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.

Structural and optical properties of dilute InAsN grown by molecular beam epitaxy

We perform a structural and optical characterization of InAs1¿xNx epilayers grown by molecular beam epitaxy on InAs substrates x 2.2% . High-resolution x-ray diffraction HRXRD is used to obtain information about the crystal quality and the strain state of the samples and to determine the N content of the films. The composition of two of the samples investigated is also obtained with time-of-flight secondary ion mass spectroscopy ToF-SIMS measurements. The combined analysis of the HRXRD and ToF-SIMS data suggests that the lattice parameter of InAsN might significantly deviate from Vegard"s law. Raman scattering and far-infrared reflectivity measurements have been carried out to investigate the incorporation of N into the InAsN alloy. N-related local vibrational modes are detected in the samples with higher N content. The origin of the observed features is discussed. We study the compositional dependence of the room-temperature band gap energy of the InAsN alloy. For this purpose, photoluminescence and optical absorption measurements are presented. The results are analyzed in terms of the band-anticrossing BAC model. We find that the room-temperature coupling parameter for InAsN within the BAC model is CNM=2.0 0.1 eV.

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index.

BACKGROUND: Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. DESIGN AND METHODS: Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. RESULTS: Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01). CONCLUSIONS: TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.

Model for the emission of Si+ ions during oxygen bombardment of Si(100) surfaces

The variation in the emission of Si+ ions from ion-beam-induced oxidized silicon surfaces has been studied. The stoichiometry and the electronic structure of the altered layer has been characterized using x-ray photoelectron spectroscopy (XPS). The XPS analysis of the Si 2p core level indicates the strong presence of suboxide chemical states when bombarding at angles of incidence larger than 30 °. Since the surface stoichiometry or degree of oxidation varies with the angle of incidence, the corresponding valence-band structures also differ among each other. A comparison between experimental measurements and theoretically calculated Si and SiO2 valence bands indicates that the valence bands for the altered layers are formed by a combination of those two. Since Si-Si bonds are present in the suboxide molecules, the top of the respective new valence bands are formed by the corresponding 3p-3p Si-like subbands, which extend up to the Si Fermi level. The changes in stoichiometry and electronic structure have been correlated with the emission of Si+ ions from these surfaces. From the results a general model for the Si+ ion emission is proposed combining the resonant tunneling and local-bond-breaking models.

Contribution of the gap junction proteins Connexin40 and Connexin43 to the control of blood pressure

Summary Cells in tissues and organs coordinate their activities by communicating with each other through intercellular channels named gap junctions. These channels are conduits between the cytoplasmic compartments of adjacent cells, allowing the exchange of small molecules which may be crucial for hormone secretion. Renin is normally secreted in a regulated manner by specific cells of the juxtaglomerular apparatus located within the renal cortex. Gap junctional communication may be requisite to maintain an accurate functioning in coordination of renin-producing cells, more especially as renin is of paramount importance for the control of blood pressure. Connexin43 (Cx43) and Cx40 form gap junctions that link in vivo the cells of the juxtaglomerular apparatus. Cx43 links the endothelial cells, whereas gap junctions made of Cx40 connect the endothelial cells, the renin secreting cells, as well as the endothelial cells of to the renin-secreting cells of the afferent arteriole. The observation that loss of Cx40 results in chronic hypertension associated with altered vasomotion and signal conduction along arterioles, has lead us to suggest that connexins may contribute to control blood pressure by participating to the integration of various mechanical, osmotic and electrochemical stimuli involved in the control of renin secretion and by mediating the adaptive changes of the vascular wall induced by elevated blood pressure and mechanical stress. We therefore postulated that the absence of Cx40 could have deleterious effects on the coordinated functioning of the renin-containing cells, hence accounting for hypertension. In the first part of my thesis, we reported that Cx40-deficient mice (Cx40) are hypertensive due to increased plasma renin levels and numbers of renin-producing cells. Besides, we demonstrated that prostaglandins and nitric oxide, which are possible mediators in the regulation of renin secretion by the macula densa, exert a critical role in the mechanisms controlling blood pressure ín Cx40 knockout hypertensive mice. In view of previous studies that stated avessel-specifc increase in the expression of Cx43 during renin-dependent hypertension, we hypothesized that Cx43 channels are particularly well-matched to integrate the response of cells constituting the vascular wall to hypertensive conditions. Using transgenic mice in which Cx43 was replaced by Cx32, we revealed that the replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin and prevent the renin-dependent hypertension which is normally induced in the 2K1C model. To gain insights into the regulation of connexins in two separate tissues exposed to the same fluid pressure, the second part of my thesis work was dedicated to the study of the impact of chronic hypertension and related hypertrophy on the expression of the cardiovascular connexins (Cx40, Cx37, Cx43 and Cx45) in mouse aorta and heart. Our results documented that the expression of connexins is differentially regulated in mouse aorta. according to the models of hypertension. Thus, blood pressure induces mechanical forces that differentially alter the expression of vascular connexins in order to respond to an adaptation of the aortic wall observed under pathological conditions. Altogether these data provide the first evidences that intercellular communication mediated by gap junctions is required for a proper renin secretion from the juxtaglomerular apparatus in order to control blood pressure.

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.

Determination of the direct band-gap energy of InAlAs matched to InP by photoluminescence excitation spectroscopy

A series of InxAl12xAs samples (0.51,x,0.55) coherently grown on InP was studied in order to measure the band-gap energy of the lattice matched composition. As the substrate is opaque to the relevant photon energies, a method is developed to calculate the optical absorption coefficient from the photoluminescence excitation spectra. The effect of strain on the band-gap energy has been taken into account. For x50.532, at 14 K we have obtained Eg05154966 meV. © 1997 American Institute of Physics.

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.

The psychostimulant modafinil enhances gap junctional communication in cortical astrocytes.

Sleep-wake cycle is characterized by changes in neuronal network activity. However, for the last decade there is increasing evidence that neuroglial interaction may play a role in the modulation of sleep homeostasis and that astrocytes have a critical impact in this process. Interestingly, astrocytes are organized into communicating networks based on their high expression of connexins, which are the molecular constituents of gap junction channels. Thus, neuroglial interactions should also be considered as the result of the interplay between neuronal and astroglial networks. Here, we investigate the effect of modafinil, a wakefulness-promoting agent, on astrocyte gap junctional communication. We report that in the cortex modafinil injection increases the expression of mRNA and protein of connexin 30 but not those of connexin 43, the other major astroglial connexin. These increases are correlated with an enhancement of intercellular dye coupling in cortical astrocytes, which is abolished when neuronal activity is silenced by tetrodotoxin. Moreover, gamma-hydroxybutyric acid, which at a millimolar concentration induces sleep, has an opposite effect on astroglial gap junctions in an activity-independent manner. These results support the proposition that astroglia may play an important role in complex physiological brain functions, such as sleep regulation, and that neuroglial networking interaction is modified during sleep-wake cycle. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Strong smooth muscle differentiation is dependent on myocardin gene amplification in most human retroperitoneal leiomyosarcomas.

Myocardin (MYOCD), a serum response factor (SRF) transcriptional cofactor, is essential for cardiac and smooth muscle development and differentiation. We show here by array-based comparative genomic hybridization, fluorescence in situ hybridization, and expression analysis approaches that MYOCD gene is highly amplified and overexpressed in human retroperitoneal leiomyosarcomas (LMS), a very aggressive well-differentiated tumor. MYOCD inactivation by shRNA in a human LMS cell line with MYOCD locus amplification leads to a dramatic decrease of smooth muscle differentiation and strongly reduces cell migration. Moreover, forced MYOCD expression in three undifferentiated sarcoma cell lines and in one liposarcoma cell line confers a strong smooth muscle differentiation phenotype and increased migration abilities. Collectively, these results show that human retroperitoneal LMS differentiation is dependent on MYOCD amplification/overexpression, suggesting that in these well-differentiated LMS, differentiation could be a consequence of an acquired genomic alteration. In this hypothesis, these tumors would not necessarily derive from cells initially committed to smooth muscle differentiation. These data also provide new insights on the cellular origin of these sarcomas and on the complex connections between oncogenesis and differentiation in mesenchymal tumors.

An analysis of inflation rates in the European Union using wavelets : strong evidence against unit roots

In this paper we assume inflation rates in European Union countries may in fact be fractionally integrated. Given this assumption, we obtain estimations of the order of integration by means a method based on wavelets coefficients. Finally, results obtained allow reject the unit root hypothesis on inflation rates. It means that a random shock on the rate of inflation in these countries has transitory effects that gradually diminish with the passage of time, that this, said shock hasn¿t a permanent effect on future values of inflation rates