Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

<p>The capacity of marine organisms to adapt to natural and anthropogenic stressors is an integral component of ocean health. Harmful algal blooms (HABs), which are one of many growing threats in coastal marine ecosystems, represent a historically present natural stressor that has recently intensified and expanded in geographic distribution partially due to anthropogenic activities. In the Gulf of Mexico, HABs of <italic>Karenia brevis</italic> occur almost annually and produce neurotoxic brevetoxins that have been associated with large-scale mortality events of many marine species, including the common bottlenose dolphin (<italic>Tursiops truncatus</italic>). The factors resulting in large-scale dolphin mortality associated with HABs are not well understood, particularly in regards to the seemingly different impacts of HABs in geographically disjunct dolphin populations. My dissertation investigates a genetic basis for resistance to HABs in bottlenose dolphins in central-west Florida and the Florida Panhandle. I used both genome-wide and candidate gene approaches to analyze genetic variation in dolphins that died putatively due to brevetoxicosis and live dolphins from the same geographic areas that survived HAB events. Using restriction site-associated DNA sequencing, I identified genetic variation that suggested both a common genetic basis for resistance to HABs in bottlenose dolphins across the Gulf coast of Florida and regionally specific resistance. Many candidate genes involved in the immune, nervous, and detoxification systems were found in close genomic proximity to survival-associated polymorphisms throughout the bottlenose dolphin genome. I further investigated two groups of candidate genes, nine voltage-gated sodium channel genes selected because of their putative role in brevetoxin binding and four major histocompatibility complex (MHC) loci selected because of their genomic proximity to a polymorphism exhibiting a strong association with survival. I found little variation in the sodium channel genes and conclude that bottlenose dolphins have not evolved resistance to HABs via mutations in the toxin binding site. The immunologically relevant MHC loci were highly variable and exhibited patterns of genetic differentiation among geographic regions that differed from neutral loci; however, genetic variation at the MHC also could not fully explain variation in survival of bottlenose dolphins exposed to HABs. In my final chapter, I consider the advantages and drawbacks of the genome-wide approach in comparison to a candidate gene approach and, as laid out in my dissertation, I recommend using both complementary approaches in future investigations of adaptation in genome-enabled non-model organisms.</p> / Dissertation

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Les cellules cancéreuses mammaires invasives expriment des canaux sodiques NaV1.5 dont l’activité semble être associée au développement métastatique. L’activité de ce canal dans les cellules MDA-MB-231 conduit à une acidification péricellulaire favorable à l’activité des cathepsines à cystéine B et S extracellulaires et à la dégradation de la matrice extracellulaire. Au cours de cette thèse, nous avons montré que l’échangeur NHE1 est le principal régulateur du pH des cellules MDA-MB-231 et que l’activité du canal NaV1.5 augmente l’activité d’efflux de protons par NHE1 vraisemblablement par modulation allostérique. NaV1.5 et NHE1 sont co-localisés dans des radeaux lipidiques et plus particulièrement dans les invadopodes des cellules MDA-MB-231. Les activités de NHE1 et NaV1.5 stimulent l’activité protéolytique des invadopodes. Enfin, l’activité du canal NaV1.5 semble moduler le cytosquelette et la morphologie des cellules cancéreuses MDA-MB-231 pour leur donner un phénotype invasif. En conclusion, NaV1.5 augmente l’activité de NHE1 dans les invadopodes stimulant ainsi l’invasivité des cellules cancéreuses mammaires. / Invasive breast cancer cells express NaV1.5 sodium channels which activity seems to be associated with metastatic progression. The activity of the channel in MDA-MB-231 cells leads to a pericellular acidification favourable for the activity of extracellular cysteine cathepsins B and S and for extracellular matrix degradation. During this thesis, we have shown that NHE1 exchanger is the main pH regulator in MDA-MB-231 cells and that the activity of NaV1.5 channels increases protons efflux activity of NHE1 possibly through allosteric modulation. NaV1.5 and NHE1 are co-localised in lipid rafts and in invadopodia of MDA-MB-231 cells. The activity of NHE1 and NaV1.5 promotes the proteolytic activity of invadopodia. Finally, the activity of NaV1.5 channels seems to modulate cytoskeleton and morphology of MDA-MB-231 cancer cells to promote the acquisition of a proinvasive phenotype. In conclusion NaV1.5 increases NHE1 activity in invadopodia to stimulate breast cancer cells invasiveness.

Lors des efflorescences de micro-algues productrices de toxines paralysantes (PST), les bivalves filtreurs peuvent bioaccumuler une grande quantité de toxines et devenir à leur tour toxiques, notamment pour l’homme. La quantité de toxines PST accumulée d’un individu à l’autre s’avère être très variable au sein même d’une population de bivalves. Ainsi, dans nos conditions expérimentales, la quantité de PST accumulées par des huîtres creuses, Crassostrea gigas, d’un même lot, exposées au dinoflagellé toxique Alexandrium minutum, variait d’un facteur 450. L’origine de cette variabilité est inconnue jusqu’alors mais l’une des hypothèses pour l’expliquer serait l’existence de plusieurs formes de canaux sodium voltage-dépendant (NaV), cible des PST, qui confèreraient aux bivalves des sensibilités différentes aux PST. L’objectif principal de cette thèse était de comprendre s’il existe une sensibilité individuelle aux PST différente entre les huîtres et si cette variabilité pouvait être due à des formes différentes de NaV.Une première partie a permis de caractériser le NaV chez C. gigas par une approche de biologie moléculaire. Deux gènes NaV ont été mis en évidence chez C. gigas : CgNaV1, codant un canal sodium et CgNaV2 codant un canal potentiellement sélectif du sodium et du calcium. L’épissage alternatif de CgNaV1 produits trois variants (A, B et C) avec des profils d’expression différents : au niveau des jonctions neuromusculaires pour CgNaV1A, dans les cellules nerveuses pour CgNaV1B et dans les deux pour CgNaV1C. L'acide aminé Q, observé dans le site de liaison aux PST (domaine II) de la séquence CgNaV1 pour les 3 variants et chez tous les individus des 4 populations étudiées, pourrait conférer aux huîtres une certaine résistance aux PST. Ainsi, les variants issus du génotypage/épissage de CgNaV1 ne seraient donc pas le point déterminant du niveau de bioaccumulation des huîtres.Une deuxième partie a permis d’étudier la sensibilité aux PST des nerfs de l’huître creuse C.gigas en relation avec l’accumulation de PST par une approche d’électrophysiologie. La sensibilité à la STX des nerfs cérébroviscéraux d'huîtres a été évaluée en étudiant leur potentiel d'action (CNAP).Il a été montré que les nerfs de C. gigas possédaient une sensibilité à la STX de l’ordre du micromolaire, ce qui leur confère une sensibilité intermédiaire parmi les bivalves. Cette sensibilité des nerfs peut varier selon la période à laquelle les huîtres ont été prélevées et potentiellement selon leur condition physiologique. Une pré-exposition des huîtres à A. minutum semble augmenter la résistance des nerfs à la STX. Cependant, aucune corrélation significative n'a été observée entre la sensibilité nerveuse à la STX et la charge en PST dans la glande digestive des huîtres.Il apparait donc que la variabilité de l’accumulation des PST par les huîtres résulterait plutôt d’une plasticité physiologique, en terme de filtration, d’ingestion et d’assimilation, que d’une sensibilité différentielle des NaV. / During bloom of microalgae producing paralytic shellfish toxins (PST), filtering bivalves can bio-accumulate a large quantity of toxins and become toxic for human consumption. The amount of accumulated PST can greatly vary from one individual to another within a bivalve population. Indeed, under our experimental conditions, the amount of accumulated PST by Pacific oysters, Crassostrea gigas, exposed to the toxic dinoflagellate Alexandrium minutum, varied by a factor of 450. To explain such variability we hypothesized the existence of several forms of voltage-gated sodium channel (NaV), target of the PST, resulting in different sensitivities to PST. The main objective of this thesis was to understand whether there are relationships between nerve sensitivity to PST, the different forms of NaV and the amount of accumulated PST.The NaV was first characterized in C. gigas by a molecular biology approach. Two NaV genes were reported in C. gigas: CgNaV1, encoding a sodium channel and CgNaV2 encoding a channel potentially selective for sodium and calcium. Alternative splicing of CgNaV1 produced three variants (A, B and C) with different expression profiles: at the neuromuscular junctions for CgNaV1A, in the nerve cells for CgNaV1B and in both for CgNaV1C. The amino acid Q observed in the binding site of PST (domain II), of the sequence CgNaV1 for the 3 variants and in all individuals from the 4 studied populations possibly provide some PST resistance to oysters. Thus, the variants resulting from the genotyping/splicing of CgNaV1 would not therefore be the determining factor of the level of bioaccumulation in oysters.A second part allowed studying the nerve sensitivity to PST of C. gigas oyster in relation to the accumulation of PST by an electrophysiology approach. The sensitivity to saxitoxin (STX, a PST) of the cerebro-visceral nerves from oysters was assessed by studying their action potential (CNAP). C.gigas nerves have been shown to have sensitivity to STX of the micromolar range, which gives them intermediate sensitivity among bivalves. This nerve sensitivity may vary depending on the period at which the oysters were collected and potentially according to their physiological condition. A preexposure of oysters to A. minutum appears to increase nerve resistance to STX. However, there was no significant correlation between STX nerve sensitivity and PST content in the oyster digestive gland.Overall, it appears that the variability of the PST accumulation by oysters would result rather from a physiological plasticity, in terms of filtration, ingestion and assimilation, than from a differential sensitivity of the NaV.

Les canaux sodiques dépendants du voltage, ou canaux Nav, jouent un rôle capital dans l'excitabilité neuronale, dans la genèse et dans la propagation des potentiels d'action. Le canal Nav1.9 se distingue par une expression restreinte aux nocicepteurs et par des propriétés électrophysiologiques uniques qui, si elles excluent sa contribution à la phase dépolarisante du potentiel d'action, lui confèreraient un rôle dans la modulation de l'excitabilité des nocicepteurs. Ce travail de thèse vise à caractériser son implication dans la physiopathologie de la douleur par une approche comportementale, moléculaire et fonctionnelle. La première partie de ce travail consiste à étudier la contribution du canal Nav1.9 à la douleur inflammatoire. Nous avons donc réalisé différents tests comportementaux chez des souris knock-out (KO) et des rats traités par antisens (knock-down) modèles de douleur inflammatoire (aigu, subaigu, chronique). L'expression du canal ainsi que ses propriétés électrophysiologiques sont ensuite analysées chez ces mêmes modèles animaux. Notre premier constat est que le canal Nav1.9 n'est pas impliqué dans la réponse à une stimulation mécanique ou thermique chaude nociceptive chez des animaux sains. En revanche, l'hypersensibilité douloureuse thermique et mécanique induite par une inflammation subaiguë (carragénine intraplantaire) ou chronique (monoarthrite) est significativement réduite chez la souris KO Nav1.9. Un résultat similaire est obtenu par traitement antisens chez le rat, sur le modèle d'inflammation subaiguë. Chez la souris, suite à l'induction d'une inflammation subaiguë, une légère diminution suivie d'une forte augmentation de l'expression protéique du canal Nav1.9 est observée dans les ganglions rachidiens innervant la patte enflammée. Une augmentation de la quantité de canaux est également observée au niveau des troncs nerveux cutanés innervant cette même zone. Les canaux néosynthétisés ne contribuent pas au courant sodique enregistré en patch clamp dans les corps cellulaires des neurones des ganglions rachidiens, mais nos données suggèrent qu'ils sont exportés en direction des terminaisons nerveuses, où ils pourraient devenir fonctionnels et augmenter l'excitabilité cellulaire. La deuxième partie de ce travail de thèse consiste à caractériser l'implication de canal Nav1.9 dans la perception du froid et dans l'hypersensibilité au froid induite par l'oxaliplatine. Nous avons en effet observé de manière inattendue que les souris KO Nav1.9 présentent des seuils de douleur au froid (<10°C) plus élevés que les souris sauvages. Ce phénomène est confirmé par plusieurs tests comportementaux chez les souris KO et chez des rats traités par antisens anti-Nav1.9. L'oxaliplatine, prescrit dans le traitement des cancers colorectaux, est connu pour induire une hypersensibilité au froid invalidante chez la majorité des patients. Nous avons donc décidé d'étudier la contribution du canal Nav1.9 à ce symptôme. Suite à une injection unique d'oxaliplatine, une forte hypersensibilité au froid apparait chez les souris dès 20°C. Nous montrons que le KO Nav1.9 permet de supprimer l'hypersensibilité au froid aux températures normalement non douloureuses (20 et 15°C, allodynie), et de réduire l'hypersensibilité aux températures douloureuses (10 et 5°C, hyperalgie). Le même effet est observé chez le rat après traitement antisens. En conclusion, ce travail permet de mettre en évidence l'intérêt du canal Nav1.9 en tant que cible pharmacologique potentielle pour le traitement de douleurs inflammatoires et de l'hypersensibilité au froid induite par l'oxaliplatine. Il est de plus intéressant de constater que les seuils de réponse à des stimuli nociceptifs ne sont pas perturbés chez les souris KO Nav1.9 saines, à l'exception de la douleur provoquée par des températures froides extrêmes. Le blocage du canal Nav1.9 aurait donc des propriétés anti-hyperalgiques plutôt qu'antalgique, ce qui est conceptuellement intéressant. / Voltage-gated sodium channels, or Nav channels, play a key role in neuronal excitability and in the emission and propagation of action potentials. Among the different Nav isoforms, Nav1.9 is only expressed in nociceptors and shows atypical electrophysiological properties which, if they exclude a possible contribution to the depolarizing phase of the action potential, could be important for the modulation of nociceptors' excitability. This study aims to characterize the Nav1.9 implication in the pathophysiology of pain using behavioral, molecular and functional approaches. The first part of this work is to assess the Nav1.9 contribution to inflammatory pain. Therefore we have performed several behavioral tests in different inflammatory pain models (acute, subacute, chronic), using knock-out (KO) mice and rats treated with antisense oligodeoxynucleotides. Nav1.9 expression and electrophysiological properties are then analyzed within the same animal models. First, we observe that Nav1.9 channels do not contribute to pain perception in response to noxious heat or pressure in healthy animals. However, thermal and mechanical pain hypersensitivity induced by subacute (intraplantar carrageenan) or chronic (monoarthritis) inflammation is significantly lowered in Nav1.9 knock-out mice. Similar results are obtained on the subacute inflammation model using a knock-down strategy in rats. A weak reduction followed by a strong increase in Nav1.9 protein expression is observed in mice dorsal root ganglions innervating the inflamed paw during subacute inflammation. We also observe an increase in Nav1.9 immunolabeling in cutaneous nerve trunks innervating this zone. Whereas the newly produced channels do not contribute to the sodium current recorded in dorsal root ganglion cell bodies, as assessed by patch clamp, our data suggest that they are transported to nerve terminals where they could become functional and increase neuronal excitability. In the second part of this study, we aim to characterize the implication of Nav1.9 channels in cold perception and in oxaliplatin-induced cold hypersensitivity. Indeed, we surprisingly observed that Nav1.9 KO mice showed higher pain thresholds to intense cold (<10°C) than wild-type mice. This observation is confirmed by several behavioral tests in KO mice and in antisense-treated rats. As oxaliplatine (a platinum salt used to treat colorectal cancer) is known to induce cold pain hypersensitivity in most of the patients, we decided to study the Nav1.9 contribution to this symptom. Following acute oxaliplatin injection, a strong cold hypersensitivity is observed in wild-type mice at 20°C and below. We show that Nav1.9 KO results in a suppression of cold hypersensitivity to non-noxious temperatures (20 and 15°C, allodynia), and a reduction of hypersensitivity to noxious cold (10 and 5°C, hyperalgesia). A similar observation is made using Nav1.9 knock-down in rats. To conclude, our data shows that Nav1.9 could be potentially a good target to treat acute to chronic inflammatory pain, as well as oxaliplatin-induced cold hypersensitivity. Furthermore, as Nav1.9 is not involved in defining pain thresholds of healthy animals (except for noxious cold), its blockade would have anti-hyperalgesic rather than analgesic effects, which is conceptually interesting.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.

Les canaux sodiques dépendants du voltage, ou canaux Nav, jouent un rôle capital dans l'excitabilité neuronale, dans la genèse et dans la propagation des potentiels d'action. Le canal Nav1.9 se distingue par une expression restreinte aux nocicepteurs et par des propriétés électrophysiologiques uniques qui, si elles excluent sa contribution à la phase dépolarisante du potentiel d'action, lui confèreraient un rôle dans la modulation de l'excitabilité des nocicepteurs. Ce travail de thèse vise à caractériser son implication dans la physiopathologie de la douleur par une approche comportementale, moléculaire et fonctionnelle. La première partie de ce travail consiste à étudier la contribution du canal Nav1.9 à la douleur inflammatoire. Nous avons donc réalisé différents tests comportementaux chez des souris knock-out (KO) et des rats traités par antisens (knock-down) modèles de douleur inflammatoire (aigu, subaigu, chronique). L'expression du canal ainsi que ses propriétés électrophysiologiques sont ensuite analysées chez ces mêmes modèles animaux. Notre premier constat est que le canal Nav1.9 n'est pas impliqué dans la réponse à une stimulation mécanique ou thermique chaude nociceptive chez des animaux sains. En revanche, l'hypersensibilité douloureuse thermique et mécanique induite par une inflammation subaiguë (carragénine intraplantaire) ou chronique (monoarthrite) est significativement réduite chez la souris KO Nav1.9. Un résultat similaire est obtenu par traitement antisens chez le rat, sur le modèle d'inflammation subaiguë. Chez la souris, suite à l'induction d'une inflammation subaiguë, une légère diminution suivie d'une forte augmentation de l'expression protéique du canal Nav1.9 est observée dans les ganglions rachidiens innervant la patte enflammée. Une augmentation de la quantité de canaux est également observée au niveau des troncs nerveux cutanés innervant cette même zone. Les canaux néosynthétisés ne contribuent pas au courant sodique enregistré en patch clamp dans les corps cellulaires des neurones des ganglions rachidiens, mais nos données suggèrent qu'ils sont exportés en direction des terminaisons nerveuses, où ils pourraient devenir fonctionnels et augmenter l'excitabilité cellulaire. La deuxième partie de ce travail de thèse consiste à caractériser l'implication de canal Nav1.9 dans la perception du froid et dans l'hypersensibilité au froid induite par l'oxaliplatine. Nous avons en effet observé de manière inattendue que les souris KO Nav1.9 présentent des seuils de douleur au froid (<10°C) plus élevés que les souris sauvages. Ce phénomène est confirmé par plusieurs tests comportementaux chez les souris KO et chez des rats traités par antisens anti-Nav1.9. L'oxaliplatine, prescrit dans le traitement des cancers colorectaux, est connu pour induire une hypersensibilité au froid invalidante chez la majorité des patients. Nous avons donc décidé d'étudier la contribution du canal Nav1.9 à ce symptôme. Suite à une injection unique d'oxaliplatine, une forte hypersensibilité au froid apparait chez les souris dès 20°C. Nous montrons que le KO Nav1.9 permet de supprimer l'hypersensibilité au froid aux températures normalement non douloureuses (20 et 15°C, allodynie), et de réduire l'hypersensibilité aux températures douloureuses (10 et 5°C, hyperalgie). Le même effet est observé chez le rat après traitement antisens. En conclusion, ce travail permet de mettre en évidence l'intérêt du canal Nav1.9 en tant que cible pharmacologique potentielle pour le traitement de douleurs inflammatoires et de l'hypersensibilité au froid induite par l'oxaliplatine. Il est de plus intéressant de constater que les seuils de réponse à des stimuli nociceptifs ne sont pas perturbés chez les souris KO Nav1.9 saines, à l'exception de la douleur provoquée par des températures froides extrêmes. Le blocage du canal Nav1.9 aurait donc des propriétés anti-hyperalgiques plutôt qu'antalgique, ce qui est conceptuellement intéressant.