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The heart is a spontaneously active organ whose contractile activity is dependent of a spontaneous intrinsic electrical activity, which refers to one of the fundamental cardiac properties: cardiac automaticity. The electrical signal is generated in the sino-atrial node tissue (SAN) and is then propagated throughout the heart, triggering cardiomyocytes contraction. Cardiac automaticity relies on the capacity of pacemaker sino-atrial node cells (SANC) to spontaneously depolarize (diastolic depolarization, DD) which brings membrane potential to the threshold of the sinoatrial action potential (AP). Complex and not entirely understood yet, diastolic depolarization results from a robust interplay of membrane ion channels activity (hyperpolarization-activated HCN4 channels, L-type Cav1.3 and T-type Cav3.1 calcium channels) and intracellular calcium dynamics with spontaneous local calcium releases (LCRs) from the sarcoplasmic reticulum via the ryanodine receptors (RyR). By triggering cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) catecholaminergic stimulation of pacemaker activity by sympathetic autonomic nervous system accelerates the firing frequency of SANC.The objective of my PhD was to determine the role of L-type Cav1.3 calcium channels in the catecholaminergic regulation of SANC. Patch-clamp experiments on isolated mouse SANC showed that pharmacologic inhibition of Cav1.3 channels completely reversed the effects of adrenergic stimulation on the frequency. Imaging of intracellular calcium further revealed that LCRs synchronization under activation of adrenergic receptors was dependent of Cav1.3 channels activity. Hence, L-type Cav1.3 calcium channels seem to be the main mediator of the positive chronotropic response of pacemaker activity in mouse SANC., Le cœur est un organe autonome dont l’activité contractile est dépendante d’une activité électrique intrinsèque spontanée. Au niveau de l’oreillette droite se situe le nœud sinoatrial (NSA) au sein duquel une impulsion électrique spontanée est initiée puis propagée à l’ensemble du tissu cardiaque provoquant alors la contraction des cardiomyocytes. La clé de l’automaticité cardiaque réside dans le fait que les cellules sinusales (SANC) ou « pacemaker » sont capables de se dépolariser spontanément (dépolarisation diastolique, DD) ce qui amène progressivement leur potentiel de membrane jusqu’au seuil de déclenchement du potentiel d’action (PA) sinusal. Complexe et encore non entièrement comprise, la dépolarisation diastolique est due à une robuste interaction entre l’activation séquentielle de canaux ioniques de la membrane cellulaire (canaux s’ouvrant par hyperpolarisation HCN4, canaux calciques de type L Cav1.3 et de type T Cav3.1) et une dynamique calcique intracellulaire avec des relargages calciques spontanés (LCRs) diastoliques par le réticulum sarcoplasmique via les récepteurs à la ryanodine (RyR). En déclenchant la voie de signalisation adénosine monophosphate cyclique/protéine kinase A (AMPc/PKA) la régulation adrénergique par le système nerveux autonome sympathique permet d’accélérer la fréquence de dépolarisation des SANC.L’objectif de ma thèse était de déterminer le rôle des canaux calciques de type L Cav1.3 dans la régulation adrénergique des SANC. La technique de patch-clamp sur SANC isolées de souris a montré que l’inhibition pharmacologique des canaux Cav1.3 inversait complètement les effets de la stimulation adrénergique sur la fréquence de dépolarisation des SANC. Les expérimentations d’imagerie calcique ont permis de révéler que la synchronisation des LCRs sous stimulation adrénergique était dépendante des canaux Cav1.3. Ainsi, les canaux calciques de type L Cav1.3 semblent être les régulateurs majoritaires de la réponse chronotrope positive chez la souris.

The membrane of cardiac ventricular myocytes is characterized by many invaginations, essential for excitation-contraction coupling mechanism, called transverse tubule (TT). The membrane of TT (TTM) contains many receptors, channels or enzymes, such as β-adrenergic receptors (β-ARs), adenylyl cyclases or L-type Ca2+ channels (LTCC). These membrane proteins are also present in the outer surface membrane (OSM), although often at a different density. Classical pharmacology allows to explore the function of a membrane protein in the whole cell membrane but not separately in OSM vs. TTM compartments.We developed a technology based on size exclusion to explore the function of β-Ars and LTCCs located in the OSM. Specific ligands of β-Ars and LTCCs have been grafted at the surface on nanoparticles and PEGylated using a covalent link between a 5000 Da PolyEthylene-Glycol (PEG) chain. The objective is to increase the size of the β-AR and LTCC ligands and prevent their access in the TT network. Using this new strategy, we are able to selectively activate or inhibit β-Ars and LTCCs present at the OSM without acting on their homologous subpopulations at the TTM and consequently study the role of these proteins in function of their location.; La membrane des myocytes ventriculaires cardiaques est caractérisée par la présence de nombreuses invaginations appelées tubules transverses (TT), essentielles pour la réalisation du couplage excitation-contraction. La membrane des TT (TTM) contient de nombreux récepteurs, canaux ou enzymes, tels que les récepteurs β-adrénergiques (β-ARs), les adénylyl cyclases ou les canaux Ca2+ voltage-dépendants de type L (LTCC). Ces protéines membranaires sont également présentes dans la membrane de surface (OSM), bien que leurs densités soient différentes. La pharmacologie classique permet d'explorer la fonction d'une protéine membranaire sur l’ensemble de la cellule, mais ne permet pas de distinguer les protéines présentes à l’OSM vs TTM. Pour ce faire, nous avons développé une technologie basée sur l'exclusion stérique afin d’explorer la fonction différentielle des β-Ars et des LTCC en fonction de s’ils sont situés dans l’OSM ou dans la TTM. Des ligands spécifiques de β-Ars et LTCC ont été greffés à la surface sur des nanoparticules et PEGylé à l’aide d’une liaison covalente avec une chaîne de polyéthylène-glycol (PEG) de 5000 Da. L'objectif est d'augmenter la taille des ligands β-AR et LTCC et d'empêcher leur accès dans le réseau TT. Grâce à cette nouvelle stratégie, nous sommes capables d'activer ou d'inhiber sélectivement les β-Ars et LTCC présents à l'OSM sans agir sur leurs homologues à la TTM et par conséquent d'étudier le rôle de ces protéines en fonction de leur localisation.

Cardiac automaticity is generated in the sino-atrial node by specialized cardiomyocytes called "pacemaker" cells. Spontaneous activity is permanently controlled by the autonomic nervous system, which regulates its frequency. Sympathetic regulation is mediated by beta1/2 receptors and cholinergic regulation by acetylcholine (ACh)-dependent activation of M2 muscarinic receptors. M2 receptors are coupled to a Gi protein. When muscarinic receptors are activated, the beta gamma subunit directly activates GIRK1/4 channels thereby opening K+ IKACh current, whereas the alpha i subunit inhibits adenylate cyclase (AC) activity. Inhibition of AC decreases intracellular cAMP production affecting directly (via the cyclic nucleotide binding domain, CNBD, of HCN channels) or indirectly via protein Kinase A (PKA) the activity of voltage-gated ion channels involved in cardiac pacing, mainly HCN4 (underlying the If current) and L-type Ca2+ channels (ICa,L). Two isoforms of L-type Ca2+ channels are expressed in pacemaker cells of the sinus node: Cav1.2 and Cav1.3. However, only Cav1.3 channels are open at low diastolic voltages, thus providing an inward current during diastolic depolarization. L-type Cav1.3 channels are therefore a potentially important effector of cholinergic regulation of cardiac pacemaker activity.I have employed the patch-clamp technique, isolated Langendorff-perfused hearts and in vivo telemetric recordings of electrocardiogram to study the role of Cav1.3 and IKACh in cholinergic regulation of pacemaker activity. I found that concomitant ablation of ICaL carried by Cav1.3 and IKACh carried by GIRK1/4 prevents cholinergic regulation after beta-adrenergic stimulation. These data show strong functional interaction between Cav1.3 and GIRK1/4 in cholinergic regulation of pacemaking. In addition, we show that Cav1.3 and Cav1.2 are both sensitive to acetylcholine, however Cav1.3 display sensitivity to low doses while Cav1.2 to high doses.In conclusion, L-type Cav1.3 channels play an important role in cholinergic regulation of pacemaking together with GIRK1/4 underlying IKACh., L’automatisme cardiaque est généré dans le nœud sino-atrial par des cardiomyocytes spécialisés appelés cellules « pacemaker ». Ces cellules sont douées d’une activité électrique spontanée qui est sous le contrôle de la régulation par le système nerveux autonome qui en régule sa fréquence. La régulation sympathique est médiée par les récepteurs beta1/2 et la régulation cholinergique par l'activation dépendante de l'acétylcholine (ACh) des récepteurs muscariniques M2. Les récepteurs M2 sont couplés à une protéine Gi. Une fois les récepteurs muscariniques activés, la sous-unité beta gamma active directement les canaux GIRK1/4 augmentant ainsi le courant K+ IKACh, tandis que la sous-unité alpha i inhibe l'activité de l'adénylate cyclase (AC). L'inhibition de l'AC diminue la production d'AMPc intracellulaire affectant directement (via le domaine de liaison des nucléotides cycliques, CNBD, des canaux HCN) ou indirectement via la protéine Kinase A (PKA) l'activité des canaux ioniques voltage-dépendants impliqués dans le pacemaking cardiaque, principalement HCN4 (responsable du courant If ) et les canaux Ca2+ de type L (ICa,L). Deux isoformes de canaux Ca2+ de type L sont exprimées dans les cellules du pacemaker du nœud sinusal : Cav1.3 et Cav1.2. Toutefois, seuls les canaux Cav1.3 sont ouverts à de potentiels de membrane diastoliques, fournissant ainsi un courant entrant pendant la dépolarisation diastolique. Les canaux de type L Cav1.3 sont donc un effecteur potentiellement important de la régulation cholinergique de l'activité du pacemaker cardiaque, mais leur rôle n’a pas été étudié directement.Les expérimentations, en patch-clamp et cœur isolé-perfusé, mais également par l’enregistrement d’électrocardiogramme in vivo par télémétrie a permis de mettre en évidence deux points importants. L’ablation concomitante de ICaL porté par Cav1.3 et de IKACh porté par GIRK1/4 empêche la régulation cholinergique de l’activité pacemaker après une stimulation beta-adrénergique. Ceci montre une interaction fonctionnelle entre Cav1.3 et GIRK1/4 dans la régulation cholinergique.Il est également apparu que les courants de type L portés par Cav1.3 et Cav1.2 sont tous les deux sensibles à l’Acétylcholine. Toutefois, Cav1.3 présente une sensibilité à de faibles doses tandis que Cav1.2 à de forte dose. Ce résultat suggère que les canaux Cav1.2 pourraient participer à la régulation cholinergique à tonus vagal élevé.En conclusion, les canaux Cav1.3 et GIRK1/4 jouent un rôle important dans la régulation cholinergique de l’automatisme cardiaque chez la souris.

Les canaux calciques CaV1.2 activés par la dépolarisation membranaire jouent un rôle majeur dans le couplage excitation-contraction en promouvant l’entrée de Ca2+ dans les cellules cardiaques. Une perturbation de la fonction de ces canaux a été associé avec l’apparition de maladies cardiovasculaires telles que les arythmies cardiaques. Ces canaux forment un complexe hétéromérique constitués de la sous-unité principale CaVa1C formant le pore et de l’association d’au moins deux sous-unités auxiliaires. L’interaction avec la sous- unité cytoplasmique CaVb est importante pour l’adressage membranaire du canal tandis que la liaison de la sous-unité extracellulaire CaVa2d1 augmente les densités de courant d’un facteur 5 à 10 et permet l’activation du canal à des potentiels physiologiques. L’ensemble de ces sous- unités est donc nécessaire pour reproduire les propriétés biophysiques du canal natif. Tandis que la modulation fonctionnelle du canal CaVa1 par CaVb a été bien caractérisée, les mécanismes de régulation par la protéine CaVa2d1 ne sont pas encore élucidés. La caractérisation structurale de CaVa2d1 a été entreprise mais l’extrême complexité d’organisation de cette protéine en raison de ces multiples modifications post-traductionnelles (seize sites de glycosylation, ponts disulfures intra- et intermoléculaires, délétion du C- terminal pour son ancrage membranaire) a limitée cette étude. Notre stratégie alternative a alors été de purifier séparément les domaines structuraux de CaVa2d1 dans le but d’étudier leurs structures 3D. La structure du domaine Cache2 obtenue à basse résolution par diffusion des rayons X aux petits angles a révélé un repliement majoritairement en feuillets-b et la présence de nombreuses régions flexibles. Cependant, la publication en 2016 de la structure du canal calcique homologue CaV1.1 purifié à partir de muscle squelettique à une résolution de 3.6 Å par cryo-microscopie électronique a fourni l’essentiel des détails structuraux sur la sous- unité CaVa2d1. Cette structure a montré que le domaine VWA de CaVa2d1 se situe à une distance atomique de CaVa1S. Dans le but de mieux comprendre comment cette association contribue à la fonction de CaVa2d1 dans les cellules cardiaques, nous a amené à étudier les déterminants moléculaires responsables de l’interaction entre CaVa1C (CaV1.2) et CaVa2d1. Nos études combinées de modélisation par homologie, électrophysiologie et co-immunoprécipitation ont montré, dans un premier temps, que les deux résidus aspartates (Asp- 180 et Asp-181) situés dans la boucle S1S2 du domaine I (IS1S2) de CaV1.2 étaient critiques pour la modulation du canal par CaVa2d1. Plus particulièrement, l’Asp-181 est crucial pour l’interaction physique avec CaVa2d1 tandis que l’Asp-180 joue un rôle important dans le déplacement du potentiel d’activation du canal vers des valeurs hyperpolarisantes. Nous avons, dans un second temps, montré que l’Asp-259 dans CaVa2d1 était nécessaire pour capter un ion Ca2+ et permettre le repliement optimal du site MIDAS (metal-ion-dependant adhesion site) sur le domaine VWA (Won Willebrand factor A) de la protéine. Dans cette conformation, la Ser-261 et la Ser-263 de CaVa2d1 peuvent alternativement établir une liaison électrostatique avec l’Asp-181 de CaV1.2 ce qui stabilise l’interface et permet la régulation du canal par CaVa2d1. Nos travaux ont révélé de nouveaux mécanismes moléculaires, par lesquels, la liaison de CaVa2d1 avec la boucle IS1S2 de CaV1.2 induit un réarrangement structural local à travers de multiples interactions fonctionnant en synergie ce qui facilite l’ouverture du canal, et indirectement l’augmentation des densités de courants à travers le pore., Voltage-gated calcium channels play a key role in the excitation-contraction coupling by promoting Ca2+ influx through CaV1.2 into the cardiac cells. A dysregulation in the function of this channel has been involved in cardiac arrhythmias. L-type voltage-gated calcium channels are formed by the main pore-forming CaVa1C subunit assembled with two auxiliary subunits. The cytoplasmic CaVb subunit strongly interacts with the intracellular loop between repeat I and II of CaVa1 and promote cell surface expression of the channel whereas the interaction with the extracellular CaVa2d1 subunit increases by 5- to 10-fold the peak current density and promote channel activation within a physiological range. The full set of subunits is required to reproduce the biophysical properties of the native channel. For L-type voltage-gated calcium channel, the mechanism whereby CaVa2d1 exerts its function is still not fully understood. A structural determination of CaVa2d1 conducted using a typical biochemical approach was initiated but many post-translational modifications (16 Nglycosylation sites, intra- and extra-disulfide bonds, glycosylphosphatidylinositol anchor) prevented the purification of the entire protein. We then devised a “divide-and-conquer” strategy that lead to the purification of the Cache2 domain of CaVa2d1. The low-resolution structure of the Cache2 domain by small angle X-rays scattering revealed a predominantly fold in b-sheet with multiple flexible regions. The publication in 2016 of the high-resolution structure of the homologous CaV1.1 channel complex at 3.6 Å of resolution solved by cryoelectronic microscopy provided crucial molecular details regarding the organization of the structural domains of CaVa2d1 but also its spatial orientation within the CaV1.1 complex. In particular, the VWA domain of CaVa2d1 appears to come within atomic distance of many extracellular loops of CaVa1 subunit. To establish whether physical interaction between the two proteins is essential to mediate the functional regulation of CaV1.2 current by CaVa2d1, we investigated the molecular determinants responsible for the interaction between CaVa1C (CaV1.2) and CaVa2d1. Using homology modeling, electrophysiology and coimmunoprecipitation experiments we demonstrated that residues in the first extracellular loop in repeat I of CaV1.2 play a dominant role in the interaction and modulation by CaVa2d1. In particular, CaV1.2 Asp-181 anchors the physical interaction with CaVa2d1 while CaV1.2 Asp- 180 mediates the channel activation in a physiological range of membrane potential. In addition, we have explored the contribution of metal ion-dependant adhesion site (MIDAS) residues within the Won Willebrand Factor-A (VWA) domain of CaVa2d1 in mediating channel modulation. Our results show that mutating side chain at position Asp-259 and Ser- 261 eliminate channel modulation although these residues are not predicted to be within atomic distance of CaVa1. Hence, we propose that CaVa2d1 Asp-259 captures the Ca2+ ion which in turn folds the MIDAS motif within the VWA domain of CaVa2d1. The folding of the MIDAS around the Ca2+ locks CaVa2d1 Ser-261 and Ser-263 within atomic distance of CaVa1C Asp-181. This network of molecular interactions stabilizes the protein-protein interface demonstrating that high affinity binding of CaVa2d1 induce a local structural rearrangement that facilitate CaV1.2 channel opening and consequently increase Ca2+ influx through the pore. Al together, the result emerging from these studies provide novel mechanism insight regarding the function of the cardiac CaV1.2 channel.

Heart automaticity is generated by a basic pacemaker mechanism not fully understood and still controversial. Pacemaker activity is initiated by specialized cardiomyocytes in the Sino-atrial node (SAN). The spontaneous phase of diastolic depolarization (DDP) characterizes SAN cells (SANc). This phase drives the membrane potential of SANc from the end of the repolarization to the threshold of the next action potential (AP). This spontaneous activity involves several ion channels on the plasma membrane and the intracellular dynamic of calcium. In terms of calcium channels, atrial and ventricular cardiomyocytes express mostly Cav1.2 whereas SANc express two additional isoforms. Specifically, in SANc are expressed Cav1.3 LTCC (L type Calcium channels) and the Cav3.1 TTCC (T type Calcium channels), which are activated during the DD. Genetically modified mice inactivated for Cav1.3, Cav3.1 and Cav1.3/Cav3.1 we generated and used as a models of study of human SAN dysfunctions. In particular, we highlighted the impairment of the pacemaker activity in these mice by optical mapping of the intact SAN, and by patch clamping and calcium imaging of isolated SANc. Coupling this approaches with pharmacological tools allowed us to evaluating the contribution of the various elements constituting to the pacemaker mechanism. This thematic of research presents major issues in terms of public health. Indeed, we need a better understanding of the pacemaker mechanism to develop pharmacological strategies against SAN dysfunction.; L’automatisme cardiaque est généré par un mécanisme fondamental partiellement compris et controversé, initié par des cardiomyocytes spécialisés dans le nœud sino-atrial (NSA). Ces cellules pacemaker (cNSA) présentent une phase spontanée de dépolarisation diastolique (DD), qui mène le potentiel de membrane de la fin de la repolarisation du potentiel d’action (PA) au seuil de déclenchement du PA suivant. Cette activité spontanée implique plusieurs canaux ioniques à la surface de la membrane plasmique et la dynamique calcique intracellulaire. Les cardiomyocytes contractiles du myocarde expriment majoritairement le canal calcique Cav1.2 tandis que les cNSA en expriment d’autres isoformes. Ce sont les canaux calciques de type L (LTCC) Cav1.3 et de type T (TTCC) Cav3.1, qui sont impliqués dans la DD. Les souris génétiquement modifiées pour Cav1.3 et/ou Cav3.1 ont des caractéristiques physiopathologiques et sont utilisées comme modèle d’étude des dysfonctions sinusales de l’homme. La cartographie optique du NSA isolé a permis de révéler une activité électrophysiologique intrinsèque altérée par les mutations. L’expérimentation en patch clamp et en imagerie calcique des cNSA isolées montrent que les mutations altèrent la mécanistique cellulaire du pacemaker. Le couplage de ces approches à l’utilisation d’outils pharmacologiques spécifiques a permis d’évaluer la contribution des différents éléments à cette mécanistique cellulaire et de préciser les controverses sur les fondements de l’automatisme cardiaque. Cette thématique de recherche présente des enjeux majeurs dans le domaine de la santé puisque les perspectives thérapeutiques et les stratégies pharmacologiques pour traiter les dysfonctions sinusales nécessitent une connaissance intégrale du mécanisme.

Heart automaticity is generated by a basic pacemaker mechanism not fully understood and still controversial. Pacemaker activity is initiated by specialized cardiomyocytes in the Sino-atrial node (SAN). The spontaneous phase of diastolic depolarization (DDP) characterizes SAN cells (SANc). This phase drives the membrane potential of SANc from the end of the repolarization to the threshold of the next action potential (AP). This spontaneous activity involves several ion channels on the plasma membrane and the intracellular dynamic of calcium. In terms of calcium channels, atrial and ventricular cardiomyocytes express mostly Cav1.2 whereas SANc express two additional isoforms. Specifically, in SANc are expressed Cav1.3 LTCC (L type Calcium channels) and the Cav3.1 TTCC (T type Calcium channels), which are activated during the DD. Genetically modified mice inactivated for Cav1.3, Cav3.1 and Cav1.3/Cav3.1 we generated and used as a models of study of human SAN dysfunctions. In particular, we highlighted the impairment of the pacemaker activity in these mice by optical mapping of the intact SAN, and by patch clamping and calcium imaging of isolated SANc. Coupling this approaches with pharmacological tools allowed us to evaluating the contribution of the various elements constituting to the pacemaker mechanism. This thematic of research presents major issues in terms of public health. Indeed, we need a better understanding of the pacemaker mechanism to develop pharmacological strategies against SAN dysfunction.; L’automatisme cardiaque est généré par un mécanisme fondamental partiellement compris et controversé, initié par des cardiomyocytes spécialisés dans le nœud sino-atrial (NSA). Ces cellules pacemaker (cNSA) présentent une phase spontanée de dépolarisation diastolique (DD), qui mène le potentiel de membrane de la fin de la repolarisation du potentiel d’action (PA) au seuil de déclenchement du PA suivant. Cette activité spontanée implique plusieurs canaux ioniques à la surface de la membrane plasmique et la dynamique calcique intracellulaire. Les cardiomyocytes contractiles du myocarde expriment majoritairement le canal calcique Cav1.2 tandis que les cNSA en expriment d’autres isoformes. Ce sont les canaux calciques de type L (LTCC) Cav1.3 et de type T (TTCC) Cav3.1, qui sont impliqués dans la DD. Les souris génétiquement modifiées pour Cav1.3 et/ou Cav3.1 ont des caractéristiques physiopathologiques et sont utilisées comme modèle d’étude des dysfonctions sinusales de l’homme. La cartographie optique du NSA isolé a permis de révéler une activité électrophysiologique intrinsèque altérée par les mutations. L’expérimentation en patch clamp et en imagerie calcique des cNSA isolées montrent que les mutations altèrent la mécanistique cellulaire du pacemaker. Le couplage de ces approches à l’utilisation d’outils pharmacologiques spécifiques a permis d’évaluer la contribution des différents éléments à cette mécanistique cellulaire et de préciser les controverses sur les fondements de l’automatisme cardiaque. Cette thématique de recherche présente des enjeux majeurs dans le domaine de la santé puisque les perspectives thérapeutiques et les stratégies pharmacologiques pour traiter les dysfonctions sinusales nécessitent une connaissance intégrale du mécanisme.

The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, and therefore is an interesting drug target to develop innovative analgesic compounds for improved chronic pain management. Despite recent advances in the identification of small organic molecules targeting the Cav3.x/T-type calcium channel family, to date specific Cav3.2 inhibitors remains to be identified. Spider venoms proved to contain a large diversity of neurotoxins including gating modifiers of calcium channels. Using recombinant Cav3.2 channels, we performed a screening of a Tarantula venom library and identified a new 28 amino acid peptide (Psp3Tx1). The synthetic form of the peptide was used to determine its selectivity profile over a panel of closely related members of the voltage gated calcium (Cav) and sodium (Nav) channels. The peptide proved to be selective for the Nav1.7 channel (largely validated target in the context of pain pathologies) with an additional effect on Cav3.2 at more elevated doses. In vivo in mice, the peptide demonstrated to be an efficient analgesic molecule with anti hyperalgesic and antiallodynic properties in the context of neuropathic pain. This peptide also possess analgesic properties in a context of spontaneous pain induced by a Nav1.7 agonist. The results presented in this thesis are encouraging for the setup of a project taking Psp3Tx1 into clinical tests.; Les canaux calciques de type T Cav3.2 sont des régulateurs clés des fonctions sensorielles, et de fait sont également des cibles intéressantes pour le développement de nouveaux composés analgésiques pour le traitement et le management de la douleur. Malgré de récentes avancées dans l’identification de petites molécules organiques ciblant la famille des canaux Cav3.x/Type T, il reste encore à identifier un inhibiteur spécifique de Cav3.2. Le venin d’araignées contient une large diversité de neurotoxines incluant des modificateurs de gating des canaux calciques. En utilisant des canaux calciques recombinants, nous avons procédé à un criblage d’une bibliothèque de venins et avons identifié un nouveau peptide de 28 acides aminés (Psp3Tx1). La forme synthétique de ce peptide a été utilisé pour déterminer son profil de selectivité sur un panel de membres proches de la famille des canaux calciques (Cav) et sodiques (Nav) dépendants du voltage. Le peptide est sélectif pour le canal Nav1.7 (une cible largement validée dans le contexte des pathologies de la douleur) avec un effet complémentaire sur Cav3.2 à de fortes doses. In vivo chez la souris, le peptide possède des propriétés analgésiques avec des effets anti-hyperalgésiques et anti-allodyniques dans un contexte de douleur neuropathique. Ce peptide possède également un pouvoir analgésique dans un contexte de douleur spontanée induit par un agoniste des canaux Nav1.7. Les résultats présentés dans cette thèse sont encourageants pour la mise en place d’un projet amenant Psp3Tx1 au niveau clinique.

The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, and therefore is an interesting drug target to develop innovative analgesic compounds for improved chronic pain management. Despite recent advances in the identification of small organic molecules targeting the Cav3.x/T-type calcium channel family, to date specific Cav3.2 inhibitors remains to be identified. Spider venoms proved to contain a large diversity of neurotoxins including gating modifiers of calcium channels. Using recombinant Cav3.2 channels, we performed a screening of a Tarantula venom library and identified a new 28 amino acid peptide (Psp3Tx1). The synthetic form of the peptide was used to determine its selectivity profile over a panel of closely related members of the voltage gated calcium (Cav) and sodium (Nav) channels. The peptide proved to be selective for the Nav1.7 channel (largely validated target in the context of pain pathologies) with an additional effect on Cav3.2 at more elevated doses. In vivo in mice, the peptide demonstrated to be an efficient analgesic molecule with anti hyperalgesic and antiallodynic properties in the context of neuropathic pain. This peptide also possess analgesic properties in a context of spontaneous pain induced by a Nav1.7 agonist. The results presented in this thesis are encouraging for the setup of a project taking Psp3Tx1 into clinical tests.; Les canaux calciques de type T Cav3.2 sont des régulateurs clés des fonctions sensorielles, et de fait sont également des cibles intéressantes pour le développement de nouveaux composés analgésiques pour le traitement et le management de la douleur. Malgré de récentes avancées dans l’identification de petites molécules organiques ciblant la famille des canaux Cav3.x/Type T, il reste encore à identifier un inhibiteur spécifique de Cav3.2. Le venin d’araignées contient une large diversité de neurotoxines incluant des modificateurs de gating des canaux calciques. En utilisant des canaux calciques recombinants, nous avons procédé à un criblage d’une bibliothèque de venins et avons identifié un nouveau peptide de 28 acides aminés (Psp3Tx1). La forme synthétique de ce peptide a été utilisé pour déterminer son profil de selectivité sur un panel de membres proches de la famille des canaux calciques (Cav) et sodiques (Nav) dépendants du voltage. Le peptide est sélectif pour le canal Nav1.7 (une cible largement validée dans le contexte des pathologies de la douleur) avec un effet complémentaire sur Cav3.2 à de fortes doses. In vivo chez la souris, le peptide possède des propriétés analgésiques avec des effets anti-hyperalgésiques et anti-allodyniques dans un contexte de douleur neuropathique. Ce peptide possède également un pouvoir analgésique dans un contexte de douleur spontanée induit par un agoniste des canaux Nav1.7. Les résultats présentés dans cette thèse sont encourageants pour la mise en place d’un projet amenant Psp3Tx1 au niveau clinique.

Le canal calcique de type-L CaV1.2 participe au couplage excitation-contraction des cardiomyocytes. Cav1.2 est composé d’une sous-unité principale CaVα1, associée aux sous-unités auxiliaires CaVβ et CaVα2δ1. Lorsque présente à la membrane, c’est CaVα2δ1 qui est responsable de moduler la densité du courant calcique. Elle ne possède qu’un seul segment transmembranaire présent du côté C-terminal, au niveau de la protéine δ, ce qui en fait une protéine transmembranaire de type I. Certaines protéines qui appartiennent à cette famille doivent être clivées au niveau du site dit « omega », une modification post-traductionnelle nécessaire à leur fonction. Une fois clivées, ces protéines sont retenues à la membrane plasmique par une ancre glycosyl-phosphatidyl-inositol (GPI). Nos études en microscopie confocale montrent que la protéine sauvage est sensible à l’action de la phospholipase C qui clive de manière spécifique les groupements phosphoinositol, ce qui est compatible avec la présence d’une ancre GPI fonctionnelle. De plus, la mutation des résidus formant le site « omega » en isoleucine au niveau des sites G1060 et G1061 prévient l’adressage membranaire de CaVα2δ1 estimé par cytométrie en flux et imagerie confocale, et réduit la modulation des courants calciques mesurés par la méthode du « patch-clamp ». Les mutants G1060I et G1061I sont aussi associés à un changement dans le patron de migration de la partie C-terminale, suggérant un processus protéolytique défecteueux. Les mutations simples des glycines en alanines préservent les propriétés de la protéine mais le double mutant G1060A/G1061A réduit significativement l’expression de CaVα2δ1 à la surface de la cellule et sa modulation sur le canal CaV1.2. Ces données suggèrent fortement que le clivage requiert spécifiquement un résidu Glycine en position 1060 ou 1061 pour produire le clivage protéolytique dominant chez CaVα2δ1, et que cet ancrage GPI est essentiel à la fonction du canal., Voltage-gated calcium channels CaV1.2 play an essential role in the regulation of cardiac excitability. Functional channels are formed by the CaVα1 subunit and the intracellular CaVβ and the extracellular CaVα2δ1 subunits. CaVα2δ1 are type I transmembrane proteins that undergo a posttranslational modification producing their association at the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. The molecular determinants required for the proteolytic cleavage of the recombinant CaVα2δ1 protein were studied using biochemical, immunocytochemical, fluorescence, and electrophysiological methods. Enzymatic treatment with a phospholipase C specific for the cleavage of phosphatidyl inositol lipids abolished the colocalisation of CaVα2δ1 with a plasma membrane marker as shown using live-cell confocal imaging. Single point mutations G1060I or G1061I in the predicted transmembrane CaVδ domain was shown to significantly reduce the cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging, and to prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2 currents. The isoleucine mutations were also associated with a change in the migration pattern of the C-terminal fragments suggesting that proteolytic processing was altered. Single glycine to alanine mutations preserved the protein properties but the double mutant G1060A/G1061A significantly impaired cell surface expression of CaVα2δ1 and its functional regulation of CaV1.2. Altogether our data support a model where one Glycine residue at position 1060 or 1061 is required to produce the dominant proteolytic cleavage of CaVα2δ1 and further suggest that the GPI-anchored form of CaVα2δ1 is essential for channel function.

The voltage gated calcium channels are involved in many cellular processes such as muscle contraction, neurotransmitter release and regulation of gene expression. These channels consist of the pore-forming subunit α1 usually associated with different regulatory subunits: α2δ, β and γ. The auxiliary subunit β (Cavβ) plays a key role in regulating membrane trafficking of the channel and its biophysical properties. Recent studies describe this subunit as a multifunctional protein that can also perform calcium channel-independent functions such as gene regulation. Four different isoforms of Cavβ are encoded by 4 different genes and are characterized by structural similarities but different tissue distribution. Cavβ4 isoform is mainly expressed in the brain and cerebellum, thus, playing an important role in the regulation of neuronal calcium currents. The importance of Cavβ4 neuronal functions has been highlighted by its R482X mutation that was associated to a form of human epilepsy.The aim of my thesis was to study the role of Cavβ4 in the control of cell division and its involvement in the Wnt signaling pathway. I also studied the influence of the R482X mutation on this new function of Cavβ4.To this end, CHO cells stably expressing Cavβ4, or its epileptic mutant (Cavβ1-481), were generated and the subcellular localization of the two proteins and their implication in the proliferation and cell cycle progression were studied. In these cells, Cavβ4 undergoes nuclear translocation and is found preferentially in the nucleoli. However, the deletion of 38 amino acids in the C-terminus domain of Cavβ4, corresponding to the R482X mutation, prevents its nucleolar translocation. In addition, the expression of Cavβ4 significantly reduces the proliferative rate of the cells. This reduction seems to be linked to Cavβ4 nuclear localization because the epileptic mutant is unable to slow down cell proliferation. On the other hand, the expression of each of these proteins is able to deregulate cell cycle progression and to alter the expression of many genes linked to the cycle.Since the Wnt/β-catenin pathway is known as one of the most important pathways controlling cell proliferation, I studied the effect of Cavβ4 expression on this signaling pathway. Indeed, Cavβ4, but not Cavβ1-481, substantially reduces the transcription of β-catenin-dependant genes and therefore slows down cell proliferation. This inhibition is due to a direct interaction between Cavβ4 and TCF4 that prevents the interaction of TCF4 with β-catenin, and thereafter negatively regulates the transcription of targeted genes.These findings suggest that Cavβ4 can play a role in the control of proliferation during development, particularly in neuronal cells.; Les canaux calciques dépendants du voltage sont impliqués dans de nombreux processus cellulaires tels que la contraction musculaire, la libération des neurotransmetteurs et la régulation de l’expression génique. Ces canaux sont constitués d’une sous-unité canalaire α1, qui permet l’entrée des ions calciques dans le milieu intracellulaire, généralement associée à différentes sous-unités auxiliaires (α2δ, γ et β) qui régulent les fonctions du canal. La sous-unité auxiliaire β (Cavβ) joue un rôle capital dans l’adressage membranaire du canal et dans la régulation de ses propriétés biophysiques. Des études récentes décrivent cette sous-unité comme une protéine multifonctionnelle capable d’accomplir des fonctions indépendantes du canal. Quatre différentes isoformes de Cavβ sont codées par 4 gènes différents et caractérisées par des similarités structurales mais une distribution tissulaire différente. L’isoforme Cavβ4 est principalement exprimée dans le cerveau et le cervelet jouant ainsi un rôle important dans la régulation des courants calciques neuronaux. L’importance des fonctions neuronales de Cavβ4 a été soulignée par le fait que la mutation R482X de Cavβ4 a été associée à une forme d’épilepsie humaine.Mon travail de thèse a porté sur l’étude du rôle de Cavβ4 dans le contrôle de la division cellulaire et son implication dans la voie de signalisation Wnt. J’ai également étudié l’influence de la mutation R482X sur cette nouvelle fonction de Cavβ4.Dans ce but, des cellules CHO exprimant de manière stable Cavβ4 ou son mutant épileptique (Cavβ1-481) ont été générées et la localisation subcellulaire des deux protéines ainsi que leur implication dans la prolifération et la progression du cycle cellulaire ont été étudiées. Dans ces cellules, Cavβ4 subit une translocation nucléaire et se retrouve préférentiellement dans les nucléoles. Néanmoins, la délétion de 38 acides aminés de l’extrémité C-terminale de Cavβ4, correspondante à la mutation R482X, empêche sa translocation nucléolaire. L’expression de Cavβ4 réduit considérablement la capacité proliférative des cellules. Cette réduction semble être dépendante de la localisation nucléaire, voir nucléolaire de Cavβ4, parce que le mutant Cavβ1-481 n’induit aucune inhibition de la prolifération. D’un autre côté, l’expression de chacune de ces protéines entraine une modification du cycle cellulaire et l’altération de l’expression de certains gènes liés au cycle.Étant donné que la voie de signalisation Wnt/β-caténine est connue comme l'une des voies les plus importantes contrôlant la prolifération cellulaire, j’ai étudié l'effet de l’expression de Cavβ4 sur cette voie. J’ai ainsi pu montrer que Cavβ4, mais pas Cavβ1-481, réduit considérablement la transcription des gènes cibles de la β-caténine et ralenti la prolifération cellulaire. Cette inhibition est due à une interaction directe entre Cavβ4 et TCF4 qui empêche l’interaction de TCF4 avec la β-caténine et prévient la transcription des gènes cibles.L’ensemble de ces résultats suggèrent que Cavβ4 peut jouer un rôle dans le contrôle de la prolifération au cours du développement, en particulier dans les cellules neuronales.

Les canaux calciques de type L CaV1.2 sont principalement responsables de l’entrée des ions calcium pendant la phase plateau du potentiel d’action des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomérique qui est composé de la sous-unité principale CaVα1 et des sous-unités auxiliaires CaVβ et CaVα2δ1. CaVβ joue un rôle déterminant dans l’adressage membranaire de la sous-unité CaVα1. CaVα2δ1 stabilise l’état ouvert du canal mais le mécanisme moléculaire responsable de cette modulation n’a pas été encore identifié. Nous avons récemment montré que cette modulation requiert une expression membranaire significative de CaVα2δ1 (Bourdin et al. 2015). CaVα2δ1 est une glycoprotéine qui possède 16 sites potentiels de glycosylation de type N. Nous avons donc évalué le rôle de la glycosylation de type-N dans l’adressage membranaire et la stabilité de CaVα2δ1. Nous avons d’abord confirmé que la protéine CaVα2δ1 recombinante, telle la protéine endogène, est significativement glycosylée puisque le traitement à la PNGase F se traduit par une diminution de 50 kDa de sa masse moléculaire, ce qui est compatible avec la présence de 16 sites Asn. Il s’est avéré par ailleurs que la mutation simultanée de 6/16 sites (6xNQ) est suffisante pour 1) réduire significativement la densité de surface de! CaVα2δ1 telle que mesurée par cytométrie en flux et par imagerie confocale 2) accélérer les cinétiques de dégradation telle qu’estimée après arrêt de la synthèse protéique et 3) diminuer la modulation fonctionnelle des courants générés par CaV1.2 telle qu’évaluée par la méthode du « patch-clamp ». Les effets les plus importants ont toutefois été obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces résultats montrent que Asn663 et à un moindre degré Asn348, Asn468 et Asn812 contribuent à la biogenèse et la stabilité de CaVα2δ1 et confirment que la glycosylation de type N de CaVα2δ1 est nécessaire à la fonction du canal calcique cardiaque de type L., L-type CaV1.2 channels play a key role in the excitation-contraction coupling in the heart. They are formed of a pore-forming CaVα1 subunit in complex with the intracellular CaVβ and the disulfur-linked CaVα2δ accessory subunits. CaVα2δ significantly increases peak current densities of CaV1.2. The mechanism underlying this effect is still under study but requires that CaVα2δ be trafficked at the cell surface. CaVα2δ contains 16 putative N-glycosylation sites. A study was carried out to identify the role of N-glycosylation in the trafficking and protein stability of the subunit CaVα2δ. Herein we show that enzymatic removal of N-glycans produced a 50 kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. Simultaneous mutation of the 16 Asn sites was required to fully account for this change in protein mobility. Nonetheless, the mutation of only 6/16 sites was sufficient to 1) significantly reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) accelerate the degradation kinetics estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational 6 Asn mutant functionally rescued CaVα2δ1. Single mutation N663Q and double mutations N348Q/ N468Q, N348Q/ N812Q, N468Q/N812Q decreased protein stability/synthesis and abolished steady-state cell surface density as well as upregulation of L-type currents. These results demonstrate that Asn663, and to a lesser extent Asn348, Asn468, and Asn812 contribute to the stability of CaVα2δ1 function and furthermore that N- glycosylation of CaVα2δ1 is essential to produce functional L-type Ca2+ channels.

Les canaux Ca2+ activés par le voltage (CaV) sont des protéines membranaires qui génèrent des courants Ca2+ dans les cellules excitables suite à une dépolarisation membranaire. Ces complexes oligomériques sont classifiés selon les propriétés structurelles de la sous-unité principale qui forme le pore du canal, soit la sous-unité CaVα1. La sous-unité auxiliaire CaVβ module l’expression membranaire et la dépendance au voltage du « gating » de la sous-unité CaVα1 des canaux HVA (« high-voltage-activated ») CaV1 et CaV2. La sous-unité CaVβ est formée par un domaine SH3 (« Src homology-3 ») connecté à un domaine GK (« guanylate kinase-like ») par le biais d’un domaine variable HOOK. Dans le but d’identifier les résidus dans la CaVβ3 qui sont responsables de la densité membranaire du CaV2.3, nous avons produit des mutants de la sous-unité auxiliaire le long de ses domaines fonctionnels. Cela dit, la délétion complète du domaine SH3 ainsi que la délétion du domaine HOOK n’ont pas modifié la densité membranaire de CaV2.3 ni ses propriétés d’activation. Cependant, la délétion de cinq résidus dans le domaine GK interrompt l’expression membranaire et l’expression fonctionnelle de CaV2.3. La mutation de résidus identifiés précédemment comme soutenant une affinité de liaison de l’ordre du nanomolaire dans le domaine GK de CaVβ n’a pas modifié de manière significative l’adressage membranaire de CaV2.3. Toutefois, les mutations de quatre résidus leucine dans les régions α3, α6, β10 et α9 du domaine GK ont grandement réduit l’adressage membranaire du canal CaV2.3. Nos résultats confirment que le domaine GK contient les déterminants moléculaires responsables de la fonction chaperone de CaVβ. Cela dit, l’adressage membranaire induit par CaVβ semble être déterminé par des éléments structuraux qui ne sont pas strictement dépendants d’une liaison à haute affinité de CaVβ sur CaVα1., Voltage-activated Ca2+ channels (CaV) are membrane proteins that play a key role in promoting Ca2+ influx in response to membrane depolarization in excitable cells. They form oligomeric complexes that are classified according to the structural properties of the pore-forming CaVα1 subunit. Auxiliary CaVβ subunits modulate cell-surface expression and voltage-dependent gating of high-voltage-activated (HVA) CaV1 and CaV2 α1 subunits. CaVβ subunits are formed by a Src homology-3 (SH3) domain and a guanylate kinase-like (GK) domain connected through a variable HOOK-domain. In order to identify the residues responsible for the CaVβ3-induced membrane density of CaV2.3, we produced mutants along CaVβ3’s fonctionnal domains. Complete deletion of the SH3 domain as well as deletion of the HOOK domain did not alter plasma membrane targeting of CaV2.3 nor its typical activation gating. In contrast, 5-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of CaV2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of CaVβ did not significantly alter cell surface density. Mutations of a quartet of leucine residues in the α3, α6, β10, and α9 regions of the GK domain, each expected to curtail protein-protein interaction, were found to significantly impair cell surface targeting of CaV2.3 channels. Altogether, our results confirm that the GK domain includes the molecular determinants carrying the chaperone function of CaVβ. However, CaVβ-induced cell surface targeting appears to be determined by structural elements that are not strictly dominated by high-affinity binding of CaVβ onto CaVα1.

La grossesse s’accompagne d’importantes modifications hormonales et hémodynamiques. Parmi celles-ci, le système rénine-angiotensine-aldostérone (SRAA) est activé très tôt durant la grossesse. De plus, cette augmentation du SRAA est accompagnée d’élévations du débit cardiaque et du volume plasmatique ainsi que des baisses paradoxales de la pression artérielle et de la résistance vasculaire périphérique. Ceci suggère que la grossesse induit un remaniement des réponses physiologiques normales au SRAA. Une résistance vasculaire à l’action des vasopresseurs est également observée durant la gestation. Ce phénomène serait causé par la modification de la fonction des canaux calciques et potassiques. De plus, il serait possiblement dû à la participation de la Na+/K+-ATPase, par son influence sur le potentiel membranaire des cellules des muscles lisses vasculaires (VSMC). La présence des récepteurs minéralocorticoïdes (MR) dans les VSMC laisse croire que l’aldostérone peut influencer le tonus vasculaire par des effets génomiques et non-génomiques. Compte tenu des connaissances actuelles, nous avons émis l’hypothèse que l’augmentation des taux sériques d’aldostérone durant la grossesse est responsable des changements hémodynamiques observés et que ces effets sont causés par l’activation des MR. Des rates gestantes ont été traitées avec du canrénoate de potassium (20 mg/kg•jr), un antagoniste des MR, durant la dernière semaine de gestation (sur 3). Sur des anneaux aortiques dénudés de leur endothélium, nous avons mesuré les réponses contractiles à la phényléphrine (PhE) et au KCl en présence d’un bloqueur des canaux calciques dépendants du voltage (VDCC), la nifédipine, et d’activateurs des canaux potassiques à large conductance (BKCa) et ceux dépendants de l’ATP (KATP), respectivement le NS-1619 et la cromakalim. Les réponses à la PhE et au KCl sont réduites à partir du 17e jour de gestation et le traitement au canrénoate augmente ces réponses dans tous les groupes. Les modulateurs de canaux ioniques atténuent les réponses à la PhE et au KCl. Cependant, le canrénoate modifie aussi les effets des modulateurs sur les aortes. Aucun effet ou une baisse des réponses est observable chez les rates non gestantes, tandis qu’une hausse de leur effet inhibiteur est notée chez les rates gestantes. Ces effets du canrénoate font croire que l’aldostérone participe à l’adaptation de la réactivité vasculaire durant la grossesse. Par ailleurs, le potentiel membranaire des VSMC pourrait être affecté dans la gestation. Pour vérifier cette hypothèse, nous avons évalué l’activité de la Na+/K+-ATPase, impliquée dans le contrôle du potentiel membranaire. Nos résultats démontrent que l’activité de la pompe est inhibée à partir du 19e jour de gestation. Cet effet est renversé par le canrénoate. Toutefois, comme le renversement de l’inhibition de la pompe est également présent chez les rates gestantes traitées avec du PST 2238, un antagoniste de l’ouabaïne sur la Na+/K+-ATPase, et que le canrénoate agit également comme agoniste partiel de la pompe, nous croyons que la diminution d’activité associée à la gestation est liée à une inhibition de la Na+/K+-ATPase par des stéroïdes cardiotoniques plutôt qu’à un effet des minéralocorticoïdes. L’augmention d’activité de la pompe liée au canrénoate s’accompagne d’une diminution de l’expression de la sous-unité α1, suggérant que la sous-unité α2 est responsable des variations de contractilité de l’aorte, puisque son expression n’est pas modifiée par le canrénoate. Les effets de la diminution de l’expression de la sous-unité α1, influencée par la signalisation du MR, restent à être déterminés. Néanmoins, nos résultats montrent que les modifications d’activité de la Na+/K+-ATPase influencent l’activité des canaux potassiques et que la pompe pourraient être un des éléments primordiaux dans le contrôle de la réactivité vasculaire durant la grossesse. Comme le canrénoate modifie la réactivité vasculaire, nous voulions déterminer ses impacts sur la pression artérielle. Des rates gestantes ont été traitées avec du canrénoate (20 ou 60 mg/kg•jr) et les paramètres hémodynamiques ont été évalués par radiotélémétrie. Aucune modification de la pression artérielle, du rythme cardiaque et de la pression pulsée ne sont mesurées chez les rates recevant le traitement. Toutefois, des augmentations de l’osmolalité, des taux sériques d’aldostérone et de corticostérone ainsi que de l’activité rénine plasmatique sont observées chez les animaux recevant 60 mg/kg•jr. Le canrénoate bloque donc le rétrocontrôle du SRAA. Par contre, les MR ne sont pas les principaux responsables du contrôle de la pression artérielle durant la grossesse. En conclusion, nous avons démontré que le traitement des rates au canrénoate influence la réactivité vasculaire de l’aorte durant la gestation. Cet effet est causé par la modification de l’activité de certains canaux ioniques (VDCC, BKCa et KATP). De plus, le canrénoate renverse l’inhibition de la Na+/K+-ATPase observée durant la gestation. Finalement, les actions locales de cet antagoniste des MR sur les vaisseaux sanguins ne se répercutent pas sur l’effet systémique global et aucune modification de la pression artérielle n’est observée. D’autres études seront toutefois nécessaires pour déterminer les voies de signalisation par lesquelles l’aldostérone module les réponses des canaux ioniques dans les VSMC., Pregnancy is accompanied by important hormonal and hemodynamic modifications. Among them, the renin-angiotensin-aldosterone system (RAAS) is activated early during pregnancy. Furthermore, this increase of RAAS is accompanied by raises of cardiac output and blood volume as well as paradoxal decreases in blood pressure and peripheral vascular resistance. These suggest that pregnancy induces reorganization of the normal physiological responses to RAAS. In addition, a decreased vascular reactivity to vasoconstrictive agents is observed during pregnancy. Modifications of calcium and potassium channels function would be implicated in this phenomenon. Furthermore, an implication of the Na+/K+-ATPase is suspected, through its influence on the membrane potential of vascular smooth muscle cells (VSMC). Aldosterone, through the presence of mineralocorticoid receptors (MR) in VSMC, could control vascular tone by its genomic and non-genomic effects. With our knowledge at this time, we submit the hypothesis that the increased serum aldosterone levels of pregnancy are responsible for the hemodynamic changes associated with pregnancy and that these effects are caused by the activation of MR. Pregnant rats were treated with potassium canrenoate (20 mg/kg•d), a MR antagonist, during the last week of pregnancy (out of 3 weeks). Vascular reactivity of endothelium-denuded aortic rings was measured. Contractile responses to phenylephrine (PhE) and KCl were studied in the presence of a voltage-dependent calcium channel (VDCC) blocker, nifedipine, as well as calcium-actived (BKCa) and ATP-dependent (KATP) potassium channels activators, NS-1619 and cromakalim respectively. Vascular responses to PhE and KCl were reduced as of the 17th day of gestation. Canrenoate increased the responses to both agonists in the aortas of all the groups. Ionic channel modulators reduced the contractile responses to PhE and KCl. However, canrenoate also modified the responses to the modulators. In the aortas of non pregnant rats, no effect or a decrease of their inhibitory effect were observed while with the pregnant rats we noticed an increased effect. These results suggest that aldosterone could be implicated in the adaptation of vascular responses to pregnancy. On the other hand, VSMC membrane potential could be affected during pregnancy. To verify this hypothesis, Na+/K+-ATPase activity was evaluated since it is implicated in its control. Our results demonstrated that the pump is inhibited as of the 19th day of pregnancy. This effect was reversed by canrenoate. However, since the reversal of the inhibition of the pump was also present in pregnant rats treated with PST 2238, an ouabain antagonist on the Na+/K+-ATPase, and because canrenoate can also act as a partial agonist of the pump, we believe that the decreased activity associated with pregnancy is linked to the inhibition of the Na+/K+-ATPase by cardiotonic steroids rather than the effect of mineralocorticoids. The increased activity of the pump by canrenoate is linked with a decreased of the α1 subunit expression. This suggests that the α2 subunit of the pump would be responsible for the variations of aortic contractility since its expression is not modified by canrénoate. The effects of the diminished expression of the α1 subunit, influenced by the MR signalization, still need to be determined. Nevertheless, our results showed that modifications of Na+/K+-ATPase activity had an impact on the activity of potassium channels and that the pump could be one of the principal elements implicated in the control of vascular reactivity during pregnancy. Since canrenoate modified vascular reactivity, we wanted to determine its impact on blood pressure. Pregnant rats were treated with canrenoate (20 or 60 mg/kg•d) and hemodynamic functions were determined by radiotelemetry. No modification was observed in blood pressure, cardiac output and pulse pressure among the treated rats. However, increases in osmolality, aldosterone and corticosterone levels as well as plasma renin activity were observed in the animal receiving the 60 mg/kg•d canrénoate. Thus, canrenoate blocks the negative feedback of the RAAS. However, MR are not the principal element responsible for the control of blood pressure during pregnancy. In conclusion, we have demonstrated that canrenoate affects aortic vascular reactivity during pregnancy. This effect is perpetuated by modifying the activity of certain ionic channels (VDCC, BKCa and KATP). Furthermore, canrenoate is able to reverse the inhibition of the Na+/K+-ATPase observed during pregnancy. Finally, the local actions of this MR antagonist on blood vessels are not reflected by the global systemic effect and no modification of the blood pressure was observed. Other studies will be necessary to determine the signaling pathways by which aldosterone modulate ionic channel responses in VSMC.

Calcium signaling is essential for all the functions of T lymphocytes, including those of Th2 cells. Th2 lymphocytes producing interleukins 4, 5 and 13 orchestrate allergic diseases including asthma. T-cell activation induces an influx of Ca(2+) from the external medium through ORAI calcium channels although other calcium channels are likely to be involved. Among them, voltage-gated calcium (Ca(v)1) channels have been reported in some T-cell subsets including Th2 cells. The inhibition of Ca(v)1 channels abrogates T-cell receptor-driven calcium influx and interleukin production by Th2 cells. From a therapeutic point of view, the inhibition of Ca(v)1 channels prevents Th2-dependent experimental allergic asthma. In this review, we will discuss the singularities of calcium responses depending upon the T-cell subset and its state of activation.

Six years ago, STIM1 (stromal interaction molecule 1) was identified as an essential component of store-operated calcium channels and in less than one year teamed up with its first partner ORAI1 in immune cells to reconstitute CRAC (calcium-release activated current) channel function. Since then, STIM1 and ORAI1 have developed an ever increasing social network and to date are now linked to nine families of proteins involved in calcium signalling. As a result of this, STIM1 and ORAI1 are now involved in three separate calcium entry pathways, Icrac, Iarc (arachidonic regulated calcium current) and voltage-dependent channels. Physiopathological roles of STIM1 and ORAI1 were first described in the immunological system but, as main actors at the central node in the calcium signalling network, there are now clear evidences that mutations in genes coding STIM1 or ORAI1 interfere with several other diseases.

This review focuses on the so-called "periodic syndromes of childhood that are precursors to migraine", as included in the Second Edition of the International Classification of Headache Disorders. Three periodic syndromes of childhood are included in the Second Edition of the International Classification of Headache Disorders: abdominal migraine, cyclic vomiting syndrome and benign paroxysmal vertigo, and a fourth, benign paroxysmal torticollis is presented in the Appendix. The key clinical features of this group of disorders are the episodic pattern and intervals of complete health. Episodes of benign paroxysmal torticollis begin between 2 and 8 months of age. Attacks are characterized by an abnormal inclination and/or rotation of the head to one side, due to cervical dystonia. They usually resolve by 5 years. Benign paroxysmal vertigo presents as sudden attacks of vertigo, accompanied by inability to stand without support, and lasting seconds to minutes. Age at onset is between 2 and 4 years, and the symptoms disappear by the age of 5. Cyclic vomiting syndrome is characterized in young infants and children by repeated stereotyped episodes of pernicious vomiting, at times to the point of dehydration, and impacting quality of life. Mean age of onset is 5 years. Abdominal migraine remains a controversial issue and presents in childhood with repeated stereotyped episodes of unexplained abdominal pain, nausea and vomiting occurring in the absence of headache. Mean age of onset is 7 years. Both cyclic vomiting syndrome and abdominal migraine are noted for the absence of pathognomonic clinical features but also for the large number of other conditions to be considered in their differential diagnoses. Diagnostic criteria, such as those of the Second Edition of the International Classification of Headache Disorders and the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition, have made diagnostic approach and management easier. Their diagnosis is entertained after exhaustive evaluations have proved unrevealing. The recommended diagnostic approach uses a strategy of targeted testing, which may include gastrointestinal and metabolic evaluations. Therapeutic recommendations include reassurance, both of the child and parents, lifestyle changes, prophylactic therapy (e.g., cyproheptadine in children 5 years or younger and amitriptyline for those older than 5 years), and acute therapy (e.g., triptans, as abortive therapy, and 10% glucose and ondansetron for those requiring intravenous hydration).

Cholinergic receptors have an essential physiological role in the central nervous system because of their implication in higher functions in the neuromuscular junction within the brain and also in the peripheral nervous system by activating nicotinic (nAChRs) or muscarinic (mAChRs) receptors. Moreover, cholinergic receptors could be recognized by animal toxins isolated from snake venoms or alkaloids having animal or vegetal origin. In this context, we aim to find such molecules in a non toxic venom fraction of Buthus occitanus tunetanus scorpion, M1, which could therefore constitute promising medical tool. We present here a physiological study in skeletal muscle cells that regroups data that have been recently published and some new results reinforcing the last ones. The global effect of M1, was firstly studied on isolated nerve-muscle preparation. In cultured myotubes, we have found that the intracellular calcium increase, induced by M1 was blocked when ryanodine or inositol 1,4,5-triphosphate receptors are inhibited. Moreover, we have shown that M1 application on myotubes, induced a membrane depolarization as seen with acetylcholine. The treatment of myotubes with alpha-bungarotoxin blocked in most parts the depolarization amplitude. Thus, these results confirm the presence of at least one component in M1 active in nAChRs.

The GABA(B) receptors belong to the family of class C metabotropic receptors. They are inhibitory receptors forming obligatory heterodimers. Their analgesic role in the dorsal horn of the spinal cord is well established since more than 25 years ago. However, Baclofen, the reference agonist of the GABA(B) receptor, proved to have little efficiency in clinics in neuropathic patients. It seems therefore useful to decipher GABA(B) functions in the nociceptive circuitry, and their regulation in conditions of chronic pain. In the present review, we will focus first on the distribution of the GABA(B) subtypes. Then, we will consider their pre- and post-synaptic functions in the dorsal horn of naïve rats. Finally, we will document the mechanisms that may lead to receptor impairment in neuropathic conditions.

In the heart, two types of calcium currents were described, the L- and T-type. In addition to these two types, a dihydropyridine-resistant Ca2+ component has been described to be up-regulated in rat ventricular cardiomyocytes during their differentiation- dedifferentiation process. The aim of our study is to examine if such calcium current component is present in human cardiomyocytes. The patch clamp technique was used to record Ca2+ current in atrial cells. In the presence of 2 microM nifedipine, residual current was activated (-2.7 +/- 0.7 pA/pF, n = 6) in the same voltage range as the L-type, nifedipine-sensitive Ca2+ current (-2.1 +/- 0.4 pA/pF, n = 6), but its steady-state inactivation was negatively shifted by 10 mV. This nifedipine-resistant Ca2+ current was completely blocked by 500 microM cadmium chloride and significantly enhanced by 1 microM isoproterenol (-7.5 +/- 0.5 pA/pF, n = 6; p0.01). These results give evidence that a nifedipine-resistant Ca2+ current, similar to the one which has been shown to be developmentally expressed in rat ventricular cardiomyocytes, is observed in human atrial cells. Its molecular identity, its expression level as well as its role in pathophysiologic conditions remain to be studied.

International audience; Voltage-dependent calcium channels constitute one of the main pathways of calcium entry into neurons. They are the principal actors of synaptic transmission by controlling the release of neurotransmitters. They also contribute to numerous other cell functions, such as gene expression or synaptogenesis. These channels, by their essential cell functions, are at the origin of numerous channelopathies resulting from mutations of the genes encoding their different subunits. Familial Hemiplegic Migraine (FHM) represents one such example of these channelopathies. In this human disease, genetic studies have demonstrated the implication of the CACNA1A gene in a type 1 form of FHM. This gene encodes for the Ca(v)2.1 subunit of P/Q calcium channels and is the target of numerous mutations affecting the properties of channel activity. The question on how discrete mutations of this gene are able to alter the activity of the channel and contribute to the physiopathology of FHM remains an open question. The functional characterization of mutated channels in various heterologous expression systems, as well as in vivo in an animal model, provides a molecular scheme of the physiopathology of FHM in which neurons, astrocytes and blood circulation act in concert.

Neuronal voltage-gated calcium channels (VGCCs) represent a major pathway of calcium entry into neuronal cells, where they play a crucial role in the molecular processes of synaptic transmission. With this fact, their activity is finely regulated to guarantee a perfect coordination between calcium influx and associated cellular processes. Hence, G-protein coupled receptors play a critical role in negative feedback to modulate VGCCs activity in response to the neuromediators release. This regulation, direct and spatially delimited, is driven by the binding of the Gβγ dimmer on different structural elements of the Cav2.x subunit, and drastically inhibits calcium currents ("ON" regulation), independently of the presence of the β subunit. The unbinding of the Gβγ dimmer following channel activation, reverse this inhibition, and induce a set of apparent phenotypic modifications of channel activity ("OFF" regulation). We put in evidence that the concept of "reluctance", described by a depolarised shift in the voltage-dependence of channel activation, historically associated with the "ON" regulation, is in fact a particular characteristic of the "OFF" regulation. On the other hand, the binding of the Gβγ dimmer directly onto the Cav2.x subunit, induces a slowing of channel inactivation kinetics, which appears as a novel characteristic of the "ON" regulation. Finally, we have characterised the major implication of fast channel inactivation in the "OFF" regulation. Channel inactivation acts as a synergistic factor to channel activation for the Gβγ removal from the channel, but defined a temporal window in the course of which the process can take place. Taken together, these results provide a better understanding in the molecular mechanisms of the regulation of synaptic activity by G-protein coupled receptors., Les canaux calciques neuronaux activés par le potentiel électrique de membrane (VGCCs) représentent une des voies majeures d'entrée du calcium dans la cellule nerveuse, où ils participent activement aux processus moléculaires de la transmission synaptique. De ce fait, leur activité est finement régulée, afin de garantir une parfaite coordination entre le flux calcique et les processus cellulaires qui lui sont associés. Aussi, les récepteurs couplés aux protéines G hétérotrimériques (RCPGs) occupent un rôle central dans le rétrocontrôle négatif de l'activité des VGCCs suite à la libération de neuromédiateurs. Cette régulation, directe et spatialement délimitée, est conduite par le dimère Gβγ, dont la fixation sur différents déterminants structuraux de la sous-unité Cav2.x conduit à l'inhibition drastique du courant calcique (régulation "ON"), indépendamment de la présence d'une sous-unité β. Le décrochage du dimère Gβγ, en réponse à l'activation du canal, reverse cette inhibition, et induit un ensemble de modifications phénotypiques apparentes de l'activité du canal (régulation "OFF"). Aussi, nous avons mis en évidence que la notion de "reluctance", décrite par le shift dépolarisant de la courbe d'activation du canal, communément accepté comme un caractère de la régulation "ON", ne traduit en définitive qu'une caractéristique particulière de la régulation "OFF". En revanche, la fixation du dimère Gβγ sur la sous-unité Cav2.x, est à l'origine d'un ralentissement de la cinétique d'inactivation du canal, et représente un caractère nouveau de la régulation "ON". Enfin, nous avons caractérisé l'implication majeure de l'inactivation rapide du canal, dans le caractère "OFF" de cette régulation. L'inactivation se présente comme un catalyseur du décrochage du dimère Gβγ, et délimite une fenêtre temporelle durant laquelle le processus peut avoir lieu. Ensemble, ces résultats permettent une meilleure compréhension des mécanismes moléculaires à la base de la régulation directe de l'activité synaptique par les récepteurs couplés aux protéines G hétérotrimériques.

Pregabalin is a novel central nervous system (CNS) drug with no interaction at benzodiazepine or GABA receptor. Its mechanism of action is correlated with its high affinity for the alpha/delta submit of the voltage-dependant CNS calcium channel. Pregabalin is rapidly absorbed with at least 90% bioavailable irrespective of dose, does not bind to plasma proteins and is excreted virtually unchanged by the kidneys. Pharmacokinetics are linear and predictable across the therapeutic dose range (150-600 mg/ day). Pregabalin is indicated, like gabapentin, in the treatment of neuropathic pain syndromes like post-herpetic neuralgia (PHN) and diabetic polyneuropathy (DPN). Efficacy in other neuropathic pain syndromes need further investigations. This paper emphasizes advantages and disadvantages on a clinical point of view.

International audience; Voltage-dependent calcium channels represent a major pathway of calcium entry into neurons, where they participate actively to cell excitability and to the molecular processes of synaptic transmission. For that reason, they have been the direct or indirect pharmacological targets of analgesics and this long before their implication in the physiology of nociception had been demonstrated. These last years, the still more refined molecular characterization of these channels and their associated regulatory subunits and the demonstration of their implication in nociceptive processes indicates that these structures are prime pharmacological targets for the management of pain. Herein, we detail the recent breakthroughs on calcium channel structure, function and pharmacology, review the implication of calcium channels in the transmission of nociception, and evaluate their importance as targets for the treatment of pain perception. The search for specific inhibitors of voltage-dependent calcium channels appears as a prelude to the development of new promising analgesic molecules.

This review of literature concerns the different autoantibodies directed against membrane receptors and ion channels. The target antigens, the associated pathologies, the pathogenesis and the methods of detection of these autoantibodies will be addressed. Some of these autoantibodies are thought to be closely related to the autoimmune disease whereas for some others their pathogenesis role is still unclear. Overall, the roles of antibodies are different between diseases, but the presence of such autoantibodies support the basis of intervening immunotherapy, antibody titers predicted the activity of the diseases and some of them are very specific and become the useful markers for the diagnosis. Some autoantibodies are detected routinely as the antiacetylcholine receptor, voltage-gated potassium and calcium channels autoantibodies whereas most of them are detected very rarely and only by specialized laboratories. This review will be divided in three parts with the following classification: the first group of autoantibodies directed against membrane receptors included receptors with an enzymatic activity (mostly tyrosine kinase) with one transmembrane domain, receptors associated to G protein with seven transmembrane domains, ion channels and receptors associated to the membrane by the glycosyl phosphatidyl inositol and the second group of intracellular receptor autoantibodies directed to the estrogens, androgens, lamin and kinesin receptors.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune channelopathy in which patients produce autoantibodies directed against voltage-gated calcium channels. Autoantibodies down-regulate calcium channels resulting in reduced transmitter release, which in turn leads to muscular weakness and autonomic dysfunction. LEMS is paraneoplastic in 60-70% of patients, most frequently associated with small cell lung carcinoma (SCLC). SCLC lines express many neuronal and neuroendocrine proteins including neuronal calcium channels of the Cav2 family (P/Q and N-type channels). It is thus likely that the paraneoplastic form of LEMS is the consequence of an anti-tumoral immune response and the production of antibodies that cross-react with identical or homologous antigens in nerve terminals. Neurological symptoms generally appear several Months before detection of the tumor. Consequently correct diagnosis of LEMS is crucial as it can allow early treatment of a particularly aggressive carcinoma. Based on published studies, our laboratory has set-up serological assays for LEMS autoantibodies as an aid to diagnosis. Calcium channels in detergent extracts of rat brain or cerebellum membranes were labeled with radioligands specific for N-type (125I-omega conotoxin GVIA) or P/Q-type (125I-omega conotoxin MVIIC) calcium channels. Autoantibodies that immunoprecipitate the ligand/channel complex can thus be titrated. Analysis of 31 LEMS sera revealed the presence of anti-N type channel antibodies in 58% and anti-P/Q type channel antibodies in 74% of patients with titres ranging from 90 to 2950 pM. Only 5 patients were seronegative in both tests, thus a combination of the two assays reliably detected autoantibodies in 26/31 (84%) patients.

Understanding of the properties of normal and diseased voltage-dependent calcium channels has greatly improved these last Years after the extensive development of the patch-clamp and molecular biology studies and the functional expression strategies. The calcium channel diversity is based on the expression of numerous genes that encode pore channel subunits (10 genes) and auxiliary/regulatory subunits (16 genes). In addition, most of these genes are subject to alternative splicing. The study of calcium channels has also benefited from the discovery of genetic diseases linked to calcium channel mutations: the calcium channelopathies. The review describes the recent data and working hypothesis that address the challenging question of how the calcium channel diversity occurs and how alterations in channel function lead to selective cellular dysfunction.

Although cannabis has been used as a medicine for several centuries, the therapeutic properties of cannabis preparations (essentially haschich and marijuana) make them far most popular as a recreational drugs.Scientific studies on the effects of cannabis were advanced considerably by the identification in 1964 of cannabinoid D9-tetrahydrocannadinol (THC), recognized as the major active constituent of cannabis. Cloning of the centrally located CB1 receptor in 1990 and the identification of the first endogenous ligand of the CB1 receptor, anandamide, in 1992 further advanced our knowledge.Progress has incited further research on the biochemistry and pharmacology of the cannabinoids in numerous diseases of the central nervous system. In the laboratory animal, cannabinoids have demonstrated potential in motion disorders, demyelinizing disease, epilepsy, and as anti-tumor and neuroprotector agents. Several clinical studies are currently in progress, but therapeutic use of cannabinoids in humans couls be hindered by undesirable effects, particularly psychotropic effects. CB1 receptor antagonists also have interesting therapeutic potential.

Alzheimer's disease has definitively emerged from its ghetto and has been identified as a (priority) public health concern in view of the increasing age of the population. Considerable advances have been made in this disease over the last 15 Years, with progress in the following fields: knowledge of the underlying aetiopathogenetic, genetic and biochemical mechanisms; semiological, clinical and paraclinical approaches; creation of early diagnostic centres and multidisciplinary care networks; therapy available to patients or currently under development. The four existing acetylcholinesterase inhibitors having confirmed symptomatic action in patients with mild to moderate Alzheimer's disease have now been joined by memantine (Ebixa), a non-competitive agonist of N-methyl-D-aspartate (NMDA) receptors. One pathogenic mechanism of Alzheimer's disease appears to be hyperactivity of the glutaminergic neurons. Various preclinical studies have shown that memantine (Ebixa) inhibits glutaminergic hyperactivity in Alzheimer's disease through modulation of NMDA receptors. Since the early 1990s, several controlled clinical trials in patients with moderate to severe Alzheimer's disease (3MMSEor =14) have demonstrated the efficacy of memantine on cognitive criteria (cognitive evaluation of severe dementia) (Severe Impairment Battery--SIB), functional criteria (Functional Assessment Stage--FAST) and global clinical criteria (Clinician's Interview-Based Impression of Change--CIBIC-Plus). The data from these studies together with clinical experience of memantine in Germany since 1982 confirm the safety of use and good tolerability profile of this medication at the recommended dosages (10 to 30 mg/day). Treatment with memantine reduces the global costs of the disease by lightening the burden on helpers and delaying institutionalisation of patients. These different studies have resulted in approval of memantine in this particular indication by the European Medicines Agency. The efficacy of memantine in mild to moderate Alzheimer's disease is currently being assessed. The preliminary results also appear to militate in favour of the efficacy of the drug in certain forms of vascular dementia. Finally, the good safety profile of combined use of this drug with antiacetylcholinesterases opens up a realistic perspective of bitherapy in Alzheimer's disease.

Calcium influxes are of fundamental importance in eukaryotic cell functions. These calcium influxes are carried by different classes of membrane proteins that allow regulated calcium entry. If in excitable cells, such as neurones or muscle, voltage-dependent calcium channels represent the main source of calcium influx, other proteins are needed to assume such a function in non-excitable cells. In these, a sustained calcium influx is observed, secondary to phospholipase C activation, IP3 synthesis and internal calcium release. The identity of proteins implicated in this second messenger calcium-driven influx, as well as the mechanisms of activation of these channels have long been debated. In recent years, genes encoding a new kind of cationic channels called TRP channels have been identified. This molecular work has set the basis for further functional studies and helped to gain crucial information on the mechanisms by which extracellular calcium can penetrate into non-excitable cells. This review will present the most recent advances obtained on the molecular diversity of TRP channels and their mode of gating.

In amphibian embryos the central nervous system derives from the dorsal region of the ectoderm. Molecular studies led to the formulation of the "neural default model" in which neural development is under the inhibitory control of members of the BMP family. These growth factors also act as epidermis inducers. The neural fate is revealed by factors secreted by the Spemann Organizer such as noggin, chordin, follistatin, Xnr3 and cerberus which act by blocking BMP signalling. We propose a new model for neural cell determination in which a signalling pathway controlled by an increase in intracellular calcium suppresses the epidermis fate and activates the neural fate instead. This increase in calcium is due to an influx through calcium channels of the L-type, expressed in ectodermal cells during gastrulation. The possible involvement of a calcium-dependent phosphatase (calcineurin) to inhibit the epidermis fate and a calcium-calmodulin kinase (CaMkinase II) which activates the neural fate is discussed.

Local anaesthetics used for regional anaesthesia inhibit impulse depolarisation through blockade of sodium channels. Regional anaesthesia accounts for 20% of all anaesthetic procedures performed in France. Spinal anaesthesia is the most common technique used peroperatively, while epidural anaesthesia is used in labouring women and for postoperative pain control. Nerve stimulation allows a safer an easier approach to peripheral blocks. Regional anaesthesia is mainly indicated for limb or eye surgery, and for mildly invasive surgical procedures. Regional anaesthetic techniques are the most efficient to control postoperative pain and to facilitate patient rehabilitation. The risk related to the practice of regional anaesthesia compares with that of general anaesthesia and is thus very low. Main complications are epidural haematoma, cardiac arrest due to inadvertent intravascular injection of local anaesthestics, and peripheral nerve damage. Regional anaesthesia requires the same technical environment and careful monitoring as general anaesthesia.

Spontaneous activities are a major semeiologic sign in electromyography. The present article deals with the different aspects recorded in practice in normal and pathological cases. There are two types of spontaneous activities, those related to motor unit hyperactivity (fasciculations and myokymia) and those related to the hyperactivity of one or more muscle fibers: fibrillations, positive sharp waves, myotonic discharges and complex repetitive discharges. In the first case the lesion is located in the axone and in the second in the membrane of the muscle fibers; All theories related to the cells' abnormalities share a common feature: spontaneous activities result from abnormal firing of the membrane action potential of muscular fibers. This functional abnormality may results from different types of lesions within the cells' membrane and determines the aspects of spontaneous activities. Impaired function of muscular cells' membranes can be produced by denervation or lesion of the membrane structure itself. The latter can be multiple and linked with the membrane proteins (such as laminine or dystrophin as in AIDS diseases) or with ion channel disturbances. Multiple membrane cell alterations may produce the same kind of spontaneous activity; for instance, myotonic discharges have the same morphology in Thomsen and Steinert's disease despite their different mechanisms and fibrillations seen in denervations and myopathies. The practical consequences are discussed and a new classification of these spontaneous activities is proposed.

In several cardiovascular diseases, including hypertension, atherosclerosis and restenosis, an uncontrolled proliferation of smooth muscle cells from the arterial wall participates in the vascular hypertrophy that is often observed to be associated with these diseases. In this article, the mechanisms of smooth muscle cell activation and proliferation are briefly reviewed, focusing on the predominant role played by Ca2+ ions and the voltage-dependent Ca2+ channels in cellular Ca2+ homeostasis. In addition, the major alterations in pathways involved in smooth muscle cell activation and proliferation that have been observed in the cardiovascular diseases under discussion are reviewed. The effects of amlodipine on the molecular/cellular pathways discussed above are then presented. Finally, we discuss recent experimental results obtained with amlodipine, which make it an excellent candidate for the treatment of those cardiovascular diseases.

The possible involvement of calcium and potassium channels in mediating the vascular actions of methyclothiazide (MCTZ), a thiazide diuretic, was investigated in isolated aortic rings from 12 week-old hypertensive rats. MCTZ (10(-4) M) inhibits the contractile response induced by addition of Ca2+ to a depolarizing solution, the maximal contracture is reduced by 87.16 +/- 6.4%. Furthermore this inhibitory effect was unaffected by charybdotoxine a selective blocker of calcium-activated K+ channels (Kca). This suggesting that MCTZ inhibits voltage-gated Ca2+ channels and blunts the Ca2+ entry into vascular smooth muscle cells. This inhibition was partially attenuated by either mechanical removal of the endothelium or N omega-nitro-L-arginine (NOLA) treatment, suggesting that MCTZ effects are also mediated by an endothelium-dependent mechanism involving endothelium-dependent relaxing factor (EDRF)/nitric oxide (NO) release. Taken together, these observations could point to a role of voltage-gated Ca2+ channels and endothelial release of EDRF/NO in the antihypertensive action of MCTZ.

Animal venoms contain various toxins which act on ion-channels, responsible for either sodium, potassium, calcium or chloride permeation. Structure determination of these toxins demonstrate that they are organised around two different structural motifs: potassium and sodium channel effectors are organised around an alpha-helix connected by two disulfide bridges to a two- or three-stranded beta sheet whereas calcium channels effectors are structured around an "Inhibitory Cystine Knot" motif made of a dense disulfide-rich core from which emerge several loops. Analysis of local structural modifications allows us to understand the structural basis of the selectivity of these effectors towards the various ion channels. This is the first step in the design of new synthetic molecules which are potent therapeutic drugs for diseases involving ion channel dysfunctioning.

Malignant hyperthermia susceptibility (MHS), a skeletal muscle disorder, is mostly inherited as an autosomal dominant trait. Exposure of susceptible individuals to volatile halogenated anaesthetics can lead to a MH episode resulting in irreversible tissue damages or to the patient's death if not immediately reversed by dantrolene treatment. A MH episode is characterised by a combination of hyperthermia, skeletal muscle rigidity and hypermetabolism. Porcine stress syndrome has proved to be a valuable model for physiopathological studies of MHS. Malignant hyperthermia syndrome is associated with a failure of the calcium homeostasis in muscular fibres. Dysfunction of the calcium channels: the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), which are involved in the release of the Ca2+ stored in sarcoplasmic reticulum has been clearly demonstrated. A biochemical test based on the analysis of the in vitro contracture response of muscular fibres to caffeine and halothane was developed to define the MHS status of patients. Although the genetic analysis of MHS has beneficiated from recent progresses, genetic testing is still far to answer to all testing situations. If in swine, hyperthermia syndrome was always associated with a unique mutation of the RyR1 gene, genetic analysis is far more complicated in human: i) more than 20 different MHS mutations in the RyR1 gene have been described; ii) a mutation of the gene encoding the dihydropyridine receptor has been identified; iii) 4 other potential MHS loci have been reported.

In adult rat pinealocytes, acetylcholine activates nicotinic receptors whose stimulation causes a depolarization of the cells, opening of voltage-gated cation channels of the L-type and sub- sequent increase in the intracellular calcium ion concentration. These events trigger a release of glu- tamate that, by its action on metabotropic glutamate type 3 receptors, activates an inhibitory cyclic AMP cascade and suppresses norepinephrine-induced melatonin biosynthesis. The nicotinic res- ponse is fully developed in the third postnatal week. Prior to this timepoint, rat pinealocytes possess functional muscarinic receptors whose activation causes a rise in the intracellular calcium ion concen- tration through a calcium release from thapsigargin-sensitive intracellular calcium stores and an opening of store-operated calcium channels. This cascade may influence tissue differentiation and matu- ration of the melatonin pathway. The demonstration of functional cholinoreceptors and the ontoge- netic switch from muscarinic to nicotinic signalling in rat pinealocytes supports the concept that pineal functions in mammals are influenced by neuronal inputs other than the sympathetic innerva- tion which serves as the major regulatory system. © Inra/Elsevier, Paris

Cyclosporin A (CyA) induced vasoconstriction and impaired relaxation to agonists has been related in some models to its vehicle: the cremophor (CRE). There is no data concerning the effect of CyA and its vehicle CRE on the pulmonary veins. Therefore, the present study was designed to characterize the effect of CyA and CRE on isolated pulmonary veins.Third-order canine pulmonary veins (n = 6) were suspended in organ chambers for measurement of isometric force. Segments were exposed to cumulative doses (10(-9) to 10(-4) M) of CyA in its vehicle CRE, and to CRE alone.CyA induced an ehdothelium-independent vaasoconstriction similar to CRE, maximal constriction (Emax) were: 1.8 +/- 0.3 g versus 2.1 +/- 0.4 g respectively (p = NS). This vasoconstriction observed with CRE was not affected by indomethacine (a cyclooxygenase inhibitor), and by pinane thromboxane (a thromboxane antagonist). However, the vasoconstriction was decreased by diltiazem (10(-5) M), a calcium channel blocker, Emax were: 2.1 +/- 0.4 g and 0.8 +/- 0.04 g respectively for CRE and CRE with diltiazem (p0.05).CyA vehicle CRE induces an endothelium-independent vasoconstriction in isolated third order pulmonary veins in the dog. This vasoconstriction is not related to a cyclooxygenase product, but is partially mediated by a calcium channel activation in vascular smooth muscle.

The staphylococcal bi-component leukotoxins constitute a family included in the super-family of the beta-sheet-structured pore-forming toxins. They may be produced by Staphylococcus aureus and by Staphylococcus intermedius and their target cells vary according to the molecules. The mode of action proceeds by the sequential binding of the class S proteins, then by that of the class F proteins at the surface of the membranes. Then, the activation of cellular calcium-channels precedes the pore formation which seems to be sensitive to several monovalent cations. The cell response is inflammatory and includes the neosynthesis as well as the secretion of leukotriene B4, interleukin -8, histamine. The injection of leukotoxins to rabbits generates cell chemotaxis , vasodilatation, and tissue necrosis. The association of the production of leukotoxins with clinical syndromes concerns several aspects of the pathology of S. aureus, and confers to these leukotoxins an important role of virulence factors.

Peptidic toxins extracted from spider, marine snails or snakes venoms, have considerably helped the pharmacological characterization of calcium channels. They have successfully been used for calcium channels mapping. However, the actual situation remains unclear. Genetic investigations demonstrated the existence of a great number of types or sub-types of calcium channels. In recent year a large number of toxins have been purified. Many of these toxins have specific actions on calcium channels and have been used as powerful tools in pharmacological approaches of calcium channels. However the pharmacology of the calcium channels remains very limited, many of them are waiting for the discovery of pharmacological tools allowing their molecular approach in order to determinate their biological implications. In this paper we describe the different families of calcium channels and toxins that interact with these channels. We also recapitulate the "non defined" calcium channels i.e. calcium channel which does not correspond to a N, L, P/Q, R or T type channel and for which no effector are available. We report the discovery and characterization of mapacalcine, a toxin extracted for a marine sponge, as an example of an approach of an undefined calcium channels first characterized by electrophysiological techniques and for which a specific toxin has been purified allowing its pharmacological approach. We also state the possible role of calcium channel toxins in the domain of therapeutic applications.

The hormone-mediated intercellular Ca2+ waves were analyzed in multiplets of rat hepatocytes by video imaging of fura2 fluorescence. These multicellular systems are composed of groups of several cells (doublets to quintuplets) issued from the liver cell plate, a one cell-thick cord of about 20 hepatocytes long between portal and centrolobular veins. When the multiplets were homogeneously bathed with the glycogenolytic agonists vasopressin, noradrenaline, angiotensin II and ATP, they showed highly organized Ca2+ signals. Surprisingly, for a given agonist, the primary rises in intracellular Ca2+ concentration ([Ca2+]i) originated invariably in the same hepatocyte, then was propagated in a sequential manner to the nearest connected cells (cell 2, then 3, cell 4 in a quadruplet, for example). The sequential activation of the cells appeared to be an intrinsic property of multiplets of rat hepatocytes. The same sequence was observed at each train of oscillations occurring between cells. The order of [Ca2+]i responses was modified neither by repeated additions of hormones nor by the hormonal dose. The mechanical disruption of an intermediate cell did not prevent the activation of the next cell. These results suggest that each hepatocyte in the multiplet displays its own sensitivity to the hormone and that a gradient of sensitivity between each cell could be responsible for directing the intercellular Ca2+ wave. To test this hypothesis, we selectively isolated rat hepatocytes from periportal (PP) and perivenous (PV) areas of the liver cell plate. Periportal (PP) and perivenous (PV) rat hepatocyte suspensions were loaded with quin2/AM and hormonal responses were studied in a spectrofluorimeter. Noradrenaline, angiotensin II, and vasopressin-induced [Ca2+]i rises were greater in PV than in PP hepatocytes. In contrast, PP cells were more responsive than PV cells to ATP. The function of the InsP3 receptor (InsP3R) was also studied by measuring the InsP3-mediated 45Ca2+ release from permeabilized PP and PV hepatocytes. In permeabilized PP and PV hepatocytes, internal Ca2+ stores displayed the same loading-kinetics, the responses to InsP3 were similar, and the sizes of InsP3-sensitive compartment were not different. In a further study, we investigated by video microscopy in fura2-loaded multicellular systems of rat hepatocytes, the mechanisms controlling intercellular propagation of the Ca2+ wave and coordination of Ca2+ signals induced by the different hormones. Using focal microperfusion which allows local perfusion of any cell of the multiplet, rapid agonist removal during the Ca2+ response and microinjection, we found that second messengers and [Ca2+]i rises in one hepatocyte cannot trigger Ca2+ responses in connected adjacent cells, suggesting that diffusion across gap junctions, while required for coordination, is not sufficient by itself for the propagation of the intercellular Ca2+ wave. In addition, focal microperfusion and intermediate cell disruption experiments revealed very fine functional differences (hormonal delay, frequency of [Ca2+]i oscillations) between hormone-induced Ca2+ signals, even between two adjacent connected hepatocytes. Recent unpublished results performed in suspensions of PP and PV rat hepatocytes supported the view of a major role played by vasopressin receptors (V1a) in genesis and orientation of the Ca2+ wave. Vasopressin binding sites, V1a mRNAs detected by RNAse Protection Assay, and vasopressin-induced InsP3 production, were more abundant in PV than in PP cells. A gradient of hormone receptors could orientate the propagation of the Ca2+ wave in multicellular systems and in liver cell plate. These results suggest that the intercellular Ca2+ wave in multicellular systems of rat hepatocytes is propagated through mechanisms involving at least three factors. (ABSTRACT TRUNCATED)

Calcium channel blockers are also termed calcium antagonists or calcium entry blockers. The use of calcium antagonists for the management of hypertension is well established. Their control of vascular tone is related to their interaction with the alpha 1 subunit of L-type calcium channels. This interaction is not simple since prolonged depolarisation promotes the inactivated state of the channels resulting in a change of affinity which is different for various molecules so far considered. The isoforms of alpha 1 subunits and the duration of the stimulus required to activate heart or vessels are important parameters to be considered with the nature of the molecule. Those parameters influence the vascular selectivity which is quantified as the ratio of the concentrations required to reduce by 50% the contraction of heart and of vessels. This selectivity is an important component in the therapeutic action. Another component of this action is the prevention of structural changes noted in heart and arteries. As well as lowering blood pressure, calcium channel blockers have also been found to exert blood pressure independent effects. For instance, they reduce cardiac and vascular hypertrophy and avoid renal damage. In the stroke-prone rat, such protective effects are accompanied by reduction of the salt-dependent overexpression of the gene of endothelin-1 and of fetal genes associated with cardiac hypertrophy. This paper summarizes available information about those components and discuss their significance.