Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)]; transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20 mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH. (c) 2011 Elsevier Ltd. All rights reserved.

Expression and function of colonic epithelial K(v)LQT1 K+ channels

1. K(V)LQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential Defects in ion channels have been demonstrated in cardiac arrhythmia. This channel is inhibited potently by the chromanol 293B, The same compound has been shown to block cAMP-dependent electrolyte secretion in rat and human colon, Therefore, it was suggested that a K+ channel similar to K(V)LQT1 is expressed in the colonic epithelium. 2, In the present paper, expression of K(V)LQT1 and its function in colonic epithelial cells is described. Reverse transcription-polymerase chain reaction analysis of rat colonic mucosa demonstrated expression of K(V)LQT1 in both crypt cells and surface epithelium. When expressed in Xenopus oocytes, K(V)LQT1 induced a typical delayed activated K+ current. 3, As demonstrated, the channel activity could be further activated by increases in intracellular cAMP. These and other data support the concept that K(V)LQT1 is forming a component of the basolateral cAMP-activated Kf conductance in the colonic epithelium.

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium.

The SV channel encoded by the TPC1 gene represents a Ca(2+)- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1, named fou2 for fatty acid oxygenation upregulated 2, results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca2+ signals and cytosolic Ca2+ is required for SV channel function, we here studied the Ca(2+)-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca2+ sensitivity and Ca2+ impermeability. K+ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca2+ reached the 0.1-mm level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca2+ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca2+. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca(2+) before SV channel activity vanishes and K(+) homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca2+ signals resulting in overproduction of jasmonate.

Ca(2+) signaling by T-type Ca(2+) channels in neurons.

Among the major families of voltage-gated Ca(2+) channels, the low-voltage-activated channels formed by the Ca(v)3 subunits, referred to as T-type Ca(2+) channels, have recently gained increased interest in terms of the intracellular Ca(2+) signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca(2+) channels act as an important Ca(2+) source in a wide range of neuronal cell types. The work is focused on T-type Ca(2+) channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca(2+) entering through T-type Ca(2+) channels have been described. Notably, Ca(2+) influx through T-type Ca(2+) channels is the predominant Ca(2+) source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca(2+) signaling through T-type Ca(2+) channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions.

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.

Guard cell-specific calcium sensitivity of high density and activity SV/TPC1 channels.

The slow vacuolar (SV) channel, a Ca2+-regulated vacuolar cation conductance channel, in Arabidopsis thaliana is encoded by the single-copy gene AtTPC1. Although loss-of-function tpc1 mutants were reported to exhibit a stoma phenotype, knowledge about the underlying guard cell-specific features of SV/TPC1 channels is still lacking. Here we demonstrate that TPC1 transcripts and SV current density in guard cells were much more pronounced than in mesophyll cells. Furthermore, the SV channel in motor cells exhibited a higher cytosolic Ca2+ sensitivity than in mesophyll cells. These distinct features of the guard cell SV channel therefore probably account for the published stomatal phenotype of tpc1-2.

The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane.

Land plants are prone to strong thermal variations and must therefore sense early moderate temperature increments to induce appropriate cellular defenses, such as molecular chaperones, in anticipation of upcoming noxious temperatures. To investigate how plants perceive mild changes in ambient temperature, we monitored in recombinant lines of the moss Physcomitrella patens the activation of a heat-inducible promoter, the integrity of a thermolabile enzyme, and the fluctuations of cytoplasmic calcium. Mild temperature increments, or isothermal treatments with membrane fluidizers or Hsp90 inhibitors, induced a heat shock response (HSR) that critically depended on a preceding Ca(2+) transient through the plasma membrane. Electrophysiological experiments revealed the presence of a Ca(2+)-permeable channel in the plasma membrane that is transiently activated by mild temperature increments or chemical perturbations of membrane fluidity. The amplitude of the Ca(2+) influx during the first minutes of a temperature stress modulated the intensity of the HSR, and Ca(2+) channel blockers prevented HSR and the onset of thermotolerance. Our data suggest that early sensing of mild temperature increments occurs at the plasma membrane of plant cells independently from cytosolic protein unfolding. The heat signal is translated into an effective HSR by way of a specific membrane-regulated Ca(2+) influx, leading to thermotolerance.

Architectural and Functional Similarities between Trimeric ATP-Gated P2X Receptors and Acid-Sensing Ion Channels

ATP-gated P2X receptors and acid-sensing ion channels are two distinct ligand-gated ion channels that assemble into trimers. They are involved in many important physiological functions such as pain sensation and are recognized as important therapeutic targets. They have unrelated primary structures and respond to different ligands (ATP and protons) and are thus considered as two different ion channels. As a consequence, comparisons of the biophysical properties and underlying mechanisms have only been rarely made between these two channels. However, the recent determination of their molecular structures by X-ray crystallography has revealed unexpected parallels in the architecture of the two pores, providing a basis for possible functional analogies. In this review, we analyze the structural and functional similarities that are shared by these trimeric ion channels, and we outline key unanswered questions that, if addressed experimentally, may help us to elucidate how two unrelated ion channels have adopted a similar fold of the pore.

Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acide-Sensing Ion Channels)

RESUME: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse Les canaux sodiques ASICs (Acid-Sensing Ion Channels) participent à la signalisation neuronale dans les systèmes nerveux périphérique et central. Ces canaux non voltage dépendants sont impliqués dans l'apprentissage, l'expression de la peur, la neurodégénération consécutive à une attaque cérébrale et la douleur. Les bases moléculaires sous-tendant leur activité ne sont pas encore totalement comprises. Ces canaux sont activés par une acidification du milieu extracellulaire et régulés, entre autres, par des ions tels que le Ca2+, le Zn2+ et le CI". La cristallisation de ASIC inactivé a été publiée. Le canal est un trimére de sous-unités identiques ou homologues. Chaque sous-unité a été décrite en analogie à un avant bras, un poignet et une main constituée d'un pouce, d'un doigt, d'une articulation, une boule β et une paume. Nous avons appliqué une approche bioinformatique systématique pour identifier les pH senseurs putatifs de ASICIa. Le rôle des pH senseurs putatifs a été testé par mutagénèse dirigée et des modifications chimiques combinées à une analyse fonctionnelle afin de comprendre comment les variations de ρ H ouvrent ces canaux. Les pH senseurs sont des acides aspartiques et glutamiques éparpillés sur la boucle extracellulaire suggérant que les changements de pH contrôlent l'activation et l'inactivation de ASIC en (dé)protonant ces résidus en divers endroits de la protéine. Par exemple lors de l'activation, la protonation des résidus à l'interface entre le pouce, la boule β et le doigt d'une même sous-unité induit un mouvement du pouce vers la bouie β et le doigt. De même lors de l'inactivation du canal les paumes des trois sous-unités formant une cavité se rapprochent. D'après notre approche bioinformatique, aucune histidine n'est impliquée dans la détection des variations de pH extracellulaire c'est-à-dire qu'aucune histidine ne serait un pH-senseur. Deux histidines de ASIC2a lient le Zn2+ et modifient l'affinité apparente du canal pour les protons. Une seule des deux est conservée parmi tous les ASICs, hASICIa H163. Elle forme un réseau de liaison hydrogène avec ses voisins conservés. L'étude détaillée de ce domaine, Pinterzone, montre son importance dans l'expression fonctionnelle des canaux. La perturbation de ce réseau par l'introduction d'un résidu hydrophobe (cystéine) par mutagénèse dirigée diminue l'expression du canal à la membrane plasmique. La modification des cystéines introduites par des réactifs spécifiques aux groupements sulfhydryle inhibe les canaux mutés en diminuant leur probabilité d'ouverture. Ces travaux décrivent les effets de l'acidification du milieu extracellulaire sur les canaux ASICs. ABSTRACT: Study of pH-dependent activation and inactivation of ASIC channels Benoîte BARGETON, Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1G05 Lausanne, Switzerland The ASIC (Acid-Sensing Ion Channels) sodium channels are involved in neuronal signaling in the central and peripheral nervous system. These non-voltage-gated channels are involved in learning, the expression of fear, neurodegeneration after ischemia and pain sensation. The molecular bases underlying their activity are not yet fully understood. ASICs are activated by extracellular acidification and regulated, eg by ions such as Ca2+, the Zn2+ and CI". The crystallization of inactivated ASIC has been published. The channel is a trimer of identical or homologous subunits. Each subunit has been described in analogy to a forearm, wrist and hand consisting of a thumb, a finger, a knuckle, a β-ball and a palm. We applied a systematic computational approach to identify putative pH sensor(s) of ASICIa. The role of putative pH sensors has been tested by site-directed mutagenesis and chemical modification combined with functional analysis in order to understand how changes in pH open these channels. The pH sensors are aspartic and glutamic acids distributed throughout the extracellular loop, suggesting that changes in pH control activation and inactivation of ASIC by protonation / deprotonation of many residues in different parts of the protein. During activation the protonation of various residues at the interface between the finger, the thumb and the β-ball induces the movement of the thumb toward the finger and the β-ball. During inactivation of the channel the palms of the three subunits forming a cavity approach each other. No histidine has been shown to be involved in extracellular pH changes detection, i.e. no histidine is a pH- sensor. Two histidines of ASIC2 bind Zn2+ and alter the apparent affinity of channel for protons. Only one of the two His is conserved among all ASICs, hASICIa H163. This residue is part of a network of hydrogen bonding with its conserved neighbors. The detailed study of this area, the interzone, shows its importance in the functional expression of ASICs. Disturbance of this network by the introduction of hydrophobic residues decreases the cell surface channel expression. Chemical modification of the introduced cysteines by thiol reactive compounds inhibits the mutated channels by a reduction of their open probability. These studies describe the effects of extracellular acidification on ASICs. RESUME GRAND PUBLIC: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse La transmission synaptique est un processus chimique entre deux neurones impliquant des neurotransmetteurs et leurs récepteurs. Un dysfonctionnement de certains types de synapses est à l'origine de beaucoup de troubles nerveux, tels que certaine forme d'épilepsie et de l'attention. Les récepteurs des neurotransmetteurs sont de très bonnes cibles thérapeutiques dans de nombreuses neuropathologies. Les canaux ASICs sont impliqués dans la neurodégénération consécutive à une attaque cérébrale et les bloquer pourraient permettre aux patients d'avoir moins de séquelles. Les canaux ASICs sont des détecteurs de l'acidité qui apparaît lors de situations pathologiques comme l'ischémie et l'inflammation. Ces canaux sont également impliqués dans des douleurs. Cibler spécifiquement ces canaux permettrait d'avoir de nouveaux outils thérapeutiques car à l'heure actuelle l'inhibiteur de choix, l'amiloride, bloque beaucoup d'autres canaux empêchant son utilisation pour bloquer les ASICs. C'est pourquoi il faut connaître et comprendre les bases moléculaires du fonctionnement de ces récepteurs. Les ASICs formés de trois sous-unités détectent les variations de l'acidité puis s'ouvrent transitoirement pour laisser entrer des ions chargés positivement dans la cellule ce qui active la signalisation neuronale. Afin de comprendre les bases moléculaires de l'activité des ASICs nous avons déterminé les sites de liaison des protons (pH-senseurs), ligands naturels des ASICs et décrit une zone importante pour l'expression fonctionnelle de ces canaux. Grâce à une validation systématique de résultats obtenus en collaboration avec l'Institut Suisse de Bioinformatique, nous avons décrit les pH-senseurs de ASICIa. Ces résultats, combinés à ceux d'autres groupes de recherche, nous ont permis de mieux comprendre comment les ASICs sont ouverts par une acidification du milieu extracellulaire. Une seconde étude souligne le rôle structural crucial d'une région conservée parmi tous les canaux ASICs : y toucher c'est diminuer l'activité de la protéine. Ce domaine permet l'harmonisation des changements dus à l'acidification du milieu extracellulaire au sein d'une même sous-unité c'est-à-dire qu'elle participe à l'induction de l'inactivation due à l'activation du canal Cette étude décrit donc quelle région de la protéine atteindre pour la bloquer efficacement en faisant une cible thérapeutique de choix.

Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons.

Extracellular acidification has been shown to generate action potentials (APs) in several types of neurons. In this study, we investigated the role of acid-sensing ion channels (ASICs) in acid-induced AP generation in brain neurons. ASICs are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family and are transiently activated by a rapid drop in extracellular pH. We compared the pharmacological and biophysical properties of acid-induced AP generation with those of ASIC currents in cultured hippocampal neurons. Our results show that acid-induced AP generation in these neurons is essentially due to ASIC activation. We demonstrate for the first time that the probability of inducing APs correlates with current entry through ASICs. We also show that ASIC activation in combination with other excitatory stimuli can either facilitate AP generation or inhibit AP bursts, depending on the conditions. ASIC-mediated generation and modulation of APs can be induced by extracellular pH changes from 7.4 to slightly <7. Such local extracellular pH values may be reached by pH fluctuations due to normal neuronal activity. Furthermore, in the plasma membrane, ASICs are localized in close proximity to voltage-gated Na(+) and K(+) channels, providing the conditions necessary for the transduction of local pH changes into electrical signals.

Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone

The vasorelaxing activity of rotundifolone (ROT), a major constituent (63.5%) of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g). In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 µg/ml) inhibited the contractile effects of 1 µM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means ± SEM = 184 ± 6, 185 ± 3 and 188 ± 19 µg/ml, N = 6, respectively). In aortic rings pre-contracted with 1 µM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 ± 7 µg/ml, N = 6). Furthermore, ROT inhibited (pD2 = 6.04, N = 6) the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 µM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM). In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a) voltage-dependent Ca² channels, and b) intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

β-Citronellol, an alcoholic monoterpene with inhibitory properties on the contractility of rat trachea

β-Citronellol is an alcoholic monoterpene found in essential oils such Cymbopogon citratus (a plant with antihypertensive properties). β-Citronellol can act against pathogenic microorganisms that affect airways and, in virtue of the popular use of β-citronellol-enriched essential oils in aromatherapy, we assessed its pharmacologic effects on the contractility of rat trachea. Contractions of isolated tracheal rings were recorded isometrically through a force transducer connected to a data-acquisition device. β-Citronellol relaxed sustained contractions induced by acetylcholine or high extracellular potassium, but half-maximal inhibitory concentrations (IC50) for K+-elicited stimuli were smaller than those for cholinergic contractions. It also inhibited contractions induced by electrical field stimulation or sodium orthovanadate with pharmacologic potency equivalent to that seen against acetylcholine-induced contractions. When contractions were evoked by selective recruitment of Ca2+ from the extracellular medium, β-citronellol preferentially inhibited contractions that involved voltage-operated (but not receptor-operated) pathways. β-Citronellol (but not verapamil) inhibited contractions induced by restoration of external Ca2+ levels after depleting internal Ca2+ stores with the concomitant presence of thapsigargin and recurrent challenge with acetylcholine. Treatment of tracheal rings with L-NAME, indomethacin or tetraethylammonium did not change the relaxing effects of β-citronellol. Inhibition of transient receptor potential vanilloid subtype 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) receptors with selective antagonists caused no change in the effects of β-citronellol. In conclusion, β-citronellol exerted inhibitory effects on rat tracheal rings, with predominant effects on contractions that recruit Ca2+ inflow towards the cytosol by voltage-gated pathways, whereas it appears less active against contractions elicited by receptor-operated Ca2+ channels.

Couplage entre les régions IIS4S5 et IIIS6 lors de l’activation du canal calcique CaV3.2

Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2.

Effects of neuropathy on high-voltage-activated Ca2+ current in sensory neurones

Voltage-dependent Ca2+ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the CaV2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca2+ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from CaV2.3(−/−) knock-out and wild-type (WT) mice. DRG neurones from CaV2.3(−/−) mice had significantly reduced sensitivity to SNX-482 versusWTmice. DRGs from CaV2.3(−/−) mice also had increased sensitivity to the CaV2.2 VDCC blocker -conotoxin. In WT mice, PSNL caused a significant increase in -conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In CaV2.3(−/−) mice, PSNL caused a significant reduction in -conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca2+ current were not accompanied by effects on voltagedependence of activation in either CaV2.3(−/−) or WT mice. These data suggest that CaV2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca2+ current in these cells. In WT mice, PSNL caused adaptive changes in CaV2.2- and CaV2.3-mediated Ca2+ currents, supporting roles for these VDCCs in nociception during neuropathy. In CaV2.3(−/−) mice, PSNL-induced changes in CaV1 and CaV2.2 Ca2+ current, consistent with alternative adaptive mechanisms occurring in the absence of CaV2.3 subunits.

Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis ( 2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a > 1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha 2 delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S(35)methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.