Phoneutria nigriventer toxin 1: a novel statedependent inhibitor of neuronal sodium channels that interacts with mu-conotoxin binding sites

ABSTRACT A toxin was purified to homogeneity from the venom of the South American armed spider Phoneutria nigriventer and found to have a molecular mass of 8600 Da and a C-terminally amidated glycine residue. It appears to be identical to Toxin 1 (Tx1) isolated previously from this venom. Tx1 reversibly inhibited sodium currents in Chinese hamster ovary cells expressing recombinant sodium (Na v 1.2) channels without affecting their fast biophysical properties. The kinetics of inhibition of peak sodium current varied with membrane potential, with on-rates increasing and off-rates decreasing with more depolarized holding potentials in the Ϫ100 to Ϫ50 mV range. Thus, the apparent affinity of Tx1 for the channel increases as the membrane is depolarized. A mono[ 125 I]iodo-Tx1 derivative displayed high-affinity binding to a single class of sites (K D ϭ 80 pM, B max ϭ 0.43 pmol/mg protein) in rat brain membranes. Solubilized binding sites were immunoprecipitated by antibodies directed against a conserved motif in sodium channel ␣ subunits. 125 I-Tx1 binding was competitively displaced by conotoxin GIIIB (IC 50 ϭ 0.5 M) but not by 1 M tetrodotoxin. However, the inhibition of 125 I-Tx1 binding by conotoxin GIIIB was abrogated in the presence of tetrodotoxin (1 M). Patch-clamp and binding data indicate that P. nigriventer Tx1 is a novel, state-dependent sodium-channel blocker that binds to a site in proximity to pharmacological site 1, overlapping conotoxin but not tetrodotoxin binding sites

Régulation différentielle du canal sodium dépendant du potentiel Nav.1.1 par la calmoduline et le calcium (une cible potentielle de mutations pro-épileptiques)

L isoforme Nav1.1 du canal sodium dépendant du potentiel est localisé dans le compartiment somato-dendritique mais également dans le segment initial et le long de l axone des interneurones à parvalbumine de l hippocampe et du cortex. L identification de nombreuses mutations de ce canal impliquées dans des syndromes épileptiques de sévérité variable souligne son rôle important dans le contrôle de l excitabilité. Nous avons mis en évidence que Nav1.1 est régulé de manière différentielle par la calmoduline (CaM) et le calcium dans la lignée stable HEK-Nav1.1. La CaM augmente l amplitude du courant sodium uniquement en présence de calcium. Elle déplace également la courbe d activation vers des potentiels hyperpolarisés et accélère l inactivation indépendamment du calcium. Nos travaux suggèrent que le lobe C de la CaM interagit avec la partie proximale du motif IQ dans le domaine C-terminal même en l absence de calcium et que l intégrité du motif IQ est indispensable à cette liaison. En présence de calcium les deux lobes de la CaM interagissent avec le domaine C-terminal du canal avec un site de fixation pour le lobe N dans la partie distale du motif IQ. Contrairement à la CaM, le calcium ne module pas la densité du courant Nav1.1 et déplace positivement la courbe d inactivation. Nous avons également observé une régulation opposée à celle de la CaM : un déplacement positif de l activation qui est contrecarré lorsque le canal est coexprimé avec la CaM ainsi qu un ralentissement de l inactivation. Cette modulation pourrait être médiée par la fixation directe du calcium sur un motif EF du domaine C-terminal que nous avons mise en évidence. Des mutations du canal identifiées chez des patients épileptiques et localisées dans ce domaine C-terminal pourraient perturber la régulation du canal par ces deux effecteurs. La mutation W1801G abolit la liaison calcium-indépendante de la CaM et perturbe le repliement du domaine C-terminal. D autres mutations augmentent la fixation de la CaM en présence de calcium et la diminuent en l absence de calcium. Nous avons construit le canal Nav1.1 étiqueté dans une boucle extracellulaire pour pouvoir détecter les cellules exprimant le canal et étudier d éventuelles perturbations de son transport à la membrane. L introduction d une étiquette dans le premier domaine extracellulaire ne permet pas de détecter l expression de surface du canal, mais donne des courants présentant les mêmes caractéristiques biophysiques que le canal non étiqueté. Le couplage N-terminal de la GFP au canal permet de visualiser les cellules transfectées. Le canal GFP-Nav1.1 exprime des courants dans la lignée tsA201. L introduction de la résistance à la Tetrodotoxine (TTX) par mutation ponctuelle conserve environ 80% du courant sodium après application de 5 M TTX. L expression du canal GFP-Nav1.1-TTXr sera testée dans différents types neuronaux en présence de TTX pour inhiber les conductances sodiques endogènes et plus particulièrement dans des neurones excitateurs et inhibiteurs afin de : 1) déterminer si la régulation de Nav1.1 par la CaM et le calcium persiste et si elle varie suivant le type neuronal, 2) étudier l impact des mutations sur le fonctionnement et les régulations du canal ainsi que sur l excitabilité neuronale.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

Axon Physiology

International audienceAxons are generally considered as reliable transmission cables in which stable propagation occurs once an action potential is generated. Axon dysfunction occupies a central position in many inherited and acquired neurological disorders that affect both peripheral and central neurons. Recent findings suggest that the functional and computational repertoire of the axon is much richer than traditionally thought. Beyond classical axonal propagation, intrinsic voltage-gated ionic currents together with the geometrical properties of the axon determine several complex operations that not only control signal processing in brain circuits but also neuronal timing and synaptic efficacy. Recent evidence for the implication of these forms of axonal computation in the short-term dynamics of neuronal communication is discussed. Finally, we review how neuronal activity regulates both axon morphology and axonal function on a long-term time scale during development and adulthood

Kv3.1b is a novel component of CNS nodes

We herein demonstrate that Kv3.1b subunits are present at nodes of Ranvier in the CNS of both rats and mice. Kv3.1b colocalizes with voltage-gated Na � channels in a subset of nodes in the spinal cord, particularly those of large myelinated axons. Kv3.1b is abundantly expressed in the gray matter of the spinal cord, but does not colocalize with Na � channels in initial segments. In the PNS, few nodes are Kv3.1b-positive. During the development of the CNS, Kv3.1b clustering at nodes occurs later than that of Na � channels, but precedes the juxtaparanodal clustering of Kv1.2. Moreover, in myelin-deficient rats, which have severe CNS dysmyelination, node-like clusters of Kv3.1b and Na � channels are observed even in regions devoid of oligodendrocytes. Ankyrin G coimmunoprecipitates Kv3.1b in vivo, indicating that these two proteins may interact in the CNS at nodes. 4-Aminopyridine, a K � channel blocker, broadened the compound action potential recorded from adult rat optic nerve and spinal cord, but not from the sciatic nerve. These effects were also observed in Kv3.1-deficient mice. In conclusion, Kv3.1b is the first K � channel subunit to be identified in CNS nodes; but Kv3.1b does not account for the effects of 4-aminopyridine on central myelinated tracts. Key words: Shaw; potassium channels; oligodendrocyte; Schwann cells; myelin; multiple sclerosi

A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels

International audienceMutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2 A294V). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2 A294G) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2 A294V alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2 A294V channel alone. Although the total Kv7.2 A294V expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' hetero-zygous genotype i.e., Kv7.2 A294V /Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2 A294V , the current density of homomeric Kv7.2 A294G was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2 A294G /Kv7.2/Kv7.3 and Kv7.2 A294G /Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a dominant-negative effect on wild-type subunits but alters the preferential axonal targeting of heteromeric Kv7 channels. Our data suggest that the disease severity is not necessarily a consequence of a strong inhibition of M current and that additional mechanisms such as abnormal subcellular distribution of Kv7 channels could be determinant

A highly virulent variant of HIV-1 circulating in the Netherlands

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence