Systematic investigations through several experimental techniques have revealed that hippocampal pyramidal neurons express voltage gated ion channels (VGICs) with well-defined gradients along their dendritic arbor. These actively maintained gradients in various dendritic VGICs effectuate several stereotypic, topographically continuous functional gradients along the topograph of the dendritic arbor, and have been referred to as intraneuronal functional maps. The prime goal of my thesis was to understand the emergence and homeostasis of the several coexistent functional maps that express within hippocampal pyramidal neurons. In the first part of the thesis, we focus only on spatial interactions between ion channels and analyzed the role of such interactions in the emergence of functional maps. We developed a generalized quantitative framework, the influence field, to analyze the extent of influence of a spatially localized VGIC cluster. Employing this framework, we showed that a localized VGIC cluster could have spatially widespread influence, and was heavily reliant on the specific physiological property and background conductances. Using the influence field model, we reconstructed functional gradients from VGIC conductance gradients, and demonstrated that the cumulative contribution of VGIC conductances in adjacent compartments plays a critical role in determining physiological properties at a given location. These results suggested that spatial interactions among spatially segregated VGIC clusters are necessary for the emergence of the functional maps. In the second part of the thesis, we assessed the specific roles of only kinetic interactions between ion channels in determining physiological properties by employing a single-compartmental model. In doing this, we analyzed the roles of interactions among several VGICs in regulating intrinsic response dynamics. Using global sensitivity analysis, we showed that functionally similar models could be achieved even when underlying parameters displayed tremendous variability and exhibited weak pair-wise correlations. These results suggested that that response homeostasis could be achieved through several non-unique channel combinations, as an emergent consequence of kinetic interactions among these channel conductances. In the final part of the thesis, we analyzed the combined impact of both spatial and kinetic interactions among ion channel conductances on the emergence and homeostasis of functional maps in a neuronal model endowed with extensive dendritic arborization. To do this, we performed global sensitivity analysis on morphologically realistic conductance-based models of hippocampal pyramidal neurons that coexpressed six functional maps. We found topographically continuous functional maps to emerge from disparate model parameters with weak pair-wise correlations between parameters. These results implied that individual channel properties need not be set at constant values in achieving overall homeostasis of several coexistent functional maps. We suggest collective channelostasis, where several channels regulate their properties and expression profiles in an uncorrelated manner, as an alternative for accomplishing functional map homeostasis. Finally, we developed a methodology to assess the contribution of individual channel conductances to the various functional measurements employing virtual knockout simulations. We found that the deletion of individual channels resulted in variable, measurement-and location-specific impacts across the model population.

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.

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.

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 canaux potassiques (Kv) dépendants du voltage sont des protéines transmembranaires qui permettent le flux passif d’ions potassium à travers une membrane plasmique lorsque celle-ci est dépolarisée. Ils sont constitués de quatre domaines périphériques sensibles au voltage et un domaine central, un pore, qui délimite un chemin hydrophile pour le passage d’ions. Les domaines sensibles à la tension (VSD) et le pore sont couplés, ce qui signifie que l’activation des VSD déclenche l’ouverture du pore, et qu’un pore ouvert favorise l’activation des VSD. Le phosphatidylinositol-4,5-bisphosphate (PIP2) est un lipide mineur du feuillet interne de la membrane plasmique. Ce lipide fortement chargé négativement module le fonctionnement de plusieurs canaux ioniques, y compris les membres de la famille Kv. En particulier, l’application de ce lipide à Kv1.2 et Kv7.1, deux canaux homologues, augmente leur courant ionique. Cependant, alors que Kv1.2 est capable de s’ouvrir en l’absence de PIP2, dans le cas de Kv7.1, ce lipide est absolument nécessaire pour l’ouverture du canal. En outre, dans Kv1.2, PIP2 induit une perte de fonction, qui est manifesté par un mouvement retardé des VSD. Jusqu’à présent, les mécanismes sous-jacents à de telles modulations des canaux Kv par PIP2 restent inconnus. Dans ce travail, nous tentons de faire la lumière sur ces mécanismes en utilisant des simulations de dynamique moléculaire (DM) combinées avec une approche expérimentale, entreprise par nos collaborateurs. En utilisant des simulations de DM sans contrainte, nous avons identifié les sites potentiels de liaison du PIP2 au Kv1.2. Dans l’un de ces sites, PIP2 interagit avec le canal de sorte à former des ponts salins dépendants de l’état du canal, soit avec le VSD soit avec le pore. Sur la base de ces résultats, nous proposons un modèle pour rationaliser les données expérimentales connues. En outre, nous avons cherché à évaluer quantitativement la perte de fonction induite par la présence de PIP2 au voisinage du VSD du Kv1.2. En particulier, nous avons calculé l’énergie libre des deux premières transitions le long de l’activation du VSD en présence et en l’absence de ce lipide. Nous avons constaté que PIP2 affecte à la fois la stabilité relative des états du VSD et les barrières d’énergie libre qui les séparent. Enfin, nous avons étudié les interactions entre PIP2 et un autre membre de la famille Kv, le canal Kv7.1 cardiaque. Dans le site de liaison de PIP2 que nous avons identifié pour ce canal, l’interaction entre les résidus positifs de Kv7.1 et le lipide sont dépendants de l’état du VSD, comme dans le cas de Kv1.2. On montre que cette interaction est importante pour le couplage entre les VSD et le pore, couplage qui est par ailleurs affaibli à cause de la répulsion électrostatique entre quelques résidus positifs. Ces résultats et prédictions ont été vérifiés par les données expérimentales obtenues par nos collaborateurs / Voltage-gated potassium (Kv) channels are transmembrane proteins that enable the passive flow of potassium ions across a plasma membrane when the latter is depolarized. They consist of four peripheral voltage sensor domains, responding to the applied voltage, and a central pore domain that encompasses a hydrophilic path for passing ions. The voltage sensors and the pore are coupled, meaning that the activation of the voltage sensors triggers the pore opening, and the open pore promotes the activation of the voltage sensors. Phosphatidylinositol-4,5-bisphosphate (PIP2) is a minor lipid of the inner plasma membrane leaflet. This highly negatively charged lipid was shown to modulate the functioning of several ion channels including members of the Kv family. In particular, application of this lipid to Kv1.2 and Kv7.1, two homologous channels, enhances their ionic current. However, while Kv1.2 is able to open without PIP2, in the case of Kv7.1, this lipid is absolutely required for opening. Additionally, in Kv1.2, PIP2 induces a loss of functioning, which is manifested by delayed motions of the voltage sensors. So far, the mechanism underlying the Kv channels modulation by PIP2 remains unknown. In the present manuscript, we attempt to shed light on this mechanism using molecular dynamics (MD) simulations combined with experiments, which was undertaken by our collaborators. Using unconstrained MD simulations, we have identified potential PIP2 binding sites in Kv1.2. In one of these sites, PIP2 interacts with the channel in a state-dependent manner forming salt bridges either with the voltage sensor or with the pore. Based on these findings, we propose a model rationalizing the known experimental data. Further, we aimed to estimate the loss of functioning effect induced by PIP2 on the Kv1.2 voltage sensors. In particular, we have calculated the free energy of the first two transitions along the activation path in the presence and absence of this lipid. We found that PIP2 affects both the relative stability of the voltage sensor states and the free energy barriers separating them. Finally, we studied the interactions between PIP2 and another member of the Kv family, the cardiac channel Kv7.1. In the PIP2 binding site that we have identified for this channel, the interaction between positive residues of Kv7.1 and the lipid was state-dependent, as in the case of Kv1.2. This state-dependent interaction, however, is prominent for coupling between the voltage sensors and the pore, which is otherwise weakened due to electrostatic repulsion of some positive residues. These findings are in a good agreement with the experimental data obtained by our collaborators

Modification of Hodgkin and Huxley’s experimentally derived set of nonlinear differential equations was implemented to accurately simulate the action potential of the Hirudo Medicinalis’s Retzius cell in MATLAB under analogous conditions to those found in the Retzius cell environment. The voltage-gated sodium and potassium channel responses to changes in membrane potential, as experimentally determined by Hodgkin and Huxley, were manipulated to suit simulation parameters established by electrophysiological Retzius cell recordings. Application of this methodology permitted additional accurate simulation of the Hirudo Medicinalis’s P cell under analogous conditions to those found in the P cell environment. Further refinement of this technique should allow for the voltage-gated behavioral based simulation of action potential waveforms found in variety of neurons under simulation conditions analogous to the nerve cell environment.

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.

KCNQ1 is a homotetrameric voltage-gated potassium channel expressed in cardiomyocytes and epithelial tissues. However, currents arising from KCNQ1 have never been physiologically observed. KCNQ1 is able to provide the diverse potassium conductances required by these distinct cell types through coassembly with and modulation by type I transmembrane β-subunits of the KCNE gene family. KCNQ1-KCNE K+ channels play important physiological roles. In cardiac tissues the association of KCNQ1 with KCNE1 gives rise to IKs, the slow delayed outwardly rectifying potassium current. IKs is in part responsible for repolarizing heart muscle, and is therefore crucial in maintaining normal heart rhymicity. IKschannels help terminate each action potential and provide cardiac repolarization reserve. As such, mutations in either subunit can lead to Romano-Ward Syndrome or Jervell and Lange-Nielsen Syndrome, two forms of Q-T prolongation. In epithelial cells, KCNQ1-KCNE1, KCNQ1-KCNE2 and KCNQ1-KCNE3 give rise to potassium currents required for potassium recycling and secretion. These functions arise because the biophysical properties of KCNQ1 are always dramatically altered by KCNE co-expression. We wanted to understand how KCNE peptides are able to modulate KCNQ1. In Chapter II, we produce partial truncations of KCNE3 and demonstrate the transmembrane domain is necessary and sufficient for both assembly with and modulation of KCNQ1. Comparing these results with published results obtained from chimeric KCNE peptides and partial deletion mutants of KCNE1, we propose a bipartite modulation residing in KCNE peptides. Transmembrane modulation is either active (KCNE3) or permissive (KCNE1). Active transmembrane KCNE modulation masks juxtamembranous C-terminal modulation of KCNQ1, while permissive modulation allows C-terminal modulation of KCNQ1 to express. We test our hypothesis, and demonstrate C-terminal Long QT point mutants in KCNE1 can be masked by active trasnsmembrane modulation. Having confirmed the importance the C-terminus of KCNE1, we continue with two projects designed to elucidate KCNE1 C-terminal structure. In Chapter III we conduct an alanine-perturbation scan within the C-terminus. C-terminal KCNE1 alanine point mutations result in changes in the free energy for the KCNQ1-KCNE1 channel complex. High-impact point mutants cluster in an arrangement consistent with an alphahelical secondary structure, "kinked" by a single proline residue. In Chapter IV, we use oxidant-mediated disulfide bond formation between non-native cysteine residues to demonstrate amino acid side chains residing within the C-terminal domain of KCNE1 are close and juxtaposed to amino acid side chains on the cytoplasmic face of the KCNQ1 pore domain. Many of the amino acids identified as high impact through alanine perturbation correspond with residues identified as able to form disulfide bonds with KCNQ1. Taken together, we demonstrate that the interaction between the C-terminus of KCNE1 and the pore domain of KCNQ1 is required for the proper modulation of KCNQ1 by KCNE1, and by extension, normal IKs function and heart rhymicity.

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 de potassium voltage-dépendant (Kv) sont des tétramères séparés en 12 familles. Chaque sous-unité est composée de six segments transmembranaires (S1-S6). Les quatre premiers (S1-S4) forment le senseur de voltage dont le rôle est de détecter des variations en potentiel membranaire grâce à des acides aminés chargés. Ces acides aminés vont bouger et ce mouvement va être transmis au second domaine, celui du pore (S5-S6). Les domaines du pore des quatre sous-unités vont se combiner pour créer le pore. Ces sous-unités peuvent former des canaux homomériques où chaque sous-unité est identique ou des canaux hétéromériques avec des membres de la même famille. Kv6.4 (KCNG4) est un membre de la famille de sous-unité silencieuse Kv6. Les familles de sous-unités silencieuses incluent également Kv5, Kv8 et Kv9. Ils ne peuvent pas former d’homomères. À la place, il doit former des hétéromères avec Kv2. Les canaux Kv2.1/Kv6.4 ont des propriétés différentes, lorsque comparées aux homomères de Kv2.1, particulièrement avec un décalage de l’inactivation vers les négatifs. Avec la technique du « cut-open voltage clamp fluorometry » (COVCF), nous avons pu déterminer que l’absence d’une charge positive à la position Kv6.4-Y345 est responsable pour une partie du décalage tout en étant capable de réduire ce décalage avec la mutation Kv6.4-Y345R. Nous avons également pu produire l’effet inverse dans Kv2.1 avec Kv2.1-R306Y. Également, nous avons déterminé que la mutation Kv6.4-L360P trouvée chez des patients souffrant de migraines mène à cette pathologie à cause d’un problème de trafic où les sous-unités mutées ne peuvent pas atteindre la surface et produire des canaux fonctionnels. Ce problème est causé par un bris dans l’hélice alpha du segment S4-S5. Uniquement des homomères de Kv2.1 se rendent à la surface ce qui réduit l’excitabilité membranaire. Nous proposons que lorsqu’exprimée dans le ganglion trigéminal, cette mutation mène à des migraines. / Voltage-gated potassium channels (Kv) are tetramers split into 12 families. Each subunit is composed of six transmembrane helices (S1-S6). The first four of those (S1-S4) form the voltage sensor domain whose role it is to detect variations in the membrane potential through charged amino acids. The movement of those amino acids will be transmitted to the second domain, the pore domain (S5-S6). The pore domain of all four subunits will combine to form the ion conducting pore. These subunits can form homomers where all four subunits are identical or heteromers with members of the same family. Kv6.4 (KCNG4) is a member of the silent subunit family Kv6, which also includes Kv5, Kv8 and Kv9. They cannot form functioning homomers. Instead, they form heteromers with Kv2. Kv2.1/Kv6.4 channels have different properties when compared to Kv2.1 homomers, particularly a negative shift of the voltage dependence of inactivation. With the cut-open voltage clamp fluorometry (COVC) technique, we were able to determine that the absence of a gating charge at position Kv6.4-Y345 is responsible for part of this shift. We were able to recover part of this shift with the mutation Kv6.4-Y345R. We were also able to produce the inverse effect in Kv2.1 with the mutation Kv2.1-R306Y. Also, we determined that the mutation Kv6.4-L360P. which is found in patients suffering from migraines, leads to this condition because of a trafficking defect caused by the mutation stopping the subunits from reaching the membrane and making functional channels. The defect is caused by a kink in the alpha helix of the S4-S5 linker. Only Kv2.1 homomers reach the membrane which reduces membrane excitability. We propose that when expressed in the trigeminal ganglion, this mutation leads to migraines because of this trafficking defect.