Les neurones sensoriels myentériques (étude in situ par la technique de Patch Clamp)

Les principaux courants ioniques des neurones myentériques AH in situ ont été étudiés en patch clamp dans le duodenum de cobaye. Ih régule la résistance d'entrée des neurones AH. La conotoxine GVIA, mais pas l'agatoxine IVA, abrège le potentiel d'action et bloque l'AHP qui est donc couplée aux canaux Ca2+ de type N. Nous avons aussi découvert un courant Na+ résistant à la TTX (TTX-R INa). TTX-R INa s'active et s'inactive lentement et possède une composante persistante. La substitution du Cl-intracellulaire par le F- hyperpolarise les paramètres dépendants du voltage de TTX-R INa, lui conférant les propriétés de NaN/NaV1.9 dans les DRG. Des expériences de RT-PCR, single cell RT-PCR et immunohistochimie indiquent que l'ARNm et la sous-unité NaV1.9 sont présents dans les neurones AH. Dans les neurones AH, NaN présente aussi une inactivation originale associant une diminution ultra lente ( max=100 s) de NaN à un ralentissement des cinétiques d'activation (m) et d'inactivation (hf).AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Modulation du courant Nav1.9 par l'activation des récepteurs NK3 aux neurokinines dans les neurones afférents primaires du système nerveux entérique de cochon d'inde

Dans le système nerveux entérique (SNE) de cochon d Inde, les neurones afférents primaires intrinsèques (IPANs) expriment sélectivement le canal Nav1.9 qui produit un courant Na+ résistant à la TTX, lent et persistant (INav1.9). Ils communiquent entre eux et avec les interneurones ou motoneurones par des PPSEs lents induits principalement par l activation des récepteurs NK3 (rNK3) aux neurokinines. Leur activation sensorielle déclenche des réflexes moteurs et sécréteurs. Ce travail de thèse, basé essentiellement sur l enregistrement en patch clamp d IPANs de duodenum de cobaye in situ, montre que l activation des rNK3 par l agoniste sélectif senktide, augmente INav1.9 Cet effet, transitoire du fait de la désensibilisation des rNK3, implique la PKC et résulte d un déplacement négatif de la dépendance au potentiel du canal, Il permet à INav1.9 de générer des plateaux dépolarisants et de diminuer le seuil de genèse des potentiels d action. Pour rechercher l impact fonctionnel du couplage Nav1.9/rNK3, nous avons entamé une étude comparative de la motricité du colon de souris sauvages et Nav1.9-/-. Après avoir vérifié la présence de Nav1.9 dans les neurones du colon de souris nous montrons, dans un premier temps, que la fréquence des complexes migrants myoélectriques est fortement réduite par un antagoniste des rNK3. Le couplage Nav1.9/NK3r favorise vraisemblablement la transmission synaptique dans le SNE. Ceci souligne que dans ce système, Nav1.9 et rNK3 pourraient avoir un rôle physiologique, alors qu on attribuait de rôle pour rNK3 qu en conditions extrêmes et que dans les neurones nociceptifs, Nav1.9 n agit que dans la douleur inflammatoire.In the enteric nervous system (ENS) of the guinea pig, the intrinsic primary afferent neurons (IPANs) selectively express the Nav1.9 channel, which produces a TTX-resistant, slow and persistent Na+ current (INav1.9). They communicate between themselves, and with interneurons and motoneurons by slow EPSPs that are mainly induced by neurokinin NK3 receptors (NK3r) stimulation. Their sensory activation triggers motor and secretory reflexes. This work, mainly performed by in situ IPANs patch clamp recording in the guinea pig duodenum, shows that rNK3 activation by senktide, a selective agonist, increases INav1.9. This effect, transient because of the rNK3 desensitization, involves PKC and is due to a negative shift of the voltage dependence of channel gating processes. It allows INav1.9 to generate plateau potentials and to reduce the action potential threshold. In order to examine the functional consequences of the Nav1.9/rNK3 coupling, we started a comparative study of colon motility in wild type and Nav1.9-/- mice. After having confirmed that Nav1.9 is expressed in mouse colon neurons, in a first step we show that myoelectric migrant complexe frequency is significantly reduces by a rNK3 antagonist. Nav1.9/rNK3 coupling seems to favor synaptic transmission in the ENS. It underlines that, in this system, Nav1.9 and rNK3 could play a physiological role, whereas rNK3 was though to act only in extreme conditions in gut motility and Nav1.9 is involved only in inflammatory pain in nociceptive neurons KeyAIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

Activation of neurokinin 3 receptor increases Na(v)1.9 current in enteric neurons.

International audienceThe intrinsic primary afferent neurons (IPANs) of the guinea pig enteric nervous system express Na(v)1.9 sodium channels that produce a persistent TTX-resistant current having a low activation threshold and slow gating kinetics. These neurons receive slow EPSPs induced mainly by the activation of neurokinin 3 receptors (NK3r). Here, we demonstrate that senktide, a specific NK3r agonist, potentiates the Na(v)1.9 current (I(Nav1.9)) in IPANs. Using whole-cell patch-clamp recordings from IPANs in duodenum longitudinal muscle/myenteric plexus preparations, we show that short (1-5 s) and long (up to 1 min) applications of senktide, increase the I(Nav1.9) peak current up to 13-fold. The effect, blocked by a NK3r antagonist SB235375 is transient, lasting approximately 2 min and is due to a negative shift of the activation voltage by approximately 20 mV and of fast inactivation by approximately 10 mV. As a consequence, the window current resulting from the product of the activation and fast inactivation curves is shifted and enlarged. The transient effect of senktide is likely to be due to the fast desensitization of NK3r. Protein kinase C (PKC) activation with phorbol or oleoyl acetylglycerol also increases I(Nav1.9), although persistently, by inducing similar voltage-dependent changes. Current-clamp experiments showed that I(Nav1.9) modulation by senktide lowers action potential threshold and increases excitability. The increase in I(Nav1.9) by NK3r activation is also likely to amplify slow EPSPs generated in the IPANs. These changes in excitability potentially have a profound effect on the entire enteric synaptic circuit and ultimately on gut motility and secretion

Effects of K + Channel Blockers on Developing Rat Myelinated CNS Axons: Identification of Four Types of K + Channels

International audienc

Encoding properties induced by a persistent voltage-gated muscarinic sodium current in rabbit sympathetic neurones

A time- and voltage-dependent Na+-selective current termed INa,M is activated by muscarinic agonists or splanchnic nerve stimulation in sympathetic neurones of rabbit coeliac and superior mesenteric ganglia. The firing patterns induced by INa,M were investigated in patch-clamped neurones within intact ganglia, and compared with those generated by a neuronal model including INa,M.INa,M was characterized by voltage-dependent low-threshold activation and high-threshold inactivation functions. The overlapping functions produced a persistent U-shaped current between −100 and −20 mV, which peaked at the cell resting potential. The activation and inactivation kinetics were fitted to single exponentials with time constants of ≈100 and 400 ms, respectively.Activating INa,M with muscarinic agonists or nerve stimulation depolarized and fired the neurones. The depolarization was paralleled by an apparent increase in input membrane resistance. The model showed that this paradox resulted from the turning off of INa,M during resistance tests, which also accounted for the all-or-none slow hyperpolarizing responses to current pulses.INa,M gave the neurones an N-shaped I-V relationship capable of producing complex firing patterns. Under given conditions, carbachol-treated neurones could either fire regularly or remain silent at ≈-80 mV, i.e. they displayed bistability. Transitions from one state to the other were triggered with short current pulses. The transitions resulted from the turning on and off of INa,M.Firing reduced INa,M, an effect abolished by blocking Ca2+ channels or adding BAPTA (40 mM) to the pipette. The Ca2+-related negative regulation of INa,M may have mediated endogenous bursting activity. Burst firing was generated by the model upon introducing Ca2+ regulation of INa,M.The results demonstrate that INa,M gives prevertebral sympathetic neurones a wide repertoire of firing patterns: pacemaker-like properties, bistability and burst firing capability. They suggest that the INa,M-related encoding properties may provide sympathetic neurotransmission with new potentialities

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. : Clues to design analogs with enhanced selectivity

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity