies should investigate the molecular mechanisms resulting in this circadian rhythmicity.

**Keywords:** circadian variation; inflammation; oxidative stress; scorpion envenomation

*6.2. Synthesis and Characterization of μ-Conotoxins as Molecular Probes for the Detection of Voltage-Gated Sodium Channel Pore Blockers*

## **Rómulo Aráoz 1,2,\*, Giovanny Covaleda-Cortés 1, Denis Servent 1,2**

<sup>1</sup> Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SIMoS, 91191 Gif-sur-Yvette, France. <sup>2</sup> CNRS, ERL9004, 91191 Gif-sur-Yvette, France.

\* Correspondence: romulo.araoz@cea.fr

**Abstract:** Voltage-gated sodium channels (Nav) play critical functional roles by controlling, in excitable and non-excitable cells, the action potential initiation/propagation, cell motility, and proliferation. In humans, there are nine subtypes of Nav channels—1.1 to 1.9—with Nav1.4 being the muscle subtype. Nav channel dysfunctions are associated with neurological, cardiovascular, muscular and psychiatric disorders. Marine phytoplankton and freshwater cyanobacteria are able to synthesize a series of neurotoxins targeting Nav channels, among them, saxitoxin, a potent site-1 pore blocker classified as a chemical bioweapon because of its high lethality for humans. Receptor binding assays are suitable analytical techniques for ligand screening owing to their extremely high resolution and sensitivity, their fast analysis and easy automation, which facilitates the development of high-throughput protocols. There is, however, a lack of toxin-tracers for Nav toxins detection. The marine cone snail of the genus *Conus* produces a series of conotoxins for prey hunting, among them, μ-conotoxin peptides, with an exquisite affinity for Nav channels. Wild-type and biotinylated synthesis of five μ-conotoxins was performed using a Protein Technologies Prelude synthesizer. Biotinylation was performed on-column after Fmoc deprotection of the N-terminal residue of the μ-conotoxins. After refolding, the resulting conopeptides were purified by reverse-phase HPLC. The affinity of each μ-conotoxin for the Nav1.4 channel was determined. Two biotinylated μ-conopeptides with high affinity for Nav channels were identified as potential toxin tracers for the detection of site-1 Nav pore blockers by ligand binding assay.

**Keywords:** cyanotoxin; ligand-binding assay; μ-conopeptide; phycoyoxin; sodium channel **Acknowledgments:** This research was supported by the NRBC-E Program project MUL-TITOX (Fiche N◦ H35 to RA). The authors acknowledge the INTERREG Atlantic Area (ALERTOX-NET EAPA\_317/2016 project to DS), and the LABEX LERMIT (DETECTNEU-ROTOX project, CDE 2017–001173—RD 91 to RA)

*6.3. Multi-Scale Evaluation of Spider Toxins as Potential Anti-Nociceptive Agents: Example of Cyriotoxin-1a*

**Evelyne Benoit 1,\*, Michel De Waard 2, Rémy Béroud 3, Michel Partiseti 4, Denis Servent <sup>1</sup>**

<sup>1</sup> Service d'Ingénierie Moléculaire pour la Santé, ERL CNRS/CEA 9004, Gif-sur-Yvette, France.

<sup>2</sup> L'institut du thorax, INSERM UMR 1087/CNRS UMR 6291, Nantes, France.

<sup>3</sup> Smartox Biotechnology, Saint-Egrève, France.

<sup>4</sup> Sanofi R & D, Integrated Drug Discovery—High Content Biology, Vitry-sur-Seine, France. \* Correspondence: evelyne.benoit@cea.fr

**Abstract:** Our expertise is the identification and structural and functional characterization of original natural toxins targeting receptors and ion channels, which may have applications in human health. In this context, and in collaboration with *L'institut du thorax*, Smartox Biotechnology and Sanofi R & D, we are interested in the multi-scale evaluation (from the cell in vitro to the organism in vivo) of spider toxins as potential anti-nociceptive agents, as illustrated by the example of Cyriotoxin-1a (CyrTx-1a). NaV1.7 channel subtype is highly expressed in the dorsal root ganglia (DRG) of the sensory nervous system and plays a central role in the pain signaling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target. We used high-throughput screening of the venom library, using automated patchclamp experiments on human voltage-gated sodium channel subtypes, and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that were purified, sequenced and chemically synthesized. Analgesic effects were evaluated in mice in vivo. We identified and further characterized CyrTx-1a, a novel peptide isolated from *Cyriopagopus schioedtei* spider venom. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV1.1–1.3 and 1.6–1.7 in the low nanomolar range, compared to the micromolar range for hNaV1.4–1.5 and 1.8. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse DRG neurons in vitro and approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability in vivo. CyrTx-1a exhibited an analgesic effect in mice by significantly increasing reaction time in the hot-plate assay.

**Keywords:** anti-nociceptive toxin; mouse dorsal root ganglia neuron; mouse tactile and hot pain sensitivity; pain; voltage-gated sodium channel subtype

*6.4. Imaging of Muscular Nicotinic Receptor Distribution Using a Synthetic Fluorescent Analogue of α-Bungarotoxin*

**Oliver Brun 1,5,6,\*, Claude Zoukimian 3, Barbara Ribeiro De Oliveira Mendes 1, Jérôme Montnach 1, Michel Ronjat 1,2, Rémy Béroud 3, Didier Boturyn 4, Frédéric Lesage 5,7, Michel De Waard 1,2,3**

<sup>1</sup> L'institut du thorax, INSERM, CNRS, UNIV NANTES, Nantes, France.

<sup>2</sup> LabEx "Ion Channels, Science & Therapeutics", Valbonne, France.

<sup>3</sup> Smartox Biotechnology, 6 rue des Platanes, F-38120 Saint-Egrève, France.

<sup>4</sup> Department of Molecular Chemistry, Univ. Grenoble Alpes, CNRS, 570 rue de la chimie, CS 40700, Grenoble 38000, France.

<sup>5</sup> Research Center, Montreal Heart Institute, Montreal, QC, Canada.

<sup>6</sup> Faculty of Medicine, Université de Montréal, Montreal, QC, Canada.

<sup>7</sup> Department of Electrical Engineering, École Polytechnique de Montréal, Montréal, QC, Canada.

\* Correspondence: oliver.brun@univ-nantes.fr

**Abstract:** Characterizing the distribution of drug receptors through optical imaging can be challenging, as the availability of suitable fluorescent probes is limited. Over history, natural toxins have benefited science through an ever-growing source of biologically active peptides, many of which show great potential as probes to study the distribution of their associated receptors. Here, we aim at developing a synthetic, fluorescent analog of α-bungarotoxin (α-BgTx), a highly potent, selective and irreversible inhibitor of the

muscle-type nicotinic receptors (mnAChR), through the addition of a Cy5 tag. We first performed functional validation of this Cy5-α-BgTx analog by high-throughput patch-clamp (SyncroPatch364, Nanion), looking at the inhibition of acetylcholine-mediated currents on TE671 cells. It, therefore, appeared that the addition of the Cy5 tag still generates a peptide with nanomolar affinity. In order to further ensure that natural binding properties of the peptide were preserved, we synthesized a peptide analog of the α-BgTx binding site on the mnAChR (BSpep). Preincubation of Cy5-α-BgTx with BSpep resulted in the capture of the α-BgTx and complete blockage of its inhibitory effect. The fluorescent toxin was then used to study the distribution of mnAChR on fixed TE671 cells, allowing us to visualize both its intra and extracellular distribution. Thus, we were able to synthesize a functional, synthetic analog of α-BgTx that can be used to localize mnAChRs, highlighting the potential of toxin-derived biopeptide engineering as a tool for receptor imaging.

**Keywords:** α-bungarotoxin; peptide engineering; receptor imaging

*6.5. Proteomic Analysis of the Predatory Venom of Conus striatus Reveals Population-Specific Glycosylation Patterns in Kappa A-Conotoxins*

#### **Claudia Murraciole-Bich, Sébastien Dutertre \***

Institut des Biomolécules Max Mousseron (IBMM), Université de Montpellier, CNRS, ENSCM, Montpellier, France.

\* Correspondence: sebastien.dutertre@umontpellier.fr

**Abstract:** Animal venoms are a rich source of pharmacological compounds with ecological and evolutionary significance, as well as with therapeutic and biotechnological potentials. Among the most promising venomous animals, cone snails produce potent neurotoxic venom to facilitate prey capture and defend against aggressors. The main objective of this study was to investigate and decipher the composition of the predatory venom injected by *Conus striatus*, one of the largest and most widely distributed piscivorous species. Standard LC-MS analysis provided an overview of native venom compounds, whereas LC-MS/MS followed by bioinformatic analysis identified many conotoxin sequences. Kappa A-conotoxins, which contain three disulfide bridges and complex glycosylation, are by far the dominant compounds in predatory venom. Remarkably, different and populationspecific glycosylation patterns were detected in specimens from the Pacific and Indian oceans, providing a new level of intraspecific variations for the first time.

**Keywords:** conotoxin; *Conus striatus*; glycosylation; KappA-conotoxin

*6.6. Why Is Androctonus australis Venom from Far more Studied Than A. mauritanicus One? Does the Answer Reside in the Venom Composition?*

**Lou Freuville 1,\*, Fernanda Gobbi Amorim 1, Alain Brans 2, Rudy Fourmy 3, Aude Violette 3, Loïc Quinton <sup>1</sup>**

<sup>1</sup> Laboratory of Mass Spectrometry, MolSys Research Unit, University of Liège, Liège, Belgium.

<sup>2</sup> Centre for Protein Engineering, University of Liège, B 4000 Liège, Belgium.

<sup>3</sup> Alphabiotoxine Laboratory sprl, Barberie 15, Montroeul-au-bois 7911 Belgium.

\* Correspondence: lfreuville@uliege.be

**Abstract:** Scorpions are characterized by the great complexity and diversity of their venom, which has evolved over 400 million years into a well-elaborated library of powerful toxins mostly targeting, with high specificity, ion channels. Moreover, protein or non-protein compounds found in such venoms display antibacterial, antiviral or again anticancer activity, converting them from toxic molecules to therapeutic and benefic ones. Even if scorpions constitute a target of choice for many researchers, the high number of identified species (>2000) imposes that some of them remain little-known. It is even still the case for the Buthidae family, which constitutes the most human medically relevant species, due to the high concentration of potent ion-channel toxins in their venoms. As an example,

despite their phylogenetical proximity, *Androctonus australis* is the heart of 365 publications (Pubmed), whereas *A. mauritanicus* is only considered in five of them. In this work, we aim at comparing the venom profiles of these scorpions at the protein level. A first macroscopic overview of the protein content of each venom was evaluated by 1D SDS-PAGE and showed no notable difference. During the second time, the crude venoms of the two scorpions were analyzed with bottom-up proteomics. Toxins were classically reduced to remove the disulfide bonds, alkylated to avoid their reformation and finally digested with trypsin. The LC-MS/MS spectra were realized with M-Class UPLC coupled to a Q-Exactive spectrometer. Data analysis was performed with the bioinformatics dedicated software "Peaks X+", with the use of an extracted database from Uniprot built with the keyword "Buthidae". As preliminary results, *A. mauritanicus* presents 163 Top proteins against 142 for *A. australis* with this database. They share 100 peptides and 63 proteins in common. A table with the comparison of the classification of proteins in each species is presented below (Table 1). In the light of the protein classification, some groups of non-toxins are unexplored in the scorpion venoms, such as the Kunitz-type family and Cysteine-rich venom proteins. Therefore, the proteomic approaches can still open perspectives for the study of these groups, which is relevant to highlight them for future explorations. Comparing a wellstudied scorpion species with another less studied but phylogenetically close is interesting to highlight the evolution, diversity and complexity of venom.



**Keywords:** *Androctonus mauritanicus*; LC-MS/MS; scorpion venom

#### *6.7. New Proteomic Approach for Inventorying the Snake Venom Arsenal*

**Fernanda Gobbi Amorim 1,\*, Damien Redureau 1, Nicholas Casewell 2, Loïc Quinton <sup>1</sup>**

<sup>1</sup> Laboratory of Mass Spectrometry, MolSys Research Unit, University of Liège, Liège, Belgium. <sup>2</sup> Centre for Snakebite Research and Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK.

\* Correspondence: fgamorim@uliege.be

**Abstract:** Multi-Enzymatic Limited Digestion (MELD) is a new methodology that applies synergic and time-limited digestion of multiple enzymes, representing a versatile yet straightforward approach for a new generation of proteomics methodology (MORSA et al., 2019). However, until now, this approach has not been applied to venomic studies, which may be useful to define the venom composition. The generation of a higher number of peptides per protein during the MELD digestion increases the quality of protein sequencing and, consequently, identification. Herein we applied the MELD strategy for the venomics of two snake species: *Echis ocellatus* (EoV) and *Dendroaspis polylepis* (DpV). Ten micrograms of each venom were reduced/alkylated followed by two different digestion protocols: (1) trypsin and (2) MELD (Trypsin, GluC and Chymotrypsin). The digested materials were analyzed in a Q-Exactive™ Plus Mass Spectrometer with protein identification performed by Peaks Studio X+ using the Uniprot and transcriptomic databases. MELD showed more peptides/proteins identified for both venoms compared to the trypsin protocol. In EoV, 82.8% were identified as toxins and 17.2% as non-toxins, compared to the trypsin protocol, which resulted in 69.2% toxins, 24% non-toxins and 6.9% for cellular components (CC). In DpV, MELD showed coverage of 26.2% for toxins, 39.9% for non-toxins and 33.9% for CC, while for trypsin, we obtained 23.3% for toxins, 37.4% for non-toxins and 39.3% for CC. MELD was able to identify new components in both venoms. Fifty-one percent of the EoV were metalloproteinases (MP), while DpV showed a high content of nerve growth factor (22%), which was not identified using the transcriptome database. The highest number of mass spectra was obtained for a metalloproteinase (tr|Q2UXQ4) for *E. ocellatus*, in which the MELD approach led to four times more mass spectra. For *D. polylepis*, dendrotoxin I (P00979) showed the highest number of mass spectra, and the trypsin-based approach yields two times more mass spectra. As the number of mass spectra gives a rough evaluation of the toxin concentration, the two cited toxins are among the most produced in the venoms. MELD presented a different coverage according to the presence of high molecular mass content in the venom arsenal. This strategy can be applied to identify new groups of venom components. It represents an innovative strategy for venomics, opening new perspectives for sequencing and inventorying the venom arsenal.

**Keywords:** proteomics; snake; toxin; venom

*6.8. Nano-Rattlers against Cancer: Snake Venom-Loaded Nanoparticles against Tumoral Cell Lines* **Jorge Jimenez-Canale 1,\*, Nayelli-Guadalupe Teran-Saavedra 1, Enrique-Fernando Velazquez-Contreras 1, Martin-Samuel Hernandez-Zazueta 2, Hector-Manuel Sarabia-Sainz 3, Daniel Fernandez-Quiroz 4, Alexel-Jesus Burgara-Estrella 5, Jose-Andrei Sarabia-Sainz <sup>5</sup>**

<sup>1</sup> Departamento de Investigacion en Polimeros y Materiales, Universidad de Sonora, blvd. Luis Encinas s/n, CP 83000, Hermosillo, Sonora, Mexico.

<sup>2</sup> Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, blvd. Luis Encinas s/n, CP 83000, Hermosillo, Sonora, Mexico.

<sup>3</sup> Departamento de Ciencias del Deporte y de la Actividad Fisica, Universidad de Sonora, blvd. Luis Encinas s/n, CP 83000, Hermosillo, Sonora, Mexico.

<sup>4</sup> Departamento de Ingenieria Quimica y Metalurgia, Universidad de Sonora, blvd. Luis Encinas s/n, CP 83000, Hermosillo, Sonora, Mexico.

<sup>5</sup> Departamento Investigacion en Fisica, Universidad de Sonora, blvd. Luis Encinas s/n, CP 83000, Hermosillo, Sonora, Mexico.

\* Correspondence: jorgejimzc@gmail.com

**Abstract:** Research in drug delivery systems has led to the development and improvement of materials that affect the pharmaceutical effect of bioactive components, broadening the options of treatment for several diseases, including cancer. Chitosan (Cs) has been firmly established as a biocompatible and biodegradable low-toxic polymer able to form complexes with bioactive agents, making them promising drug delivery vehicles. Additionally, some snake venom toxins such as phospholipases A2 (PLA2s), serine proteinases (SVSPs) and metalloproteinases (SVMPs) were reported to present cytotoxic activity in different tumor cell lines, making them an auspicious option to be used as cancer pharmaceuticals. In the present study, we identified the major proteins in the venom of a northern black-tailed rattlesnake (*Crotalus molossus molossus*) and cytotoxic activity against T-47D breast carcinoma cells were evaluated. Afterward, the venom was loaded into a Cs NPs system through the ionotropic gelation process with tripolyphosphate (TPP), obtaining particles of 415.9 ± 21 nm and zeta potential of +28.3 ± 1.1 mV. The venom-loaded Cs-ALG complex was able to deliver the venom into the breast carcinoma cells through endocytosis, inhibiting their viability and inducing morphological changes. Although more studies are required, we suggest the potential use of *C. m. molossus* venom toxins entrapped within polymer nanoparticles for the future development and research of tumor pharmaceuticals.

**Keywords:** chitosan; nanomedicine; nanoparticle; snake venom

*6.9. Adenosine Amplifies Granulopoiesis and Systemic Inflammatory Response after Scorpion Envenomation*

**Asma Kaddache \*, Louiza Bechohra, Fatima Laraba-Djebari, Djelila Hammoudi-Triki**

Department of Cellular and Molecular Biology, University of Science and Technology Houari Boumediene (USTHB), BP 32 El-Alia, Bab Ezzouar, Algiers, Algeria. \* Correspondence: akaddache@usthb.dz

**Abstract:** Scorpion envenomation (SE) triggers granulopoiesis, resulting in heightened production of neutrophils that, in turn, exacerbate tissue damage. However, the mechanisms that sustain their production and recruitment to the injured tissue are unclear. Experimental evidence suggests that alarmines, inter alia adenosine, can modulate proliferation and differentiation of hematopoietic progenitors as well as mature neutrophils mobilization. Indeed, adenosine accumulates in the extracellular space in response to cell damage and can modulate immune cells through its transmembrane receptors A1, A2A, A2B, and A3. However, despite increasing data incriminating adenosine in many models of inflammation, little is known about its effect in the inflammatory response caused by the venom of *Androctonus australis hector* (Aah). **Aim**—In this study, we focused on deciphering molecular signals that promote neutrophil production and recruitment after SE by targeting adenosine as a potential candidate. **Methods**—Using a mouse model of envenomation and a pharmacological strategy, we prevented adenosine signaling by 1,3,7-triméthylxanthine, a nonselective inhibitor of adenosine A1 and A2A receptors. We first characterized the effect of this treatment on granulopoiesis in the bone marrow and then evaluated the expanse of inflammation to various tissues, mainly of the cardiorespiratory system. To this end, medullar MPO activity was assessed as a marker of progenitor cells proliferation and differentiation. In parallel, differential count of leucocytes and histopathological analysis of heart and lungs were carried out. **Results**—Inhibition of the action of adenosine helped to attenuate venom-induced granulopoiesis and reduced the accumulation of neutrophils in the blood. The damages caused by neutrophils in the lungs and heart were improved to a large extent. **Conclusion**—These results highlight the involvement of adenosine in the inflammatory response induced after SE via its A1 and A2A receptors and could help identify a new target to modulate inflammation and prevent cardiorespiratory failure in envenomed patients.

**Keywords:** adenosine; granulopoiesis; scorpion envenomation

#### *6.10. Comparative Study of Two Thrombin-Like Molecules from Vipera lebetina Venom*

#### **Amel Kadi-Saci \*, Fatima Laraba-Djebari \***

University of Science and Technology Houari Boumediene (USTHB), Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP 32 El-Alia, Bab Ezzouar, Algiers, Algeria. \* Correspondence: amel\_saci@yahoo.fr; flaraba@hotmail.com; flaraba@usthb.dz

**Abstract:** Snake venoms contain a wide range of components that are able to initiate or inhibit several steps of coagulation or platelet aggregation. Two thrombin-like enzymes, VLCV and VLCII (45 and 60 kDa, respectively) with fibrinogenolytic and pro-aggregating activities, are purified from *Vipera lebetina* venom. These molecules of interest are involved in platelet adhesion to fibrinogen by activating signaling pathways via αIIbβ3 integrins and thus binding to fibrinogen. Results also showed that both molecules present a high pro-aggregating effect with a similar mechanism of thrombin. The incubation of heparin with VLCV or VLCII has no inhibition on induced platelet aggregation by these molecules, suggesting that both enzymes have similar functional characteristics. The use of PMSF also has no effect on the activity of both molecules, indicating that the induced platelet aggregation by VLCV or VLCII involves an independent mechanism of catalytic activity. Results also indicate that both molecules VLCII and VLCV present a mechanism of action involving the thrombin and/or ADP pathways via PAR1/PAR4/P2Y12 receptors, as well as the αIIbβ3 integrin, leading to adhesion and platelets activation. Both thrombin-like VLCII and VLCV could be used as potential targets for the development of new drugs.

**Keywords:** biomolecule; coagulation; pro-aggregating; snake venom; thrombin-like

*6.11. A comparative Study of the Histological Changes Induced by the Venoms of the Moroccan Snakes Cerastes cerastes and Naja haje*

**Soukaina Khourcha 1,2,\*, Salma Chakir 1,2, Ayoub Lafnoune 1, Bouchra Darkaoui 1, Khadija Daoudi 1, Fatima Chgoury 1, Abdelaziz Hmyene 2, Naoual Oukkache <sup>1</sup>**

<sup>1</sup> Laboratory of Venoms and Toxins, Institut Pasteur of Morocco, 1 place Louis Pasteur, Casablanca 20360, Morocco.

<sup>2</sup> Laboratory of Biochemistry, Environment and Food Technology, Faculty of Sciences and Technologies, BP 146, Mohammedia 20650, Morocco.

\* Correspondence: khourcha.soukaina9@gmail.com

**Abstract:** In Morocco, ophidian envenomations are perpetrated by eight species of snakes belonging to the families of vipers and elapids, including *Cerastes cerastes* (Cc) and *Naja haje* (Nh), respectively. The present work offers a comparative study between the two venoms from a toxicological and physiopathological standpoint. We first determined the toxicity of the Cc and Nh venoms by an LD50 test and then carried out an anatomopathological study on Swiss mice in order to detect the signs of envenomation by each venom. The organs of the envenomed mice were then removed for a histological study to determine the main systemic alterations induced by the venoms. The results of our comparative study showed large disparities between the two venoms in terms of toxicity, revealing that the Nh venom is more toxic than the Cc venom. This disparity is also evident in the MHD test and the histopathological study that shows that the Nh venom exerts a cytotoxic activity on the brain, heart, lungs, liver and kidneys, while that of Cc leads to the formation of hemorrhagic foci and lesions on the liver, kidneys and heart.

**Keywords:** characterization; histology; physiopathology; snake; toxicity; venom

*6.12. Modulation of Induced Neuro–Immuno–Inflammatory Response by K+ Neurotoxin: Involvement of Serotonin and Histamine Receptors*

**Amina Ladjel-Mendil 1,\*, Marie-France Martin-Eauclaire 2, Fatima Laraba-Djebari <sup>1</sup>**

<sup>1</sup> University of Science and Technology Houari Boumediene (USTHB), Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP 32, El–Alia Bab Ezzouar, 16111 Algiers, Algeria.

<sup>2</sup> Aix-Marseille University, CRN2M CNRS UMR 7290, IFR Jean-Roche, Faculté de Médecine Secteur Nord, boulevard Pierre Dramard, 13916 Marseille cedex 20, France. \* Correspondence: mendilamina@yahoo.fr

**Abstract:** The neuro–immuno–inflammatory response triggered by neurotoxins is a key event in the pathogenesis of scorpion envenomation. In the present study, this response was evaluated in cardiac, pulmonary and brain tissues of intoxicated mice with kaliotoxin; a neurotoxin derived from *Androctonus australis hector* scorpion venom. The involvement of serotoninergic and histaminergic pathways in the systemic inflammatory response following kaliotoxin administration was also investigated. Obtained results revealed that the injected kaliotoxin by intracerebroventricular route induces an important immuneinflammatory response in brain, cardiac and pulmonary tissues. This response is mainly characterized by local features such as edema formation, inflammatory cell infiltration and imbalanced redox status. These effects are correlated with severe tissue alterations and a concomitant increase in metabolic enzymes in sera. Pretreatment of mice with antagonists of serotonin (5HT) and histamine (H1) receptors markedly attenuated these alterations in all the studied tissues. Serotonin and histamine-specific receptors seem to be the main pharmacological targets involved in the neural and systemic inflammatory processes. These findings could help to understand better the role of serotonin and histamine in scorpion venom-induced inflammatory response and pave the way to new therapies targeting 5HT and H1 receptors in order to attenuate the induced neuro–immuno–inflammatory disturbances that may be encountered in severe grades of scorpion envenoming.

**Keywords:** 5HT and H1 receptors; immunoinflammatory response; kaliotoxin; oxidative stress; tissue injury

*6.13. High-Throughput Screening of Spider Venoms for Identification of Active Compound in Voltage-Gated Sodium Channel*

**Ludivine Lopez 1,2,\*, Jérôme Montnach 1, Sébastien Nicolas 1, Lucie Jaquillard 2, Rémy Béroud 2, Michel De Waard <sup>1</sup>**

<sup>1</sup> L'institut du thorax, INSERM UMR 1087/CNRS UMR 6291, LabEx "Ion Channels, Science & Therapeutics", F-44007 Nantes, France.

<sup>2</sup> Smartox Biotechnology, 6 rue des Platanes, F38120 Saint Egrève, France.

\* Correspondence: ludivine.lopez@univ-nantes.fr

**Abstract:** Dysfunctions of voltage-gated sodium channels (Nav) have been associated with many pathologies such as cardiac diseases, neuropathic pain and epilepsy. In order to study the role of these channels in diseases or to restore their function, selective molecules targeting ion channels are needed. Only a few molecules selective to a single sodium channel isoform have been discovered. This makes these channel types important targets in drug discovery. Animal venoms, and especially spider venoms, contain various peptides that target ion channels. By screening spider venoms on Nav isoforms, we aimed to identify new peptidic toxins targeting specifically one of them. We performed a screening of about thirty spider venoms on Nav1.3, Nav1.4, Nav1.5 and Nav1.6 isoforms using the automated patch-clamp (APC) technique (SyncroPatch364, Nanion). All venoms were preliminarily fractionated in 64 fractions and tested on each Nav. Fractions of interest are those that reduce sodium peak current (by at least 30%), slow-down inactivation or increase late sodium current. False-positive fractions were excluded based on the detection of material in HPLC or mass spectrometry. The primary screening identified 220 fractions active on at least one isoform. Some of these fractions were then selected for purification and tested again on the automatic patch-clamp (74 for Nav1.3, 42 for Nav1.4, 28 for Nav1.5 and 23 for Nav1.6). The most interesting peptides were sequenced, synthesized and characterized. This study suggests that among a large number of toxins present in venoms, close to 50% of them target sodium channels with specificity for each sodium channel isoform.

**Keywords:** 5HT screening; automated patch-clamp; spider toxin

*6.14. Gambierol Action on K<sup>+</sup> Currents and Catecholamine Release in Cultured Single Chromaffin Cells from Fetal Rat Adrenal Medulla*

**Jordi Molgó 1,2,\*, Roland Bournaud 2, Sébastien Schlumberger 2, Makoto Sasaki 3, Haruhiko Fuwa 4, Denis Servent 1, Evelyne Benoit 1,2**

<sup>1</sup> Université Paris-Saclay, CEA, INRAE, Institut des sciences du vivant Frédéric Joliot, Département Médicaments et Technologies pour la Santé (DMTS), Service d'Ingénierie Moléculaire pour la Santé (SIMoS), ERL CNRS 9004, F-91191 Gif-sur-Yvette, France.

<sup>2</sup> CNRS, Laboratoire de Neurobiologie Cellulaire et Moléculaire-UPR 9040, F-91198 Gif-sur-Yvette, France.

<sup>3</sup> Tohoku University, Graduate School of Life Sciences, Sendai, Japan.

<sup>4</sup> Chuo University, Faculty of Science and Engineering, Department of Applied Chemistry, 1-13-27 Kasuga, Bunkyo, Tokyo 112-8551, Japan.

\* Correspondence: jordi.molgo@cea.fr

**Abstract:** Gambierol, characterized by a transfused octacyclic polyether core, was first isolated and chemically typified from cultured *Gambierdiscus toxicus* dinoflagellates collected in French Polynesia. Subsequently, distinct groups in Japan and the USA, using various strategies, achieved their total chemical synthesis. This allowed detailed studies on its mode of action. Gambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells, as well as in motor nerve terminals of the skeletal neuromuscular junction. In the present study, we investigated first the effects of nanomolar concentrations

of gambierol on K<sup>+</sup> current of cultured chromaffin cells from fetal rat adrenal medulla using perforated patch-clamp current recordings. Our results show that gambierol only blocked a small component of the total K<sup>+</sup> current and affected neither calcium-activated K+ (KCa) nor ATP-sensitive K<sup>+</sup> (KATP) channels, as revealed using apamin and iberiotoxin (selective KCa channel blockers) and glibenclamid (KATP channel blocker). After inhibiting KATP and KCa channel activation, the gambierol concentration blocking 50% of the K+ current component (IC50) was 7.6 ± 1.1 nM. The repolarizing phase of all-or-none elicited action potentials, recorded under current-clamp conditions (triggered by 1-ms depolarizing pulses), was sensitive to the action of gambierol but insensitive to the action of apamin and iberiotoxin, indicating that KCa channels do not participate in the modulation of action potential duration triggered by short depolarizing pulses. The use of simultaneous patch-clamp and single-cell amperometry allowed controlling the membrane potential and detecting exocytosis events (with a carbon electrode polarized to +650 mV to allow the oxidation of released catecholamines). Such recordings revealed that gambierol did not modify the membrane potential following 14-s depolarizing current steps and did not significantly increase the number of exocytotic catecholamine release events with respect to controls. The addition of KCa channel blockers (in the continuous presence of gambierol) enhanced the membrane depolarization by about 15 mV (during the 14 s current step), and at the same time, increased significantly the number of exocytotic events related to catecholamine secretion. Such enhanced depolarization induced by the KCa channel blockers probably brings the membrane potential above the activation threshold of high-voltage activated CaV channels, triggering both Ca2+ influx and subsequent catecholamine secretion. These results emphasize the diversity of KV channels in chromaffin cells from fetal rat adrenal medulla and highlight the modulatory role played by KCa channels in the control of exocytosis in the absence of splanchnic innervation.

**Keywords:** catecholamine release; cultured chromaffin cell; fetal rat adrenal medulla; gambierol; potassium current

*6.15. Sprouting and Convergent and Stable Polyinnervation Characterize Human Orbicularis oculi Muscles Treated for Blepharospasm with Repeated Botulinum Type A Neurotoxin Injections*
