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.

Metal-Nitride-oxide-semiconductor light-emitting devices for general lighting.

The potential for application of silicon nitride-based light sources to general lighting is reported. The mechanism of current injection and transport in silicon nitride layers and silicon oxide tunnel layers is determined by electro-optical characterization of both bi- and tri-layers. It is shown that red luminescence is due to bipolar injection by direct tunneling, whereas Poole-Frenkel ionization is responsible for blue-green emission. The emission appears warm white to the eye, and the technology has potential for large-area lighting devices. A photometric study, including color rendering, color quality and luminous efficacy of radiation, measured under various AC excitation conditions, is given for a spectrum deemed promising for lighting. A correlated color temperature of 4800K was obtained using a 35% duty cycle of the AC excitation signal. Under these conditions, values for general color rendering index of 93 and luminous efficacy of radiation of 112 lm/W are demonstrated. This proof of concept demonstrates that mature silicon technology, which is extendable to lowcost, large-area lamps, can be used for general lighting purposes. Once the external quantum efficiency is improved to exceed 10%, this technique could be competitive with other energy-efficient solid-state lighting options. ©2011 Optical Society of America OCIS codes: (230.2090) Electro-optical devices; (150.2950) Illumination.

The effects of electron-hole separation on the photoconductivity of individual metal oxide nanowires

The responses of individual ZnO nanowires to UV light demonstrate that the persistent photoconductivity (PPC) state is directly related to the electron¿hole separation near the surface. Our results demonstrate that the electrical transport in these nanomaterials is influenced by the surface in two different ways. On the one hand, the effective mobility and the density of free carriers are determined by recombination mechanisms assisted by the oxidizing molecules in air. This phenomenon can also be blocked by surface passivation. On the other hand, the surface built-in potential separates the photogenerated electron¿hole pairs and accumulates holes at the surface. After illumination, the charge separation makes the electron¿hole recombination difficult and originates PPC. This effect is quickly reverted after increasing either the probing current (self-heating by Joule dissipation) or the oxygen content in air (favouring the surface recombination mechanisms). The model for PPC in individual nanowires presented here illustrates the intrinsic potential of metal oxide nanowires to develop optoelectronic devices or optochemical sensors with better and new performances.

Configurational statistical model for the damaged structure of silicon oxide after ion implantation

A configurational model for silicon oxide damaged after a high-dose ion implantation of a nonreactive species is presented. Based on statistics of silicon-centered tetrahedra, the model takes into account not only the closest environment of a given silicon atom, but also the second neighborhood, so it is specified whether the oxygen attached to one given silicon is bridging two tetrahedra or not. The frequencies and intensities of infrared vibrational bands have been calculated by averaging over the distributions and these results are in agreement with the ones obtained from infrared experimental spectra. Likewise, the chemical shifts obtained from x-ray photoelectron spectroscopy (XPS) analysis are similar to the reported values for the charge-transfer model of SiOx compounds.

Raman microstructural analysis of silicon-on-insulator formed by high dose oxygen ion implantation: As-implanted structures

A microstructural analysis of silicon-on-insulator samples obtained by high dose oxygen ion implantation was performed by Raman scattering. The samples analyzed were obtained under different conditions thus leading to different concentrations of defects in the top Si layer. The samples were implanted with the surface covered with SiO2 capping layers of different thicknesses. The spectra measured from the as-implanted samples were fitted to a correlation length model taking into account the possible presence of stress effects in the spectra. This allowed quantification of both disorder effects, which are determined by structural defects, and residual stress in the top Si layer before annealing. These data were correlated to the density of dislocations remaining in the layer after annealing. The analysis performed corroborates the existence of two mechanisms that generate defects in the top Si layer that are related to surface conditions during implantation and the proximity of the top Si/buried oxide layer interface to the surface before annealing.

Radionuclides and heavy metal contents in phosphogypsum samples in comparison to cerrado soils

Phosphogysum (PG) or agricultural gypsum, a solid waste from the phosphate fertilizer industry, is used as soil amendment, especially on soils in the Cerrado region, in Brazil. This material may however contain natural radionuclides and metals which can be transferred to soils, plants and water sources. This paper presents and discusses the results of physical and chemical analyses that characterized samples of PG and compares them to the results found in two typical soils of the Cerrado, a clayey and sandy one. These analyses included: solid waste classification, evaluation of organic matter content and of P, K, Ca, Mg, and Al concentrations and of the mineralogical composition. Natural radionuclides and metal concentrations in PG and soil samples were also measured. Phosphogypsum was classified as Class II A - Not Dangerous, Not Inert, Not Corrosive and Not Reactive. The organic matter content in the soil samples was low and potential acidity high. In the mean, the specific 226Ra activity in the phosphogypsum samples (252 Bq kg-1) was below the maximum level recommended by USEPA, which is 370 Bq kg-1 for agricultural use. In addition, this study verified that natural radionuclides and metals concentrations in PG were lower than in the clayey Oxisol of Sete Lagoas, Minas Gerais, Brazil. These results indicated that the application of phosphogypsum as soil amendment in agriculture would not cause a significant impact on the environment.

Surface collective oscillations of metal clusters and spheres: Random-phase-approximation sum-rules approach

Using the once and thrice energy-weighted moments of the random-phase-approximation strength function, we have derived compact expressions for the average energy of surface collective oscillations of clusters and spheres of metal atoms. The L=0 volume mode has also been studied. We have carried out quantal and semiclassical calculations for Na and Ag systems in the spherical-jellium approximation. We present a rather thorough discussion of surface diffuseness and quantal size effects on the resonance energies.

Static dipole polarizability of alkali-metal clusters: Electronic exchange and correlation effects

Nonlocal approximations for the electronic exchange and correlation effects are used to compute, within density-functional theory, the polarizability and surface-plasma frequencies of small jelliumlike alkali-metal clusters. The results are compared with those obtained using the local-density approximation and with available experimental data, showing the relevance of these effects in obtaining an accurate description of the surface response of metallic clusters.

Barrier for the reaction X20+ + X20+ -> X402+ in alkali-metal clusters related to electron density at the bond midpoint of the supermolecule (X20+)2

Using the extended Thomas-Fermi version of density-functional theory (DFT), calculations are presented for the barrier for the reaction Na20++Na20+¿Na402+. The deviation from the simple Coulomb barrier is shown to be proportional to the electron density at the bond midpoint of the supermolecule (Na20+)2. An extension of conventional quantum-chemical studies of homonuclear diatomic molecular ions is then effected to apply to the supermolecular ions of the alkali metals. This then allows the Na results to be utilized to make semiquantitative predictions of position and height of the maximum of the fusion barrier for other alkali clusters. These predictions are confirmed by means of similar DFT calculations for the K clusters.

Deformed-jellium model for the fission of multiply charged metal clusters

A deformed-jellium model is used to calculate the fission barrier height of positive doubly charged sodium clusters within an extended Thomas-Fermi approximation. The fissioning cluster is continuously deformed from the parent configuration until it splits into two fragments. Although the shape of the fission barrier obviously depends on the parametrization of the fission path, we have found that remarkably, the maximum of the barrier corresponds to a configuration in which the emerging fragments are already formed and rather well apart. The implication of this finding in the calculation of critical numbers for fission is illustrated in the case of multiply charged Na clusters.