Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 2. Three-dimensional solution structure

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D H-1 NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and mu O-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18 +/- 0.05 Angstrom for the backbone atoms and 1.39 +/- 0.33 Angstrom for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.

Solution structure of χ-conopeptide MrIA, a modulator of the human norepinephrine transporter

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.: Schroder, C. L; Kaye, D. M.; Adams, D. I.; Alewood, P. F.; Lewis, R. J. J Biol Client 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hypl2 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency. This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by entailing the Biopolymers editorial office at biopolymers@wiley.com (c) 2005 Wiley Periodicals, Inc.

Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli

The open channel diameter of Escherichia coli recombinant large-conductance mechanosensitive ion channels (MscL) was estimated using the model of Hille (Hille, B. 1968. Pharmacological modifications of the sodium channels of frog nerve. J. Gen. Physiol. 51:199-219)that relates the pore size to conductance. Based on the MscL conductance of 3.8 nS, and assumed pore lengths, a channel diameter of 34 to 46 Angstrom was calculated. To estimate the pore size experimentally, the effect of large organic ions on the conductance of MscL was examined. Poly-L-lysines (PLLs) with a diameter of 37 Angstrom or larger significantly reduced channel conductance, whereas spermine (similar to 15 Angstrom), PLL19 (similar to 25 Angstrom) and 1,1'-bis-(3-(1'-methyl-(4,4'-bipyridinium)-1-yl)-propyl)-4,4'-bipyridinium (similar to 30 Angstrom) had no effect. The smaller organic ions putrescine, cadaverine, spermine, and succinate all permeated the channel. We conclude that the open pore diameter of the MscL is similar to 40 Angstrom, indicating that the MscL has one of the largest channel pores yet described. This channel diameter is consistent with the proposed homohexameric model of the MscL.

Erection induced by Tx2-6 toxin of Phoneutria nigriventer spider: Expression profile of genes in the nitric oxide pathway of penile tissue of mice

The peptides Tx2-5 and Tx2-6, isolated from the whole venom of ""armed-spider"" Phoneutria nigniventer venom, are directly linked with the induction of persistent and painful erection in the penis of mammals. The erection induced by Tx2-6 has been associated with the activation of nitric oxide synthases. There is a scarcity of studies focusing on the outcome of Tx2-6 at the molecular level, by this reason we evaluated the gene profile activity of this toxin at the nitric oxide (NO) pathway. After microarray analyses on cavernous tissue of mice inoculated with Tx2-6 we found that only 10.4% (10/96) of these genes were differentially expressed, showing a limited effect of the toxin on the NO pathway. We found the genes sparc, ednrb, junb, cdkn1a, bcl2, ccl5, abcc1 over-expressed and the genes sod1, s100a10 and fth1 under-expressed after inoculation of Tx2-6. The differential expressions of sparc and ednrb genes were further confirmed using real-time PCR. Interestingly, ednrb activates the L-arginine/NO/cGMP pathway that is involved in the relaxation of the cavernous body. Therefore the priapism induced by Tx2-6 is a consequence of a highly specific interference of this neurotoxin with the NO pathway. (C) 2009 Elsevier Ltd. All rights reserved.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

Introduction. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na+) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. Aim. We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. Main Outcome Measures. PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. Methods. Rat cavernosal strips were incubated with bretylium (3 x 10-5 M) and contracted with phenylephrine (PE; 10-5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10-8 M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10-6 M), a muscarinic receptor antagonist, N-type Ca2+ channel blockers (omega-conotoxin GVIA, 10-6 M) and sildenafil (3 x 10-8 M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. Results. Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca2+ channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. Conclusion. We show that PnTx2-6 slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction. Nunes KP, Cordeiro MN, Richardson M, Borges MN, Diniz SOF, Cardoso VN, Tostes R, De Lima ME, Webb RC, and Leite R. Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue. J Sex Med 2010;7:3879-3888.

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Activation of the CFTR Cl- channel inhibits epithelial Na+ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis trans-membrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl- currents. Similar to CFTR, ClC-0 Cl- currents also inhibit ENaC, as well as high extracellular Na+ and Cl- in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl-.

The changing face of ciguatera

Ciguatera is a global disease caused by the consumption of certain warm-water fish (ciguateric fish) that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. Symptoms of ciguatera include a range of gastrointestinal, neurological and cardiovascular disturbances. This review examines progress in our understanding of ciguatera from the work of Banner in the late 1950s to the present. Similarities and differences in ciguatera in the Pacific Ocean, Indian Ocean and Caribbean Sea are highlighted, and future research directions are suggested. (C) 2000 Elsevier Science Ltd. All rights reserved.

Two new classes of conopeptides inhibit the alpha 1-adrenoceptor and noradrenaline transporter

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha (1)-adrenoceptors (rho -TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi -MrIA and chi -MrIB from the mollusk-hunting C. marmoreus). rho -TIA and chi -MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le résultat d'une lésion ou d'un dysfonctionnement du système nerveux. Les symptômes qui suivent la douleur neuropathique sont sévères et leur traitement inefficace. Une meilleure approche thérapeutique peut être proposée en se basant sur les mécanismes pathologiques de la douleur neuropathique. Lors d'une lésion périphérique une douleur neuropathique peut se développer et affecter le territoire des nerfs lésés mais aussi les territoires adjacents des nerfs non-lésés. Une hyperexcitabilité des neurones apparaît au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle épinière. Le but de ce travail consiste à mettre en évidence les modifications moléculaires associées aux nocicepteurs lésés et non-lésés au niveau des DRG et des laminae I et II de la corne dorsale, là où l'information nociceptive est intégrée. Pour étudier les changements moléculaires liés à la douleur neuropathique nous utilisons le modèle animal d'épargne du nerf sural (spared nerve injury model, SNI) une semaine après la lésion. Pour la sélection du tissu d'intérêt nous avons employé la technique de la microdissection au laser, afin de sélectionner une sous-population spécifique de cellules (notamment les nocicepteurs lésés ou non-lésés) mais également de prélever le tissu correspondant dans les laminae superficielles. Ce travail est couplé à l'analyse à large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons étudié les courants électriques et les propriétés biophysiques des canaux sodiques (Na,,ls) dans les neurones lésés et non-lésés des DRG. Aussi bien dans le système nerveux périphérique, entre les neurones lésés et non-lésés, qu'au niveau central avec les aires recevant les projections des nocicepteurs lésés ou non-lésés, l'analyse du transcriptome montre des différences de profil d'expression. En effet, nous avons constaté des changements transcriptionnels importants dans les nocicepteurs lésés (1561 gènes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 gènes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-lésés (60 gènes), mais important dans leurs laminae de projection (459 gènes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observé que plusieurs processus biologiques sont modifiés. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les mécanismes de réponse au stress sont affectés. Par contre dans les neurones non-lésés seuls les processus biologiques liés au métabolisme et au développement sont modifiés. Au niveau de la corne dorsale de la moelle, nous avons observé des modifications importantes des processus immuno-inflammatoires dans l'aire affectée par les nerfs lésés et des changements associés à l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-lésés. L'analyse approfondie des canaux sodiques a démontré plusieurs changements d'expression, principalement dans les neurones lésés. Les analyses fonctionnelles n'indiquent aucune différence entre les densités de courant tétrodotoxine-sensible (TTX-S) dans les neurones lésés et non-lésés même si les niveaux d'expression des ARNm des sous-unités TTX-S sont modifiés dans les neurones lésés. L'inactivation basale dépendante du voltage des canaux tétrodotoxine-insensible (TTX-R) est déplacée vers des potentiels positifs dans les cellules lésées et non-lésées. En revanche la vitesse de récupération des courants TTX-S et TTX-R après inactivation est accélérée dans les neurones lésés. Ces changements pourraient être à l'origine de l'altération de l'activité électrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En résumé, ces résultats suggèrent l'existence de mécanismes différenciés affectant les neurones lésés et les neurones adjacents non-lésés lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle épinière qui surviennent après lésion sont probablement intégrés différemment selon la perception de signaux des neurones périphériques lésés ou non-lésés. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqués dans la genèse et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied functionál properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

Characterization of Nedd4-2 ubiquitin ligase expression in experimental neuropathic pain

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs). The ubiquitin ligase Nedd4-2 regulates sodium channels and we have previously demonstrated in expression systems that this protein decreases the Nav1.7 current. Nav1.7 is the most abundant VGSC in dorsal root ganglion (DRG) and is a major contributor to pain perception. We hypothesize that Nedd4-2 modulates Nav1.7 channel density at the neuronal cell membrane and the goal of this present experiment is to characterize Nav1.7 and Nedd4-2 expression in the context of neuropathic pain. Methods: Biotinylation, Western Blot and Immunohistochemistry experiments for Nav1.7 and Nedd4-2 were performed in HEK transfected cells or in rodent DRGs 7 days after SNI surgery. We used antibodies against Nedd4-2 and Nav1.7 and several comarkers of DRG neurons (Peripherin for nociceptors, NF-200 for large myelinated cells, ATF3 for injured neurons). Data are expressed in proportion of positive cells (%) and protein signal ratio } SEM, n = 3-4 in each condition. Results: In HEK293 cells, upon co-expression of Nedd4-2, a decrease of 50% of Nav1.7 signal at the membrane is demonstrated (p ≤0.005). Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 } 1.2%) versus sham group (43.4 } 3.5%) (p <0.005). Nedd4-2 is mainly colocalized with markers of small neurons and almost absent in large neurons. In addition, Nedd4-2 is predominantly decreased in injured ATF3 positive cells. Conclusion: Our results indicate that Nedd4-2 decreases Nav1.7 channels and currents at the cell membrane and that it is mainly expressed in nociceptors and downregulated after nerve injury. Taken together, our data suggest that the reduction of Nedd4-2, after nerve injury, modulates Nav1.7 activity and can contribute to neuropathic pain. We will further try to restore a normal level of Nedd4.2 via a gene therapy approach with viral vectors in order to soothe symptoms of neuropathic pain.

Altered distribution of juxtaparanodal kv1.2 subunits mediates peripheral nerve hyperexcitability in type 2 diabetes mellitus.

Peripheral nerve hyperexcitability (PNH) is one of the distal peripheral neuropathy phenotypes often present in patients affected by type 2 diabetes mellitus (T2DM). Through in vivo and ex vivo electrophysiological recordings in db/db mice, a model of T2DM, we observed that, in addition to reduced nerve conduction velocity, db/db mice also develop PNH. By using pharmacological inhibitors, we demonstrated that the PNH is mediated by the decreased activity of K(v)1-channels. In agreement with these data, we observed that the diabetic condition led to a reduced presence of the K(v)1.2-subunits in juxtaparanodal regions of peripheral nerves in db/db mice and in nerve biopsies from T2DM patients. Together, these observations indicate that the T2DM condition leads to potassium channel-mediated PNH, thus identifying them as a potential drug target to treat some of the DPN related symptoms.