Biologie des cônes marins
Morphologie
Cones exhibit a wide range of shapes, sizes, and colors, showcasing diverse patterns on their shells Their dimensions can vary significantly, typically ranging from 1 to several centimeters.
The Conus prometheus, found in West Africa, typically measures between 5 and 10 cm in length, although it can reach up to 30 cm Its shell exhibits various shapes, including conical, biconical, and cylindrical-conical forms, and it spirals dextrally around an axis known as the columella.
The shell features eight spires and has a narrow, elongated opening known as the peristome Its extraordinary variety of patterns and colors serves as crucial identifying characteristics for each species The beauty and diversity of these shells continue to captivate shell collectors.
Distribution géographique et bathymétrique
The Conus genus, comprising approximately 700 species, is predominantly found in marine areas between 40° North and 40° South latitudes, including the Indo-Pacific, Caribbean, West Africa, South Africa, Peru, Patagonia, and the Mediterranean (Giancarlo Paganelli, 1998-2013) A few species extend beyond these latitudes, specifically in South Africa, southern Australia, southern Japan, and the Mediterranean Sea, with the Indo-Pacific region showcasing the highest diversity.
Paciĩique (Figure 2) [1] Elle atteint une densité maximale de 40 individus/m2, mais est généralement beaucoup moins abondante.
Figure 1 Biodiversité du genre Conus (d’après Olivera, et al., 2009).
Les cones marins habitent surtout dans les boues, les bancs de sable ou coraux ó il y a des marées et des zones rocheuses dangereuses Dans cet environnement les cônes
Introduction vermivores sont très íréquents, les cônes malacophages moins abondants, et les cônes piscivores plus rares Les cônes vivent en mer dans les eaux peu proíòndes mais aussi jusqu'à
400 m de proíbndeur. Í.4 Comportement alimentaire des cônes
All cone snails are carnivorous predators equipped with highly evolved venomous systems for capturing their prey They are categorized into three groups based on their dietary habits Notably, there are piscivorous cones, such as Conus geographus.
Cone snails, such as Conus magus and Conus striatus, are primarily piscivorous, feeding on fish, while malacophagous species like Conus textile and Conus marmoreus exclusively consume mollusks Additionally, vermivorous cones, including Conus imperialis and Conus quercinus, target worms like annelids and polychaetes Research by Olivera et al (2009) indicated that 65% of cone snails are vermivorous, with 20% being piscivorous and 15% malacophagous Recent studies support these findings and suggest the existence of a fourth category of omnivorous cones, such as Conus californicus and Conus pictus These diverse feeding strategies highlight the remarkable adaptability of gastropods, with variations in hunting strategies, venom composition, and the shape of the radular tooth used for subduing and envenomating prey depending on their dietary habits.
The venomous apparatus of cone snails consists of four distinct organs: the venom gland, venom duct, radular sac, and proboscis The venom gland, a large whitish mass, plays a mechanical role by contracting to expel venom from the duct into the proboscis.
The radular sac is an angular structure (Y or L-shaped) consisting of two branches: one short and the other long The longer branch is free, curved, and blind, measuring between 2 to 15 mm in length, where the radular teeth are formed to "harpoon" and envenomate prey These teeth are made of a chitin tube that allows venom to enter at the base and flow upstream.
Figure 3 Régime alimentaire et phylogénie des cônes marins (cPaprès O livera et al., 2009). ốlande musculaire ventn sac radulaire
Figure 4 Représentation schématique d ’une coupe transversale d ’un cône C onus consors (d’après Stocklin et al., 2010) Appareil venimeux disséqué de c textile
Once the formation of radular teeth is complete, they are transferred to a shorter radular branch that opens into the pharynx in front of the glandular canal, containing about ten ready-to-use teeth The tips of these small teeth are oriented towards the pharyngeal opening Initially, the teeth are flexible and only gain rigidity during their migration between the two radular branches The morphology of these teeth varies significantly among different species.
The size of the tooth varies from a few tenths of a millimeter to over 2 cm, depending on the type of prey captured In vermivorous and malacophagous species, the teeth are relatively simple, while some piscivorous species exhibit highly elaborate barbs The radular sac opens into the pharynx, which connects the venom duct, the proboscis, and the digestive tube.
The proboscis is a flexible organ located within the rostrum This fine, extendable tube measures only a few millimeters when at rest and serves the purpose of extracting food and then transferring it to the prey.
To enhance their mobility challenges, cones either burrow into sand, hide under stones, or seek refuge among corals during the day, remaining inactive until nightfall when they forage for food Additionally, their prey-capturing strategies vary based on their specific dietary needs.
- Les cônes piscivores qui pêchent au "harpon" ou "íìlet" (Figure 6).
- Les malacophages qui attaquent "derrière" ou "en face" des proies (Figure 7).
- Les cônes vermivores qui chassent "ensemble" ou de manière "individuelle" (Figure8).
Figure 5 Représentations de dents radulaires A Dents radulaires 50x de c striatus (chasseur de poissons), B de c textiìe (chasseur de mollusques), c de
Conus pulicarius (chasseur de vers) (d’après Legall et al., 1999).
Figure 6 illustrates two distinct venom delivery strategies employed by piscivorous cone snails to capture their prey The upper section depicts the "harpoon" strategy utilized by Conus striatus, while the lower section showcases the "net" strategy used by Conus geographus, as detailed by Olivera et al (1999).
Appareil venimeux
The venom apparatus of marine cones consists of four distinct organs: the venom gland, venom duct, radular sac, and proboscis The venom gland, which is a large, whitish mass, plays a mechanical role by facilitating the expulsion of venom from the duct to the proboscis through its contractions.
The radular sac is an angular structure (Y or L-shaped) consisting of two branches: one short and the other long In the longer, free, curved, and blind branch, which measures between 2 to 15 mm, the radular teeth are formed, designed to "harpoon" and envenom prey These teeth are made of a chitin tube that allows venom to enter at the base and flow upwards.
Figure 3 Régime alimentaire et phylogénie des cônes marins (cPaprès O livera et al., 2009). ốlande musculaire ventn sac radulaire
Figure 4 Représentation schématique d ’une coupe transversale d ’un cône C onus consors (d’après Stocklin et al., 2010) Appareil venimeux disséqué de c textile
Once the formation of the radular teeth is complete, they are transferred to a shorter radular branch that opens into the pharynx, located anterior to the glandular canal, and contains about ten teeth ready for use The tips of these small teeth are oriented towards the opening in the pharynx Initially, the teeth are flexible, gaining rigidity only during their migration between the two radular branches The morphology of the teeth varies significantly among different species.
The size of the tooth varies from a few tenths of a millimeter to over 2 cm, depending on the type of prey captured In vermivorous and malacophagous species, the teeth are relatively simple, while some piscivorous species have highly elaborate barbs The radular sac opens into the pharynx, which connects the venom duct, proboscis, and digestive tube.
The proboscis is a flexible organ located inside the rostrum This fine, extensible tube measures only a few millimeters when at rest Its primary function is to extract a tooth and then hold it within the prey.
Stratégies d’envenimation
To enhance their mobility issues, cones immobilize themselves or bury into sand, under stones, or among corals during the day, remaining active only at night to forage for food Additionally, they employ various prey-capture strategies depending on their dietary needs.
- Les cônes piscivores qui pêchent au "harpon" ou "íìlet" (Figure 6).
- Les malacophages qui attaquent "derrière" ou "en face" des proies (Figure 7).
- Les cônes vermivores qui chassent "ensemble" ou de manière "individuelle" (Figure8).
Figure 5 Représentations de dents radulaires A Dents radulaires 50x de c striatus (chasseur de poissons), B de c textiìe (chasseur de mollusques), c de
Conus pulicarius (chasseur de vers) (d’après Legall et al., 1999).
Figure 6 illustrates two distinct envenomation strategies employed by piscivorous cone snails to capture their prey The upper section depicts the "harpoon" strategy utilized by Conus striatus, while the lower section showcases the "net" strategy used by Conus geographus, as detailed by Olivera et al (1999).
Figure 7 illustrates two distinct venomous strategies employed by malacophagous cones to capture their prey The upper section depicts the "rear attack" strategy utilized by the venomous cone species Conus marmoreus, while the lower section showcases the "frontal attack" method used by the normal mollusk Conus textile, as referenced in Biggs (2009).
Figure 8 illustrates two distinct venom strategies employed by vermivorous cones to capture their prey At the top, small Conus californicus attack a worm collectively, while below, Conus quercinus stings the worm and competes with smaller C californicus (Source: Olivera et al., 1999).
Venin des cônes marins
Techniques de récupération du venin
There are primarily two different techniques for harvesting venom from cone snails The first technique involves dissecting the animal to extract venom from its venom duct, which is then cut into small pieces and crushed in a solution containing 0.1% trifluoroacetic acid (TFA) or a small amount of acetonitrile (ACN) for venom extraction A major drawback of this method is that it results in the death of the studied cone snail Additionally, the harvested venom is still in development and may not exactly match the final injected product.
In 1995, Hopkins et al developed a technique for venom retrieval that involves collecting the venom at the moment of injection into the prey This method requires presenting a small fish or similar prey to the cone until it extends its proboscis.
When the cone is ready to capture its prey, a collection tube, such as an Eppendorf tube sealed with a "parafilm" membrane, is used instead of fish However, this technique has drawbacks, as it yields very small amounts of venom, requires considerable patience, and is time-consuming.
Composants du ven in
The toxicity of cone snails was first noted over 300 years ago by Dutch naturalist Rumphius However, it wasn't until the early 1970s that researchers B.M Olivera and L.J Cruz began to explore the toxic mechanisms of cone snail venom Subsequent studies utilized traditional approaches for discovering conopeptides, including bioactivity-guided testing, purification, Edman degradation, and MS/MS sequencing Advances in chromatographic separation techniques and mass spectrometry, along with nuclear magnetic resonance methods, have allowed for the isolation and characterization of venom components Cone snail venom is known for its remarkable hyper-diversity and significant quantity of bioactive substances.
11 appears as a milky white liquid containing small insoluble granules Since 2000, an integrated approach involving proteomics, transcriptomics, and bioinformatics has led to the discovery of numerous bioactive peptides derived from marine cones.
The integrated approach streamlines the initial steps found in traditional methods, resulting in significant time savings Additionally, this approach requires a smaller quantity of materials, offering advantages over conventional techniques Typically, the venom of each species contains over 1,000 peptides (conopeptides or conotoxins), as well as vasoactive proteins (such as ebumetoxin and tesseletotoxin), proteases (including acetylcholinesterases, phosphodiesterases, and phospholipases), and low molecular weight organic molecules (like quaternary ammonium and N-methylpyridinium).
Sequential workflow approaches in traditional (white background) and integrated (black background) methodologies highlight the significance of post-translational modifications (PTMs) in neuropharmacology Conotoxins, in particular, are extensively studied due to their remarkable diversity and specificity, which enable the pharmacological differentiation of various types and subtypes of neurotransmitter receptors and ion channels Additionally, the accessibility of peptide sequences through chemical synthesis further enhances their research potential.
Compared to toxins from snakes, scorpions, and spiders, conotoxins possess a unique linear peptide sequence complemented by numerous disulfide bridges, resulting in a rigid three-dimensional structure These toxins serve as an excellent foundation for studying ligand-receptor interactions and structure-activity-selectivity relationships Additionally, conotoxins are emerging as promising alternatives in therapeutic applications.
Les conopeptides et les conotoxines
D’un gène à la petite protéine ma tu re
Studies of c textile venom-derived cDNA have revealed that conopeptides are generated from prepropeptides, which undergo specific proteolytic cleavage to produce mature toxins The N-terminal of the conopeptide precursor sequence contains a highly conserved signal sequence, while the C-terminal features a highly divergent mature toxin sequence A conserved region of the propeptide separates the signal sequence from the mature toxin sequence These three distinct regions of the prepropeptide are encoded by individual exons, which are separated by relatively long introns The arrangement of the prepropeptide for most conotoxins is schematically illustrated in the accompanying figure.
11 Ọuelques séquences de précurseurs, comme celle de la conotoxine TxX de c íextile montrée dans la Figure 11, présentent un arrangement différent ó la séquence propeptide se trouve à 1’extrémité C-terminale et est considérée comme un "postpeptide" [64] En général, les régions hypervariables des conotoxines matures sont situées entre les résidus cystéine, qui sont hautement conservés dans les motifs de conotoxines Les cystéines jouent un rôle important dans l'orientation de la connectivité des ponts disulíures de conotoxines, comme décrit, par exemple, pour le peptide King Kong par Woodward et al [83].
The observed sequence divergence in mature toxins reveals that each cone snail venom contains over 1,000 different peptides, derived from a vast library of selective toxins targeting specific receptor subtypes A single amino acid change in hypervariable regions can yield peptides with the same basic backbone structure, provided the cysteine motif is preserved, while fine-tuning selectivity for various receptor subtypes During envenomation, multiple conopeptides are injected simultaneously, potentially working synergistically to achieve the desired physiological effect, which accounts for the extensive repertoire of cone snail toxins The term "toxin cabal" describes this synergistic approach of multiple injections used by cone snails to immobilize their prey This extensive repertoire offers an evolutionary advantage consistent with the "screening hypothesis" of natural product diversity, suggesting that organisms capable of producing and filtering large quantities of chemicals at a low cost are more likely to be favored for survival.
Signal de séquence Pro-peptide Pepiide miiunv
P r é c u r s e u r B n l l '.V 'Mi' MMI ỉ MI I I ■ ■ ' i 'MI ' \ M \M)I< \M K , K \ \ \ \KI)K \SI)I \ \ I I \ kÚCCSHPACSVNNPDICU t T
Précurseur MrlA 'II I ; i I M , \ \ \ I |.|< II |)I)V l’\l s s \ > ( ,\( ,ksli RỊ ,11 KNGVCCOYKLCHPC
Figure 11 Arrangement de la protéine précurseur de conotoxines génériques et des séquences (d’après les Réf [35, 37, 64]).
The schematic representation of the conotoxin precursor protein arrangement illustrates that while the mature toxin exhibits high variability, the signal sequence and propeptide region remain conserved Typically, the signal sequence comprises approximately 20 amino acids, whereas the propeptide and mature toxin sequences vary between 20 to 60 and 11 to 71 amino acids, respectively Three conotoxin precursor sequences are depicted, highlighting specific regions for the Bnl precursor, with the signal sequence marked in red and the mature toxin in bold black The presence of XG-(X l)n at the C-terminal ends, where X denotes the mature peptide sequence and XI indicates a Lys or Arg residue, with n ranging from 0 to 4 amino acids, generally suggests a C-terminal amidation processing Additionally, the arrangement of TxX dipeptides in most precursor peptides and the propeptide region is located at the C-terminal end.
The introduction is regarded as a post-peptide and is illustrated by a white box at the end of the arrangement of the generic precursor protein shown in Figure 1 Several guided cleavage signals indicate the cleavage sites marked by arrows, leading to the formation of mature conotoxins.
Familles pharmacologiques des conotoxines ou conopeptides
Inhibiteurs des canaux calcium dépendants du potentiel
NMDA receptors, characterized by their three transmembrane domains and a variable-length cytoplasmic tail, require the simultaneous binding of the glutamate agonist (to the NR2 subunit) and the glycine co-agonist (to the NR1 subunit) for proper function Differential expression of isoforms (such as NR2A-D) in the central nervous system plays a crucial role in fast excitatory neurotransmission These receptors are implicated in various acute and chronic neurological disorders, driving significant research efforts towards the development of specific drugs targeting them Recent advancements have revealed atomic-resolution structures of the extracellular domains that bind agonists, partial agonists, and antagonists, paving the way for rational drug design of new NMDA receptor antagonists Utilizing docking models, it is possible to rationally design conantokine analogs with selectivity for specific clinically relevant subunit combinations.
4.2 Inhibiteurs des canaux calcium dépendants du potentỉel: les co-conotoxines
Calcium-dependent voltage channels (Cav) are transmembrane proteins that regulate calcium entry in response to membrane depolarization, influencing various processes such as contraction, secretion, neurotransmission, and gene expression across different cell types Their activity is crucial for coupling electrical signals from the cell surface to physiological events within the cells Cav channels belong to a superfamily of transmembrane ion channel proteins, which also includes voltage-dependent potassium and sodium channels Table 8 provides information on the physiological function and pharmacology of each member of the calcium channel family.
Calcium channels are complex proteins made up of four or five distinct subunits encoded by multiple genes The largest subunit, known as the di subunit, weighs between 190 and 250 kDa and includes the conduction pore, potential detector, gating mechanism, and regulatory sites influenced by second messengers, drugs, and toxins Similar to sodium channel subunits, the subunit of voltage-dependent calcium channels is organized into four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) The S4 segment acts as the potential detector, while the loop forming the pore between the S5 and S6 transmembrane segments in each domain is crucial for ionic conductance and selectivity, with changes in just three amino acids in these loops affecting channel properties.
L III and IV convert a sodium channel into a calcium-selective channel, consisting of an intracellular subunit, a transmembrane subunit, and a complex of subunits linked by disulfide bridges Most calcium channel subtypes include these components, with a specific subunit also found in skeletal muscle calcium channels Additionally, subunits are expressed in the heart and brain While these auxiliary subunits modulate the properties of the channel complex, the pharmacological and electrophysiological diversity of calcium channels primarily arises from the existence of multiple subunits.
Tableau 8 Fonction physiologique et pharmacologie des canaux calcium (d'après la Réf [184]).
Cavl 1 1 1,0 mascle squclettiquL* tubuk-s transverscs Dĩhydropvndirxs. plx-nylalkylamines, beivothia/epiixs Cavl 2 1 myocyles cardiaques mưsculaires lisses.invocvu-s cellules endocnnes corps cellulaircs neuronaưx dendrites proximales
DihvdrupvnduxLs plx-n> lalkylamiíxs beivm hia/epiixs
Cavl 3 I Cellules endocrines corps eellulaires neuronaưx et dendrites myocytes aunculaires.cardiaques ct celluies đe stimulateur cardiaque ceỉiules ciỉiees de la coehlee
Dihvdropvndincs phenylalkylaminex hen/nthsiA-pnìcs
Cavl 4 L íige de la retme et les eellules hrpoỉaires moeile epmiere glande surrenaỉe mastoeyỉes
Dihvdroputlincx. phenvlalkvlannnex bcn/iithu/apnx-x Cav2 1 p /ọ lermmaux et des dendrites nerveuses ceỉlules neưroendocrines
Cav2 2 N ĩermmaưx et des dendrites nerveusev cellules neuroendocnrxs f')-conoĩo\rjx-( | \ IA
C í i \ 2 3 R Corps ceilulairex ncưronaux el dcndntcx SNX-4X3
Cav 3 1 T Corps ceỉỉulaires ix-uronaux cl dendntes rmocvlcs cardiaques et muscuìaires ksse.y
C a\3 2 1 Corpx ix-llulanex ncuronaux d dendrrk-s rmncytex cardiaquex cl rnuxailaircx ksxcx
C ai 3 3 I t orpx ccllulaiR-x neuronaux L-l di.-ndiiti.-x N otx
C o u p i u e e d e \ Y ỉtatMn-k i.ằniiae U>JỈ Ỉiheraỉ ton đ e riì ot n x i x * r e u k - n x n l a U ' í ì d e s t rai iv.1 Ipthni m t e gr a t i on N\ napt kỊ iie
I i b e r u t k ì ĩ i d c ĩ l m r m o i x l e ư u l a i m n d e la t r ai ^ c Mp t mn r e e u i uD o n x \ l u p ĩ t p t L - p a è c n u k c i eVlĩd KKỊIX* a u d i e r x c l ib cr a li on d e r x t ư n t u n s m e n e u í s par l cs c e i ì u l c s s e it s or x l ỉ e s l ỉb c ia UM ì d e ĩ x * UH i U an M iv r tc uỉ s d e p h n i m e e e p t e u r s ỉ i h e r a U m d c s ĩ X H B o U a i ì M i v U e u i s nafv-.liiitu.-s > a k k p x ' d e t i d n U Ị i i e s l ihe raỉ tun d e n x m i M i x *
1 i b e u i t n n de- n e i t i u U a u s n x U e u ỉ - l ĩai is íb au-N k ak k j i i ‘\ d e n d í rfkị!ji‘ ỉ i be ral kí n d e !
K e p t u v e i c p e l r m r l t, il is íh' ỈK' \ V a k kịii"' d e n d i í i k Ị i x s
Figure 17 illustrates the schematic representation of the Cavl channel structure in skeletal muscle, which is also applicable to other Cavl and Cav2 channels The alpha helices are depicted as cylinders, and the lengths of the lines roughly correspond to the lengths of the represented polypeptide segments (according to Ref [184]).
Cav channel inhibitors are being explored as potential antihypertensive, antiarrhythmic, and anticonvulsant agents, as well as for chronic pain management, with ongoing studies into other possible applications The therapeutic potential of co-conotoxins lies in their ability to selectively inhibit mammalian Cav channel isoforms, particularly Cav2.2, which is expressed in pain pathways and exhibits antinociceptive effects in pain models Research on the pharmacology of co-conotoxins has included studies on various peptides, and although these peptides are considered pore blockers, their pharmacological properties resemble those of pO-conotoxins The co-expression of alpha subunits with auxiliary subunits can significantly alter their affinity for the channel, which may be particularly relevant in pathological states like pain, where a2δ subunits are upregulated Currently, D-conotoxins are the only conotoxin category approved by the FDA, with Ziconotide (MV11A) receiving approval in 2004 for treating refractory pain However, neurological side effects associated with the doses used have limited the clinical applications of co-conotoxins.
Conotoxins with a deleterious effect on isoforms of calcium channels in mammals have been isolated primarily from marine cone snails Based on behavioral phenotypes observed after intracerebral injection in mice, these peptides have been termed "shaker" peptides Conotoxins are now recognized as some of the most selective inhibitors for neuronal calcium channel isoforms Cav2.2 and Cav2.1 Specifically, conotoxins such as Co-CV1D, 10-CVIE, Co-CVIE, Co-GYl.A, and Co-MVIA show a strong preference for the Cav2.2 channel, while Eco-MVIIC and (α)-MIIID preferentially target the Cav2.1 channel The binding site for conotoxins on Cav channel subtypes has been primarily located at the external vestibule of the channel within the S5-S6 region of domain III Preliminary docking studies of Eco-MVIA with a homology model of the CaN2.2 channel support this localization Additionally, residues outside this region, particularly Gly326, contribute to the inhibition of Cav2.2 by conotoxin, influencing the reversibility of the blockade Intracellular domains have also been identified as modulators of the binding kinetics and affinity of conotoxins for the Cav2.2 channel Furthermore, the presence of auxiliary subunits not only modifies the opening characteristics and kinetics of Cav channels but also affects the interaction between conotoxins and these channels.
Figure 18 "Docking" de conotoxines aux modeles dlioniologie du eanal calemm
E (U-MVIIA (en gris íoncé) couplé a un modè-le triiomologic de ( a,2.2 (en bmn clair) construit à partir de structure eristalline d ‘un eanal sodium baelerie-n I l‘>7|
Tableau 9 co-conotoxines ciblant les canaux Cav (formule boucle C6C5-9CC2-4C3-6C) p: piscivore; m : malacophage; * : amidation C- terminale; ND : non déterminé; o : 4-hydroxyproline.
CnVIIA C.consors p c K G K G A o c T R L M Y D - - - c c H G s c s s s K G R c* 2.2 >2.1 [201] CVIA c catus p c K s T G A s c R R T s Y D - - - c c T G s c R s G R - - c* 2.2 >2.1 [186] CVIB c catus p c K G K G A s c R K T M Y D - - - c c R G s c R s G R - - c* 2.2 ~ 2.1 >2.3 [186] CVIC c catus p c K G K G ọ s c s K L M Y D - - - c c T G s c s R R G K - c* 2.1 ~ 2.2 [186] CVID C.catus p c K s K G A K c s K L M Y D - - - c c s G s c s G T V G R c * 2.2 >2.1 [186]
CVIE-2 C.catus p c K G K G A s c R R T s Y D - - - c c T G s c R s G R - - c* 2.2 > 2.1 > 1.2 ~ 1.3 ~ 2.3 [195] CVIF C.catus p c K G K G A s c R R T s Y D - - - c c T G s c R L G R - - c* 2.2 >2.1 > 1.2 ~ 1.3 -2 3 [195] FVIA c.fuỉmen p c K G T G K s c s R [ A Y N - - - c c T G s c R s G K - - c* 2.2 > 2.1 > 3.2 [202] CiVIA c.geographus p c K s o G s s c s o T s Y N - - - c c R s c N o Y T K R c Y 2.2 >2.1 [203] GVIIA c.geographus p c K s o G T o c s R G M R D - - - c c T s c L L Y s N K c R R Y Souris~poisson> grenouille [189] GVIIB c.geographus p c K s o G T o c s R G M R D - - - c c T s c L s Y s N K c R R Y Souris-poisson > grenouille [189] MVÍIA c.magus p c K G K G A K c s R L M Y D - - - c c T G s c R s G K - - c* 2.2 > 2.1 [204]
S03 c.striatus p c K A A G K p c s R I A Y N - - - c c T G s c R s G K - - c* Nav ~ K.V ~ 2.2 > 2.1 >2.3 [206]SVIA c.strìatus p c R s s G s o c G V T s I - - - c c G R c Y R G K - - c T 2.2 > 2.1 [58]SVIB c.striatus p c K L K G Q s c R K T s Y D - - - c c s G s c G R s G K - c* 2.1 >2.2 [58]'í VIA c.tuìipa p c í s o (ỉ s s c s o T s Y N - - - c c R s c N o Y s R K c 2.2 >2.1 [207]TxVII c textile m c K ọ A D E p c D V F s L D - - - c c T G I c L G V - - - c M w Type L de mollusque [208]PnVIA C.pennaceus m 0 c L E V D Y F c Cỉ I p F A N N G L c c s G N c V F V - - - c T p Q Type N de mollusque [209]PnVIB C.pennaceus m D D D c E p p G N F c G M I K 1 G p p - c c s G w c F F A - - - c A Type N de mollusque [209]
Les conotoxines agissant sur les canaux sodium dépendants
Sodium-dependent voltage channels (Na+) play a crucial role in initiating and propagating action potentials in excitable cells, including neurons, muscles, and certain types of neuroendocrine cells These channels are composed of alpha subunits, approximately 260 kDa in size, associated with auxiliary beta subunits In the adult central nervous system (CNS), Na+ channels contain the beta subunits Pi (or p o and ị), while adult skeletal muscle Na+ channels consist solely of different subunit compositions.
Figure 19 Organisation transmembranaire de sous-unités des eanaux MKlium (d^après la Réf [211 ]).
Chr omos Nr nc 2 r T. rNax c r- ỉ ĩ ị : n u n • • - ì ' - 'N'-i 1 5 • ; CbKUĩvV,'ntI< ạ T - , ;
Figure 20 Relations phylogénétiques localisalion et lonction pli\ siologique, amsi que sensibilité et résistance à la tétrodotoxine ( I TX-S et I I X -R ) des e iirmx
The rNa 1 7 subunit Pi Ị-12Ị is essential for the functional expression of the channel However, the channel's kinetics and voltage dependence are influenced by its subunits.
The subunits of voltage-gated sodium channels (Nav) are organized into four homologous domains (I-IV), each containing six transmembrane segments (S1-S6) and a large extracellular loop connecting segments S5 and S6 These loops form a narrow external opening of the pore, while segments S5 and S6 create a wider internal opening The positively charged amino acid residues in segment S4 of each domain move across the membrane, triggering channel activation in response to membrane depolarization Additionally, a small intracellular loop connecting domains III and IV acts as an inactivation gate, altering the channel's structure and blocking the pore from the inside during sustained depolarization In mammals, nine isoforms of Nav channels have been identified and functionally expressed, exhibiting over 50% homology in their amino acid sequences within the transmembrane and extracellular domains, allowing for their alignment.
4.3.2 Inhibiteurs des canaux sodium dépendants du potentỉel À ce jour, quatre classes de conotoxines ciblant les canaux Nav (p, p.0, s et i) ont été isolées à partir du venin de cônes [48, 77, 213, 214] Bien que ces peptides de venin présentent une activité sur les mêmes cibles pharmacologiques, ils sont structurellement distincts et varient dans leur mécanisme d'action, avec deux íamilles produisant une inhibition (les p- et pO-conotoxines) et deux familles activant (5 et t-conotoxines) les canaux Nav (Tableau 10) Les p-conotoxines provoquent rinhibition des canaux Nav par un blocage direct du pore impliquant le site 1 de la sous-unité a qui est aussi le site de fixation de la tétrodotoxine (TTX) et de ses analogues Le "docking" de la P-A15-TIIIA sur un modèle d'homologie du canal Nav1.4, construit à partir de la structure cristalline d’un canal sodium bactérien est montré dans la Figure 21 En revanche, les pO-conotoxines semblent interférer avec les détecteurs de potentiel situés dans le domaine II du canal Nav pour restreindre 1'ouverture du canal Les ỗ-conotoxines provoquent une inhibition d’une partie 1’inactivation du canal Nav par leur liaison au site 6 du canal, ce qui résulte en un prolongement du potentiel d'action et des décharges neuronales persistantes tandis que les 1-conotoxines íavorisent 1'ouverture du canal Nav en déplaẹant sa courbe d’activation vers des potentiels plus négatifs que le potentiel de repos II est à noter que certaines de ces conotoxines sont des modulateurs sélectiís des sous-types de canaux Nav, en particulier Nav1.4 Nav1.2 et, plus récemment Nav1.8, et se sont révélées comme des outils précieux pour disséquer les rôles phvsioloaiques et pharmacoloaiques des canaux N a J l à N a J 9 Le potentiel therapeutique des conotoxines ciblant les canaux Na\ commence à ètre exploré. t *
Figure 21 "Docking" de la conotoxine p-TIIIA sur un modèle d'homoloaie du canal Nav1.4 construit à partir d'une structure cristalline du canal sodium baetérien (d'après la Réf [197]).
Figure 22 Relation structure-activité au lìiveau de eertaines p-conoioxines A
Séquence consensus pour les p-conotoxines B el í Structure de la pelite p-SHIA présentant les résidus importants pour sun interacúun avec le eanaỉ Na ỉ> et F
Structure de la plus grande p-THIA presentant les residus ìmportants pour son interaction avec le canal Nav.
Tableau 10 presents inhibitory conotoxins targeting specific subtypes of Nav channels, identified by the formula loop |> conotoxin CC1-8CC3-4C2-5C The table categorizes conotoxins based on their prey types, including piscivores (p), malacophages (m), and vermivores (V) It also notes characteristics such as C-terminal amidation (*), undetermined features (ND), the presence of γ-carboxy glutamic acid (E), pyroglutamic acid (z), and 4-hydroxyproline (o) Additionally, the table distinguishes between tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) conotoxins.
CHIA M ( cưí us p G R c c E G p N G - c s s R ụ c K D H A R c c * Muscle squelettique [216]
CnlIIB M ( consors p z G c c Cì E p N L - c F T R c R N N A R c - c R Q Q Amphibiens TTX-R [291
MIVIA ND ( ma^ r ì ựi c us R D C Q E K E Y c 1 V p I L G F V Y c c p G L I c G p F V - - - c V 4 > 5-8 > 7>6 -3> l>2 [224] MrVIA Ol ( ma r mo r e i t s m A c R K K w E Y c 1 V p I í G F I Y c c p G L I c G p F V - - - c V 8>7>4>2>TTX-S [225] MrVIB OI ( m a r m o r c u s m A c s K K ụ E Y c I V p 1 L G F V Y c c p G L I c G p F V - - - c V 8>4>2-3~5-7>9 [226] conotoxin-GS Ol ( g e o g r a p h u s p A c s G R G s R C O O Q c c V G L R c G R G N P Q K C Ỉ G A H E D V 4>*)>9 [227]
Among the conotoxins targeting sodium channels, the p-conotoxins are the most numerous and well-characterized, with 20 different types identified to date These p-conotoxins belong to the M superfamily of conopeptides and consist of 16 to 26 amino acid residues, featuring three conserved disulfide bridges that stabilize their three-dimensional structure Typically, p-conotoxins exhibit a net positive charge, which enhances their ability to bind through electrostatic interactions in the outer vestibule of sodium channels, thereby inhibiting ionic conductance The first discovered p-conotoxins, including p-GIIIA, p-GIIIB, and p-GIIIC, selectively target the skeletal muscle subtype.
In recent years, there has been renewed interest in the selectivity of p-conotoxins for specific subtypes of Na channels Many sequences have shown selectivity for neuronal subtypes, including p-PIHA, p-KIIIA, and p-SIIIA, among others However, no p-conotoxin has been identified with affinity for TTX-resistant mammalian Na channel subtypes such as Nav1.5, Na1.9, and Na1.8 Less sensitive isoforms include Na1.3 and Na1.7, with no p-conotoxin found to block Na1.8 Instead, p-conotoxins appear to target the skeletal muscle isoform Na1.4 and the brain isoform Na1.2 of mammals with the highest affinity The development of analogues targeting other therapeutic subtypes is currently on the rise.
pO-conotoxins belong to the superfamily of conopeptides and feature an ICK motif with appropriate disulfide bond connectivity Although only a limited number of peptides have been identified within this class, the conotoxins pO-MrVIA and pO-MrVIB have garnered particular attention due to their analgesic effects observed in animal pain models These effects are attributed to their relative selectivity for the NaV 1.8 channel and other sodium channels sensitive to the 11X expressed in dorsal root ganglion neurons.
236] Des diíĩérenees signiricatives dans leur aHìnité pour les canaux Na, 1.8 natiỉ et Na, 1.8 hétérologue exprin.es dans les neurones DR(i ont éte signalees Gelles-C, pnH.ennent
The introduction highlights the role of auxiliary subunits p, particularly pO-MrVIB, which significantly enhance the inhibition rate of the Nav1.8 channel and the potency of the toxin Notably, pO-conotoxins can differentiate between TTX-resistant channel subtypes Nav1.8 and Nav1.9, as the Nav1.9 current in DRG neurons remains unaffected by pO-MrVIB While pO-MrVIA and pO-MrVIB influence the Cav channel in mollusks, this mechanism does not seem to contribute to analgesic effects in mammals, since the Cav channel in DRG neurons is not impacted.
Unlike p-conotoxins, our understanding of the structure-activity relationships of pO-conotoxins is limited, primarily due to the unavailability of pO-MrVIA and pO-MrVIB for pharmacological evaluation, as they differ by only three residues (Ser3/Arg3; Leu14/Ile14; Val17/Ile17), leaving their effects on various Nav channel subtypes unassessed Additionally, challenges in the synthesis, correct folding, and purification of hydrophobic pO-conotoxins have hindered efforts to identify their pharmacophore through alanine scanning approaches However, recent advancements in the oxidative folding of synthetic pO-MrVIB, utilizing selective synthesis with selenocysteines, may provide a faster method for generating pO-conotoxin analogs, facilitating the identification of residues that contribute to their affinity and selectivity.
4.3.2.3 Autres conotoxines inhibant les canaux sodium dépendants du potentiel
Conotoxin GS has been identified as a member of the o-superfamily, but unlike pO-MrVIA and pO-MrVIB, it appears to share a binding site with TTX and p-GIIIA, indicating distinct structural and functional characteristics Additionally, two new conotoxins from the o-superfamily, LtVIC and LtVIIA, were discovered in Conus luteratam through DNA sequencing These recombinant peptides inhibit sodium currents in DRG neurons similarly to pO-MrVIA and pO-MrVIB, suggesting they are novel qO-conotoxins, although their subtype selectivity and structure-activity relationships require further investigation Notably, conotoxin pT-LtVD inhibits the TTX-sensitive Nav channel.
4.3.3 Activateurs des canaux sodium dépendants du potentiel : les S-conotoxines
Conotoxins, specifically ồ-conotoxins and peptides ICK, share functional similarities with scorpion toxins by targeting the site 3 of the Na+ channel's alpha subunit These toxins inhibit the rapid inactivation of Na+ channels, shifting the activation potential to more negative values, which leads to prolonged action potentials and persistent neuronal firing Structural analysis reveals that several conserved hydrophobic residues in ồ-conotoxins are located on the external surface of the peptides, suggesting potential interactions with hydrophobic residues in the S3/S4 loop of domain IV, which is part of site 6 of the Na+ channel.
On en connait peu sur la sélectivité des 5-conoto.xines vis-àvis des sous-types de canaux N av Les ô- conotoxines isolées de cônes malacophaues à 1'exception de
TAm2766 selectively targets Na channels in mollusks and is inactive on those in mammals, while ô-conotoxins from piscivorous cones appear to also inhibit Na channels in mammals Interestingly, although malacophagous cone toxins like 5-TxVIA and ô-timVIA show no activity on mammalian Na channels, they do bind to mammalian tissues, indicating subtle differences in the binding sites of ô-conotoxins lead to varying activities In fact, several ô-conotoxins demonstrate activity on mammalian Na channels Na1.2 and Na1.4.
Recent studies indicate that the peptide Ò-LVIA shows selectivity for neuronal sodium channel subtypes Nav1.2, Nav1.3, and Nav1.6, as well as for skeletal muscle (Nav1.4) and cardiac (Nav1.5) subtypes This suggests that selectivity among sodium channel subtypes may also be present in other neurotoxins.
The reminiscence of p-conotoxins highlights the challenges in studying their structure-activity relationships and selectivity for sodium channel subtypes, primarily due to the difficulties in synthesizing and purifying hydrophobic p-conotoxins and their analogs Additionally, the limited availability of correctly folded peptides has hindered progress in this field Notably, there is significant sequence homology among different p-conotoxins from cone snails, particularly within the central hydrophobic region of these peptides.
K-conotoxines agissant sur les canaux p o ta ssiu m
Les canaux potassium constituent le groupe le plus important et le plus diversiíìé de canaux ioniques, représenté par quelques 70 loci connus dans le génome des mammifères.
Potassium voltage-dependent channels (Kv) represent the largest family of approximately 40 genes in the human potassium channel repertoire This family includes the calcium-activated potassium channel (KCa), the inward rectifier channel (KIR), and the two-pore domain potassium channels (K2P) Figure 23 illustrates the phylogenetic tree of the Kv channel family, along with their designations from the International Union of Pharmacology (IUPHAR) and gene names established by the HUGO Gene Nomenclature Committee (HGNC).
Kv3.3 ỉ*.CNCì iị f— Kv4.1 rxCNO? Xf-M'
K v 1 -5 ; kc >4A* \J Kvl i ' 1> p Kvl.l' tr 1— -Kv1 — — K rằKv1 - 2 f*CH *2 -pi)i Ị f — Kv11 '*.CNA* ’ 2 p 1 lỊ
Figure 23 illustrates the phylogenetic tree of K+ channel families KJ-12, highlighting the nomenclature established by IUPHAR and HGNC, along with the chromosomal locations of the genes and other commonly used names Notably, the 6S IM channel represents the predominant class among ligand-sensitive potassium channels, influencing membrane potential.
In vertebrate motor nerve endings, there are three types of potassium channels classified as 6STM/P: fast (or transient) potassium channels, delayed (or slow) potassium channels, and calcium-activated potassium channels Pharmacological agents that inhibit fast potassium channels, such as aminopyridines and diaminopyridines, enhance the phasic release of acetylcholine (ACh) triggered by nerve stimulation This effect results from the prolongation of the presynaptic action potential, leading to an increased calcium influx.
I ongine de I augmentation de libération d ACli Au ni\eau postx\ naplique eetle augmentation de libération se traduìt par une augmentation des inílux de Na et de ( a : et de
The efflux of intracellular calcium (IC) through nicotinic receptor channels increases with higher levels of active acetylcholine (ACh) and more receptors, resulting in enhanced synaptic responses In skeletal muscle, this leads to prolonged action potentials, causing a delay in membrane repolarization and an increased release of calcium ions (Ca2+) from the sarcoplasmic reticulum, which ultimately boosts the contractile strength of skeletal muscle.
There are several families of K-conotoxins isolated from cone snail venom, including KA-conotoxins, KO-conotoxins, KM-conotoxins, KJ-conotoxin, kI2-conotoxins, contryphan-Vn, and conkunitzin-Sl, which target various subtypes of potassium channels The activity of potassium channels appears to play a crucial role in the "lightning strike" strategy of predatory cone snails However, there is limited knowledge regarding the selectivity towards potassium channel subtypes and the structure-activity relationship, despite the numerous potential therapeutic applications.
The kA-MIVA, kA-SIVA, kA-SIVB, KA-SmIVA, KA-SmlVB, kA-PIVE, kA-CCTX, and kA-PIVF conotoxins are relatively short excitatory peptides that induce powerful effects when injected intramuscularly in fish and directly applied to neuromuscular preparations in amphibians, but not in mammals It remains unclear whether the observed differences are due to pharmacodynamic effects, such as restricted access to mammalian Kv channels located in the juxtaparanodal regions of myelinated motor axons, or if they reflect significant differences in affinity for various subtypes While some of these peptides inhibit Shaker Kv channels, the precise targets and the structure-activity relationship for most KA-conotoxins remain uncertain.
The KO-conotoxin PVIIA is extracted from the venom of the piscivorous cone snail Conus purpurascens This toxin selectively blocks the Shaker-type potassium channel expressed in Xenopus oocytes at submicromolar concentrations, without affecting Cav or Nav channels It has garnered significant attention due to its cardioprotective properties demonstrated in various animal models of ischemia.
Tableau 11 illustrates the inhibition of K\ channels by k-Conotoxins (specifically, the x-A/M-conotoxin C0C6-7CC2-4C0-3C) The cysteines are highlighted in bold and shaded in gray The table categorizes species based on their dietary habits: p for piscivore, m for malaeophage, V for vermivore, and ND for non-determined Additionally, it notes specific modifications such as * for C-terminal amidation, s for glycosylated serine, E for gamma-carboxy glutamic acid, w for D-tryptophan, z for pyroglutamic acid, and o for 4-hydroxyproline.
RI 11.1 M ( ’ nnliíUus p 1 o o c c r o () k Kn - COAOACk YK () c c K s ! 2 |25í.|
SI VA A ( ' xíriatus' p / k s I V p s V 1 r r c c (i Y I) () (ì T V c o (> - - C R C ĩ N s c * 1’oisson urcnouillc 12581
Figure 24 illustrates the interaction model between conotoxin K-PVIIA and the Shaker-type potassium channel, highlighting a potential interaction site involving Lys7 and Phe9 Interestingly, the rat K v.1 channel exhibits resistance to K-PVIIA, suggesting the existence of other unidentified mammalian K.V channel isoforms that may play a role in the cardioprotective effects of K-PVIIA.
Figure 24 Modèle de 1'interaction entre la conotoxine K-PVIIA et le canal potassium de type Shaker, révélant un modèle susceptible d'interaction de la dyade comprenant la Lys7 et la Phe9 [14].
Like conotoxin K-PVIIA, conotoxin kM-RIIIJ exhibits cardioprotective effects, likely due to its strong affinity for Kv1.2 channels and Kv1.2-containing heteromultimers Kv1.1, Kv1.5, and Kv1.6 It is important to note that conotoxin kM-RIIIJ is less potent on the Kv1.2/Kv1.7 heteromultimers compared to conotoxin kM-RIIIK, indicating that different Kv channel heteromultimers are pharmacologically distinct Several residues, including Leu1, Arg10, Lys18, and Arg19, play a critical role in the activity of conotoxin kM-RIIIK on the K TShal channel.
KJ-pll4a conotoxin, a member of the J superfamily, is a unique conotoxin that inhibits the Kv1.6 channel and certain nAChR isoforms Homology modeling indicates that this peptide features a functional dyad formed by residues Lys18 and Tyr19, as well as a ring of basic residues including Arg3, Arg5, Arg12, and Arg25, resembling the pharmacophore identified in conotoxin k M-RIIIK.
Cependant il reste à déterminer si ces résidus sont impliqués dans interactions a \ec les canaux Ky comme prévu.
Conotoxins kI2-VíTx, icI2-srlla, and KỈ2-BeTX represent an intriguing class of structurally related potassium channel modulators The conotoxin kI2-VíTx specifically inhibits the Kyl.1 and Kv1.3 channels but does not affect the Kv1.2 channel In contrast, the recently identified conotoxin K'12-srlla has been recognized as an inhibitor of the Kv1.2 and Kv1.6 channels, while it does not inhibit Kv1.3.
Ky 1.3 [264] En revanche la conotoxine Kl2-BeTX atTiche une modulation inhabituelle du canal BK sensible au potentiel, en augmentant sa probabilité d 'o u \ertu re [265Ị Let ettet indépendant de 1'inactivation du canal est facilement reversible ee qui suggère une interaction avec un site de liaison extracellulaire [265] Les bases moleculaires de la sélectivité des Kl2-conotoxìnes demeurent inconnues à ce jour.
The contryphan-Vn has recently been identified as a peptide with a unique structure that contains D-tryptophan and exhibits activity on L-sensitive K channels and membrane potential, potentially due to the presence of a specific dyad However, the structural requirements for contryphan-Vn's activity on its molecular targets, as well as its selectivity towards subtypes, remain to be determined.
Conkunitzin-S1 is a 60-amino-acid peptide stabilized by disulfide bridges It inhibits the potassium channel by interacting with the ion channel pore through the amino acid K427.
4.5 Les conotoxines agissant sur les récepteurs couplés aux protéines (, ((;P ( K) et les transporteurs de neurotransmetteurs
Applications thérapeutiques
Many conopeptides target pain pathways and allow for the specific dissection of ion channels and receptors responsible for pain In 2004, the FDA approved the conopeptide CO-MV1IA, which became the first drug derived from cone snail venom to be marketed under the generic name ziconotide and the brand name Prialt® by Elan Pharmaceuticals Prialt® is a powerful analgesic, reported to be 1000 times more effective than morphine The N-type Cav channels (Cav2.2) regulate neurotransmitter release at central and peripheral synapses, with a high density in the spinal cord where nociceptive fibers terminate This has led to the exploration of N-type Cav channel antagonists, such as conotoxin CO-MV1A, for pain suppression Prialt® effectively reduces cancer-related, inflammatory, neuropathic, nociceptive, and post-operative pain, as well as fibromyalgia, and is particularly beneficial for patients experiencing pain resistant to opioids.
Tableau 13 Différents représentants majeurs des classes de conotoxines détinis par la pharmacologie * : amidation C-terminale; a : développement clinique suspendu.
Nom Famille Mode d’action Séquence Potentiel clinique Réf.
MVIIA CO Inhibiteur Cas2.2 CKGkGAKCSRLMYDCCTG
SCRSGKC* Doulcur 1 mtralheèak* pha>L í V • Ị GẠ
SII1A n lnhibiteur Nav ZNCCNGGCSSk\YCRĐHAR cc* Douleur 1 ' í-iq
MrVlB nO lnhibìteur Nav 1.8 ACSKKVVE YCIYPIIGH YCCP
CNNRFNKCV Reperrusion èarJLie]uc Ị >8] Xen2174 X Inhibiteur NET ZGVCCGYKLCH< )C' Doulcur ! tntraihècaìc pha'-c !ỉ ' r*\
4-V c 1.1 u Inhibiteur nAChR GCrSDPRCNYDHPrie* Uouiour I inirathdcaU'1 a |M'l
Adrenoceptive PNVVRCn is associated with the antagonist Con-G Conantokine, while R-NV1DA GI UAGFNT serves as a critical component in its function Additionally, Cono-C Conopressine acts as an agonist for R-vasopressine, influencing various physiological responses Furthermore, Cont-G Contuikine functions as an agonist for R-neurotensin, highlighting its role in neurochemical signaling.
Conotoxin K-PVIIA, isolated from Conus purpurascens, targets potassium channels and plays a significant role in cardiac physiology Research has demonstrated the effects of K-PVIIA in rabbits during myocardial infarction, showing that it does not induce hemodynamic alterations at tested doses In conclusion, K-PVIIA is a potent anti-infarct agent when administered just prior to reperfusion.
Xen2174 (conotoxin y-MrIA), isolated from Conus niannoru, is being developed by the Australian biotechnology company Xenome Limited for the treatment of severe cancer-related pain This compound selectively inhibits norepinephrine transporters, which are responsible for the reuptake of this neurotransmitter, thereby increasing its levels In the spinal cord, norepinephrine serves as the dominant neurotransmitter for activating descending inhibitory pain pathways.
Les a-conotoxines ciblant les récepteurs nAChR neuronaux (représentées par a-
V cl.l de Comis victoriae and a-Iml de c imperialis may be beneficial in treating diseases associated with receptor dysregulation, including Alzheimer's disease, Parkinson's disease, schizophrenia, and pain management.
Conantokinin G (Con-G), derived from the venom of Conus geographus, acts as an antagonist to NMDA receptors, specifically targeting the NR2B subunit Preclinical trials in animal models have demonstrated significant antiepileptic properties However, clinical development was halted due to adverse side effects.
Contulakine G (Cont-G) is a neurotensin receptor antagonist patented by Cognetix in 2002 under the name CGX-1160, aimed at treating both chronic and acute pain In December 2002, Cognetix announced initial results from Phase I clinical trials; however, clinical development was halted due to certain side effects.
The limited number of characterized species in relation to the richness of venoms suggests a significant potential for exploration, indicating that many molecules with diverse biological activities are yet to be discovered.
La jonction neuromusculaire et la contraction des m u scles
Potentiel membranaire
A key characteristic of living cells is the unequal distribution of negative and positive ions across the inner and outer faces of the plasma membrane This asymmetry gives rise to the membrane potential, creating an electrical potential difference between the two sides of the membrane This difference can only be balanced when ion channels allow the movement of ions that are unevenly distributed Specifically, there are more potassium (K) ions inside the cell than outside, while the situation is reversed for sodium (Na), calcium (Ca), and chloride (Cl) ions.
Chaque espèce d ’ions a donc tendance à diffuser passivement ce qui entraine 1'apparition d ‘un gradient électrique qui va à 1’encontre du gradient de concentration et qui engendre une différence de potentiel membranaire.
AChmotecute* recsplor at mótor ondpiaie
Bmdmg o(AChloôô Í6cepf0f ỡpens Ihe sodium channel
S y n a p lic v e s i c l e s con la in ín g a c e t y c h o li n e
Aằonal lemnnal ot a motornaoron Mrtochondnon Synaptic cletí
Juncltonal toids o(tha sarcotemma ai moằor and piate
F igure 26 Reprẻsentation schématique de 1’innervation motrice des muscles squelettiques, de la transmission axonale et de la transmission neuromusculaire ld après Benjamin Cummings (2001)].
Potentiel de repos et potentiel d’action
At resting potential, there are significantly more open potassium (K+) channels than sodium (Na+) channels, with even fewer calcium (Ca²+) and chloride (Cl⁻) channels available Consequently, the membrane is predominantly permeable to K+ ions, resulting in a resting potential of approximately -70 to -80 mV, which is closer to the equilibrium potential of K+ ions (~ -80 to -90 mV) than that of Na+ ions (~ +60 mV).
The action potential is initiated by a depolarizing stimulus that increases the membrane's permeability to Na+ ions through the opening of Nav channels When a neurotransmitter binds to an ionotropic receptor, it causes a temporary increase in the membrane potential from -60 mV to +30 mV Although the membrane potential quickly returns to its original value within a few milliseconds, nearby membrane areas have been activated, allowing depolarization to propagate This process begins with the opening of Nav channels, leading to Na+ ions entering the cell due to their electrochemical gradient, which locally reverses the membrane potential The Nav channels then undergo inactivation, resulting in a brief influx of positive charges, which triggers the opening of K+ channels that allow K+ ions to exit the cell, leading to membrane repolarization Additionally, the Na+/K+-ATPase pump moves Na+ ions out of the cell and K+ ions back in, briefly causing hyperpolarization, which exceeds the resting potential of the cell.
Kv se ferment également après quelques ms et la cellule nerveuse redevient de nouveau excitable.
5.3 Libération (Tacétylcholine et transmission neuromusculaire squelettique
When an action potential is generated at the cell body of a neuron, it propagates along the axon to the nerve terminal without attenuation At the neuromuscular junction, the release of acetylcholine (ACh) is dependent on the extracellular concentration of Ca2+ Upon the arrival of an action potential at the nerve terminal, membrane depolarization triggers the opening of Ca2+ channels, leading to the fusion of synaptic vesicles containing ACh with the plasma membrane This results in the synchronous release of multiple quanta of ACh into the synaptic cleft The binding of ACh to its nicotinic receptors on muscle cells opens the nicotinic channel, allowing Na+ to enter and K+ to exit, which generates a local depolarization known as the end-plate potential If this depolarization reaches a threshold, a muscle action potential is triggered, propagating along the muscle fiber and causing muscle contraction The remaining ACh is then degraded by acetylcholinesterase anchored to the basement membrane in the synaptic cleft, playing a crucial role in neuromuscular transmission.
Flgure 27 Conductances des canaux ioniques au sodium et au potassium en relation avec les étapes d'un potentiel d'action nerveux (d'après Girard Ị271 ]).
5.4 Les différents canaux ioniques au niveau des terminaisons nerveuses de vertébrés
Vertebrate nerve endings contain various types of voltage-gated ion channels, including Nav, Kv, and Cav channels These channels play a crucial role in ensuring the physiological functions of these nerve endings, particularly in the transmission of nerve impulses.
The research outlined in this manuscript focuses on isolating, chemically characterizing, and identifying the molecular targets of conopeptides derived from the venom of the malacophagous cone snail, Conus bandanus, collected in Vietnam.
Initially, we employed a proteomic approach to assess the conopeptide composition of the venom from Conus bandanus, comparing it to that of Conus marmoreus, another closely related malacophagous cone species collected from the same region in Vietnam.
We developed extraction and fractionation methods for the crude venom of Conus bandanus, alongside biological tests on invertebrates essential for identifying bioactive actions The purification through reverse-phase chromatography enabled us to isolate the toxins homogeneously for sequencing via mass spectrometry and/or Edman degradation Subsequently, their primary structure was compared to peptides from families of conotoxins already characterized using bioinformatics and the ConoServer database.
Enfín les effets de certains conopeptides naturels ont été étudiés par diíTérentes techniques dans le but d’identifíer leur(s) cible(s) physiologique(s).
Les différents canaux ioniques au niveau des terminaisons
Vertebrate nerve endings contain various types of voltage-gated ion channels, such as Nav, Kv, and Cav channels These channels play a crucial role in the physiological function of nerve endings, facilitating the transmission of nerve impulses.
The research detailed in this manuscript focuses on isolating and chemically characterizing conopeptides derived from the venom of the malacophagous cone snail, Conus bandanus, collected in Vietnam Additionally, the study aims to identify the molecular targets of these conopeptides.
Initially, we employed a proteomic approach to assess the conopeptide composition of the venom from Conus bandanus, comparing it to that of Conus marmoreus, another closely related malacophagous species collected from the same region in Vietnam.
We developed extraction and fractionation methods for the raw venom of Conus bandanus, alongside biological tests on invertebrates to identify bioactive actions Reverse phase chromatography allowed us to isolate the toxins homogeneously for sequencing via mass spectrometry and/or Edman degradation The primary structure of these toxins was then compared to peptides from previously characterized conotoxin families using bioinformatics and the ConoServer database.
Enfín les effets de certains conopeptides naturels ont été étudiés par diíTérentes techniques dans le but d’identifíer leur(s) cible(s) physiologique(s).
Préparation de deux venins et puriíication de conopeptides
Collection de cônes au Vietnam
Conus bandunus (12 + 3 specimens) and Conus mannorens (3 specimens) were collected and selected by researchers from Nha Trang University in Nha Trang Bay, Vietnam The specimens were stored at -80°C, then thawed and dissected to extract the venom duct, venom gland, radular sac, and pharynx These components were cut into small pieces, placed in a vessel with 5 mL of 0.1% TFA, and the extraction was repeated four times with 0.1% TFA in H2O at 4°C The venom was then collected, lyophilized, weighed, and stored at -20°C.
1.2 Fractionnement du venin brut et puritìcation par HPLC-RP
The HPLC system utilized features a binary pump SYS IT3M GOLDx 126 Solvent Module, a 166 Detector from Beckman Coulter, and a data acquisition and processing system, 32 Karat 5.0.
Raw venoms were directly separated using HPLC on a semi-preparative Ci8 Vydac column (300 Å, 5 µm, 10 mm i.d x 250 mm) at a flow rate of 3 mL/min and a detection wavelength of 220 nm Each fraction exhibiting biological activity was further purified on an analytical C|8 Vydac column (300 Å, 5 µm, 4.6 mm i.d x 250 mm) at a flow rate of 1 mL/min and the same detection wavelength Finally, the conopeptides were repurified to homogeneity on the same analytical column using corresponding solvent gradient programs.
1.3 Préparation et comparaison des venins bruts de c bandanus et c m arm oreus
Huit mg de chaque venin brut lyophilisé ont été dissouts dans 1 mL d'eau distillée contenant
A 0.1% TFA solution was centrifuged for 1 minute at 20 X g to separate visible granular material The supernatant was collected, filtered through a 0.5 mL Amicon Ultra 10 kDa device (Millipore Amicon®), and centrifuged at 12,000 X g for 20 minutes at 4°C Protein concentration tests were conducted using the Biorad protein assay reagent (Ref 500-).
The protein concentration estimates were obtained following the manufacturer's instructions and adapted from Bradford's method (Bradford, 1976) Measurements were conducted using a NanoDrop 2000c spectrophotometer (Thermo Scientific), with bovine serum albumin used as the standard for quantification.
50 pL de chaque extrait du venin quantiíié (0,354 pg/pL pour c bandanus et 0,169 (J.g/|aL pour c marmoreus) ont été centrifugés et séchés sous vide par un évaporateur rotatif
SpeedVac (ThermoFisher, Courtaboeuf, France) Quelques pL d'acide formique 0,1% ont ĩinalement été ajoutés au mélange pour une analyse ultérieure (Figure 30).
Figure 28, Deux images de coquilles de c bandanus (en haut à gauche) et c m arm oreus (en bas à droite). r o m * 2n ( w f è|me de HPLC de 1 équipe c0" lprc" an' : P °mPe binaire SYSTI
T , l ỉ (So ven, M„dule, détecK„; |6 6 D 'etector; cô u lter) e( d ordinateur de traitement de données
Préparation et comparaison des venins bruts de c
Huit mg de chaque venin brut lyophilisé ont été dissouts dans 1 mL d'eau distillée contenant
A 0.1% TFA solution was centrifuged for 1 minute at 20,000 x g to separate visible granular material The supernatant was collected, filtered through a 0.5 mL Amicon Ultra 10 kDa device (Millipore Amicon®), and centrifuged at 12,000 x g for 20 minutes at 4°C Protein concentration tests were conducted using the Biorad protein assay reagent (Ref 500-).
The protein concentration estimates were obtained following the manufacturer's instructions and adapted from Bradford's method (Bradford, 1976) Measurements were conducted using a NanoDrop 2000c spectrophotometer (Thermo Scientific), with bovine serum albumin serving as the standard for the assay For comparative analysis, samples ranged from 25 to
50 pL de chaque extrait du venin quantiíié (0,354 pg/pL pour c bandanus et 0,169 (J.g/|aL pour c marmoreus) ont été centrifugés et séchés sous vide par un évaporateur rotatif
SpeedVac (ThermoFisher, Courtaboeuf, France) Quelques pL d'acide formique 0,1% ont ĩinalement été ajoutés au mélange pour une analyse ultérieure (Figure 30).
Figure 28, Deux images de coquilles de c bandanus (en haut à gauche) et c m arm oreus (en bas à droite). r o m * 2n ( w f è|me de HPLC de 1 équipe c0" lprc" an' : P °mPe binaire SYSTI
T , l ỉ (So ven, M„dule, détecK„; |6 6 D 'etector; cô u lter) e( d ordinateur de traitement de données