Pyrethroids are insecticides used since the 1970s. They are favored for their low mammalian toxicity, improved environmental stability and insecticidal potency. Voltage-gated sodium channels (VGSCs) are a known target but in vitro evidence indicates that voltage-gated calcium channels (VGCCs) are also targets. Site-directed mutagenesis of CaV2.2 (N-type), altering threonine 422 to glutamate (T422E), produces a mutant channel that acts as if permanently phosphorylated. Deltamethrin increases peak current of T422E CaV2.2 compared to its antagonistic action on wild type CaV2.2 when expressed in Xenopus oocytes. Phosphorylation of wild type CaV2.2 is evoked by the phorbol ester PMA by activating endogenous protein kinase C (PKC) in oocytes. Under steady-state conditions, deltamethrin increases transient peak current and slows deactivation kinetics of the PKC phosphorylated channel thereby increasing Ca2+ influx and neurotransmitter release. Conversely, deltamethrin treatment resulted in no effect on the deactivation kinetics of the unphosphorylated or T422E channels. Under voltage-dependent conditions, deltamethrin enhances peak current, and causes a hyperpolarizing shift in activation midpoint potential of the PKC phosphorylated channel which is consistent with enhanced Ca2+ influx. The hyperpolarizing shift of activation midpoint potential was not observed when deltamethrin was applied to the T422E mutant channels indicating that the other phosphorylation sites on CaV2.2 may be playing a role in the differential effects observed in the action of deltamethrin on the unphosphorylated channel, the T422E mutant and the PMA-activated PKC phosphorylation channel.

Calcium ions (Ca2+) are admitted into presynaptic nerve terminals through voltage gated calcium channels and diffuse to bind and activate the secretory vesicle discharge mechanism. Current research favors a highly ‘modal’ organization where the release sites are activated by one or a few closely apposed channels (Stanley, 1997). To fully understand the nanophysiology of transmitter release site activation, it is necessary to determine the rate of Ca2+ flux through individual channels at normal physiological external concentrations. OBJECTIVE: To explore the relationship between CaV2.2 channel conductance and external Ca2+ across the physiological range. CONCLUSION: The conductance of the CaV2.2 channel was determined across the range of 1-100 mM [Ca2+]EXT . With 2 mM [Ca2+]EXT, the conductance was determined to be 2.76 ± 0.24 pS.

Calcium ions (Ca2+) are admitted into presynaptic nerve terminals through voltage gated calcium channels and diffuse to bind and activate the secretory vesicle discharge mechanism. Current research favors a highly ‘modal’ organization where the release sites are activated by one or a few closely apposed channels (Stanley, 1997). To fully understand the nanophysiology of transmitter release site activation, it is necessary to determine the rate of Ca2+ flux through individual channels at normal physiological external concentrations. OBJECTIVE: To explore the relationship between CaV2.2 channel conductance and external Ca2+ across the physiological range. CONCLUSION: The conductance of the CaV2.2 channel was determined across the range of 1-100 mM [Ca2+]EXT . With 2 mM [Ca2+]EXT, the conductance was determined to be 2.76 ± 0.24 pS.

Bien que la régulation des canaux calciques par les récepteurs couplés aux protéines G soit connue depuis une trentaine d'année, ce n'est que récemment qu'il a été découvert que ce phénomène pouvait passer par une interaction directe entre ces deux partenaires. Les RCPGs sont les senseurs d'un grand nombre de paramètres (des simples photons aux molécules odorantes en passant par des hormones, acides aminés et nucléotides) et ils contrôlent un grand nombre de processus cellulaires en fonction de ces différents stimuli, ce qui en fait une cible thérapeutique majeure. Une de leurs cibles est l'activité des canaux calciques voltage dépendants qui est responsable d'un grand nombre de processus tels que le contrôle du potentiel de membrane, le relargage de neurotransmetteurs, la contraction musculaire ou, bien sûr, le contrôle du taux de calcium intracellulaire qui est lui-même un second messager impliqué dans de nombreuses voies de régulations.Il nous a donc paru intéressant de se pencher plus en avant sur ces interactions et de trouver une méthode nous permettant de cribler ces interactions potentielles avec des RCPG ciblés pouvant intervenir dans une thématique de contrôle de la douleur. Pour cela nous avons développé une stratégie de FRET en temps résolu utilisant les cryptates de terres rares à déactivation lente couplés aux ligands du tag SNAP comme donneurs de fluorescence, les canaux calciques étudiées étant fusionnés avec cet épitope et l'eGFP fusionnée aux RCPGs en tant qu'accepteur. Ce test nous a permis de confirmer l'interaction entre CaV2.2 et ORL1 le récepteur de la nociceptine. Nous avons ensuite cherché à caractériser plus précisément cette interaction et nous avons déterminé quelles en étaient les séquences peptidiques responsables au sein des domaines C-terminaux de ces deux protéines grâce à des expériences de GST-pull down. Nous avons synthétisé un peptide reproduisant la séquence d'interaction d'ORL1 que nous avons couplé à la séquence TAT, le rendant ainsi capable de pénétrer les membranes cellulaires. Lorsque nous ajoutons ce peptide leurre dans les expériences de TR-FRET, l'augmentation de fluorescence observée en présence de CaV2.2-SNAP et ORL1-GFP disparait totalement alors que l'ajout d'un peptide contrôle composé des mêmes acides aminés mais présentés dans le désordre n'a aucun effet. Nous avons ensuite cherché à étudier les effets de ce peptide in vivo lors d'un protocole de tolérance à la morphine étant donné que les souris K.O. pour le gène d'ORL1 sont résistantes à l'apparition de cette tolérance. Cette stratégie de découplage CaV2.2 :: ORL1 abolit complètement le phénomène de tolérance aux effets analgésiques de la morphine par une action au niveau spinal. Ce travail peut conduire à l'utilisation d'une telle approche dans une perspective thérapeutique visant à améliorer l'utilisation de morphiniques lors du traitement des douleurs chroniques. / The regulation of the calcium channels by GPCRs has been known for almost thirty years but the direction interaction between those two proteins is a recent breakthrough. GPCRs are sensors for a great number of parameters (photons, smell molecules, hormones, amino acids, nucleotides…) and they control numerous cellular functions according to those parameters making them a major target for pharmacology. One of the GPCR's targets is the calcium channel activity which is responsible for a great number of cellular processes like control of the membrane potential, neurotransmitters or hormonal secretion, muscular contraction and, of course, control of the intracellular calcium level which is a second messenger of numerous cell-regulation pathways.It appears to us that it would be interesting to study more closely those interactions and find a way to screen the GPCR/calcium channels interactions that may occur in pain regulation. We developed a strategy of time resolved FRET, using rare earth cryptate coupled to the ligand of the SNAP tag which is fused to the calcium channel as fluorescence donor and eGFP fused GRPRs as acceptors. That test confirmed the interaction between CaV2.2 and ORL1, the nociceptin receptor. We characterized more precisely the peptide sequence of the carboxy-terminal domain of the two proteins which is responsible for the interaction using GST-pull down experiments. We synthesized a peptide reproducing the ORL1 interaction sequence coupled to the TAT sequence allowing to go through the cell membranes. When we add this decoy peptide to ours TR-FRET experiments we lose all the increase of fluorescence that we see in presence of CaV2.2-SNAP and ORL1-GFP but the adding of a control peptide made of the same peptides but scrambled didn't affect the experiment. Then we look for the effects of this peptide in vivo, during a morphine tolerance protocol as it was reported that the ORL1 knock-out mice were insensitive to this phenomenon. This strategy of uncoupling CaV2.2 and ORL1 leads to a complete suppression of the tolerance to the analgesic effects of the morphine by an action at the spinal level. This work could lead to a therapeutic use of this approach which could enhance the use of morphinic compounds in treatment of chronics pains.

Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2-CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration-approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.

Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPgammaS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation.

Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPgammaS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20080号 / 医博第4173号 / 新制||医||1018(附属図書館) / 33196 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 川口 義弥, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM