The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Microautophagy of the nucleus coincides with a vacuolar diffusion barrier at nuclear-vacuolar junctions.

Nuclei bind yeast vacuoles via nucleus-vacuole (NV) junctions. Under nutrient restriction, NV junctions invaginate and release vesicles filled with nuclear material into vacuoles, resulting in piecemeal microautophagy of the nucleus (PMN). We show that the electrochemical gradient across the vacuolar membrane promotes invagination of NV junctions. Existing invaginations persist independently of the gradient, but final release of PMN vesicles requires again V-ATPase activity. We find that NV junctions form a diffusion barrier on the vacuolar membrane that excludes V-ATPase but is enriched in the VTC complex and accessible to other membrane-integral proteins. V-ATPase exclusion depends on the NV junction proteins Nvj1p,Vac8p, and the electrochemical gradient. It also depends on factors of lipid metabolism, such as the oxysterol binding protein Osh1p and the enoyl-CoA reductase Tsc13p, which are enriched in NV junctions, and on Lag1p and Fen1p. Our observations suggest that NV junctions form in two separable steps: Nvj1p and Vac8p suffice to establish contact between the two membranes. The electrochemical potential and lipid-modifying enzymes are needed to establish the vacuolar diffusion barrier, invaginate NV junctions, and form PMN vesicles.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Blood pressure loci identified with a gene-centric array.

Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

Molecular evolutionary patterns in the Glomeromycota;

Abstract Arbuscular mycorrhizal fungi (AMF) form symbiosis with roots of approximately 80% of known land plants. These fungi play a key role in the ecology and adaptation of plants to various ecosystems.by increasing the plant resources for various nutrients. Despite their important ecological role, we still have poor understanding of their genetic structure and their molecular evolution. The work presented in this thesis aims to isolate and analyse AMF genes with various molecular techniques, in order to obtain new insights about their genetics, phylogeny and molecular evolution. Some AMF genes were shown through phylogenetic analyses to be more related with plants or mycoparasites than with other fungal organisms. These results led to the prediction that lateral gene transfers (LGT) occurred between AMF and plants during their long-term co-évolution. By phylogenetic and molecular analyses, in the chapter 2 I demonstrate that the hypothesis of LGT is most likely a consequence of analyses carried out on contaminant non AMF-DNA. In addition, various features characteristic of AMF genes have been determined, allowing researchers to scan their own sequence databases for potential non-AMF contaminants. Phylogenetic relationships of AMF with other fungi has been mostly analysed using molecular markers of ribosomal origin. In chapter 2 I successfully isolated gene encoding α- and ß-tubulins from several AMF genera. Consequently, phylogenetic analyses showed that AMF possess an unexpected relationship with ancestral aquatic fungi (chytrids). These results are consistent with the prediction stating that AMF may have played an important role in the colonisation of land by green plants through the establishment of a symbiosis and after the divergence of AMF from aquatic ancestors. In Chapter 4 I tried to isolate the entire AMF gene family encoding P-Type II ATPases, in order to determine their molecular evolution with the fungal kingdom. These genes were further analysed to detect the level of sequence polymorphism that is present within an AMF population. The results obtained show that mutational events previously thought as occurring only among divergent evolutionary lineages (gene duplications, indel mutations in coding regions) can occur within a single population of AMF. These results have far reaching consequences for our understanding of the genetics and ecology of AMF. Résumé Les champignons endomycorrhiziens arbusculaires (CEA) forment une symbiose racinaire avec environ 80% des plantes vasculaires connues. Ces champignons possèdent un rôle important dans l'écologie et l'adaptation des plantes au sein de différents écosystèmes en .augmentant leurs ressources en nutriments. Le travail présenté dans cette thèse se propose d'isoler et d'analyser certains gènes de CEA avec différentes techniques moléculaires à fin d'obtenir de pÌus amples informations concernant l'évolution moléculaire, la phylogénie et leur diversité génétique à diverses échelles taxonomiques. Certaines analyses phylogénétiques des CEA ont conduit à l'hypothèse que des transferts horizontaux de gènes (THG) ont pu avoir lieu durant leur longue co-évolution avec les plantes vasculaires. Dans le chapitre 2 de cette thèse nous démontrons par analyses moléculaire et phylogénétique que l'hypothèse de THG est une conséquence de contaminations à partir d'ADN de plante ou d'autres micro-organismes. De plus, de nombreuses caractéristiques moléculaires de CEA ont pu être déterminées, permettant la mise en place d'un plan à suivre lors de l'analyse de gènes de CEA dans les études futures. Les relations évolutives des. CEA avec d'autres champignons ont été analysées majoritairement à l'aide de marqueurs moléculaires d'origine ribosomiale. Dans les chapitres 2 et 3 j'ai isolé des gènes codant pour l'a- et la ß-tubuline chez différents genres, de CEA. Les analyses phylogénétiques ont démontré une parenté entre les CEA et des champignons aquatiques ancestraux (chytrides). Ces résultats sont en accord avec l'hypothèse selon laquelle les CEA ont probablement joué un rôle primordial dans l'établissement des plantes sur terre à travers une symbiose et suite à leur évolution à partir d'ancêtres vivant dans des milieux aquatiques: Dans le chapitre 4 j'ai isolé une entière famille de gènes chez les CEA codant des ATPases de la membrane plasmique, et étudié leur évolution moléculaire dans le règne des champignons. Ces mêmes gènes ont été analysés ultérieurement à fin de déterminer le degré de polymorphisme de séquence qui peut être présent au sein d'une population de CEA. Les résultats obtenus montrent que des évènements mutationnels considérés comme apparaissant exclusivement dans des lignées évolutives très divergentes (duplication de gènes, insertions/délétions dans des régions transcrites du génome) ont lieu sein d'une même population de CEA. Cette découverte a un impact important sur nos connaissances concernant la génétique des populations des CEA et leur écologie.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth.

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H(+)-ATPases that are required to control apoplastic pH. Our results show that H(+)-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H(+)-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.

Common variants in the ATP2B1 gene are associated with susceptibility to hypertension: the Japanese Millennium Genome Project.

Hypertension is one of the most common complex genetic disorders. We have described previously 38 single nucleotide polymorphisms (SNPs) with suggestive association with hypertension in Japanese individuals. In this study we extend our previous findings by analyzing a large sample of Japanese individuals (n=14 105) for the most associated SNPs. We also conducted replication analyses in Japanese of susceptibility loci for hypertension identified recently from genome-wide association studies of European ancestries. Association analysis revealed significant association of the ATP2B1 rs2070759 polymorphism with hypertension (P=5.3×10(-5); allelic odds ratio: 1.17 [95% CI: 1.09 to 1.26]). Additional SNPs in ATP2B1 were subsequently genotyped, and the most significant association was with rs11105378 (odds ratio: 1.31 [95% CI: 1.21 to 1.42]; P=4.1×10(-11)). Association of rs11105378 with hypertension was cross-validated by replication analysis with the Global Blood Pressure Genetics consortium data set (odds ratio: 1.13 [95% CI: 1.05 to 1.21]; P=5.9×10(-4)). Mean adjusted systolic blood pressure was highly significantly associated with the same SNP in a meta-analysis with individuals of European descent (P=1.4×10(-18)). ATP2B1 mRNA expression levels in umbilical artery smooth muscle cells were found to be significantly different among rs11105378 genotypes. Seven SNPs discovered in published genome-wide association studies were also genotyped in the Japanese population. In the combined analysis with replicated 3 genes, FGF5 rs1458038, CYP17A1, rs1004467, and CSK rs1378942, odds ratio of the highest risk group was 2.27 (95% CI: 1.65 to 3.12; P=4.6×10(-7)) compared with the lower risk group. In summary, this study confirmed common genetic variation in ATP2B1, as well as FGF5, CYP17A1, and CSK, to be associated with blood pressure levels and risk of hypertension.

Monophyly of beta-tubulin and H+-ATPase gene variants in Glomus intraradices: consequences for molecular evolutionary studies of AM fungal genes.

Arbuscular mycorrhizal fungi (AMF) are an ecologically important group of fungi. Previous studies showed the presence of divergent copies of beta-tubulin and V-type vacuolar H+-ATPase genes in AMF genomes and suggested horizontal gene transfer from host plants or mycoparasites to AMF. We sequenced these genes from DNA isolated from an in vitro cultured isolate of Glomus intraradices that was free of any obvious contaminants. We found two highly variable beta-tubulin sequences and variable H+-ATPase sequences. Despite this high variation, comparison of the sequences with those in gene banks supported a glomeromycotan origin of G. intraradices beta-tubulin and H+-ATPase sequences. Thus, our results are in sharp contrast with the previously reported polyphyletic origin of those genes. We present evidence that some highly divergent sequences of beta-tubulin and H+-ATPase deposited in the databases are likely to be contaminants. We therefore reject the prediction of horizontal transfer to AMF genomes. High differences in GC content between glomeromycotan sequences and sequences grouping in other lineages are shown and we suggest they can be used as an indicator to detect such contaminants. H+-ATPase phylogeny gave unexpected results and failed to resolve fungi as a natural group. beta-Tubulin phylogeny supported Glomeromeromycota as sister group of the Chytridiomycota. Contrasts between our results and trees previously generated using rDNA sequences are discussed.

Étude du mécanisme de transport des cations par la Na, K-ATPase et de son implication dans la migraine familiale hémiplégique de type 2

Résumé La Na,K-ATPase est une protéine transmembranaire, présente dans toutes les cellules de mammifères et indispensable à la viabilité cellulaire. Elle permet le maintien des gradients sodiques et potassiques à l'origine du potentiel membranaire en transportant 3 Na+ en dehors de la cellule contre 2 K+, grâce à l'énergie fournie par l'hydrolyse d'une molécule d'ATP. Le potentiel membranaire est indispensable au maintien de l'excitabilité cellulaire et à la transmission de l'influx nerveux. Il semblerait que la Na,K-ATPase soit liée à l'hypertension et à certains troubles neurologiques comme la Migraine Familiale Hémiplégique (1VIFH). La MFH est une forme de migraine avec aura, qui se caractérise par une hémiparésie. Cette forme de migraine est très rare. Elle se transmet génétiquement sur un mode autosomique dominant. Plusieurs mutations localisées dans le gène de la Na,K-ATPase ont été identifiées durant ces 3 dernières années. C'est la première fois qu'une maladie génétique est associée au gène de la Na,K-ATPase. La compréhension du fonctionnement de cette protéine peut donner des informations sur les mécanismes conduisant à ces pathologies. On sait que la fonction d'une protéine est liée à sa structure. L'étude de sa fonction nécessite donc l'étude de sa structure. Alors que la structure de la SERCA a été déterminée à haute résolution, par cristallographie, celle de la Na,K-ATPase ne l'est toujours pas. Mais ces 2 ATPases présentent une telle homologie qu'un modèle de la Na,K-ATPase a pu être élaboré à partir de la structure de la SERCA. Les objectifs de cette étude sont d'une part, de comprendre le contrôle de l'accessibilité du K+ extracellulaire àses sites de liaison. Pour cela, nous avons ciblé cette étude sur la 2ìème et la 31eme boucle extracellulaire, qui relient respectivement les segments transmembranaires (STM) 3-4 et 5-6. Le choix s'est porté sur ces 2 boucles car elles bordent le canal des cations formés des 4ième' Sième et 6'ème hélices. D'autre part, nous avons également essayer de comprendre les effets des mutations, liées à la Migraine Familiale Hémiplégique de type 2 (MFH2), sur la fonctionnalité de la Na,K-ATPase. Alors que les STM et les domaines cytoplasmiques sont relativement proches entre la Na,KATPase et la SERCA, les boucles extracellulaires présentent des différences. Le modèle n'est donc pas une approche fiable pour déterminer la structure et la fonction des régions extracellulaires. Nous avons alors utilisé une approche fonctionnelle faisant appel à la mutation dirigée puis à l'étude de l'activité fonctionnelle de la Na,K ATPase par électrophysiologie sur des ovocytes de Xenopus. En conclusion, nous pouvons dire que la troisième boucle extracellulaire participerait à la structure de la voie d'entrée des cations et que la deuxième boucle extracellulaire semble impliquée dans le contrôle de l'accessibilité des ions K+àses sites de liaison. Concernant les mutations associées à la MFH2, nos résultats ont montré une forte diminution de l'activité fonctionnelle de la pompe Na,K, inférieure aux conditions physiologiques de fonctionnement, et pour une des mutations nous avons observés une diminution de l'affmité apparente au K+ externe. Nous poumons faire l'hypothèse que l'origine pathologique de la migraine est liée à une diminution de l'activité de la pompe à Na+. Summary The Na,K-ATPase is a transmembrane protein, present in all mammalian cells and is necessary for the viability of the cells. It maintains the gradients of Na+ and K+ involved in the membrane potential, by transporting 3Na+ out the cell, and 2K+ into the cell, using the energy providing from one ATP molecule hydrolysis. The membrane potential is necessary for the cell excitability and for the transmission of the nervous signal. Some evidence show that Na,K-ATPase is involved in hypertension and neurological disorders like the Familial Hemiplegic Migraine (FHM). La FHM is a rare form of migraine characterised by aura and hemiparesis and an autosomal dominant transmission. Several mutations linked to the Na,KATPase gene have been identified during these 3 last years. It's the first genetic disorder associated with the Na,K-ATPase gene. Understand the function of this protein is important to elucidate the mechanisms implicated in these pathologies. The function of a protein is linked with its structure. Thus, to know the function of a protein, we need to know its structure. While the Ca-ATPase (SERCA) has been crystallised with a high resolution, the structure of the Na,K-ATPase is not known. Because of the great homology between these 2 ATPases, a model of the Na,K-ATPase was realised by comparing with the structure of the SERCA. The aim of this study is on one side, understand the control of the extracellular K+ accessibility to their binding sites. Because of theirs closed proximity with the cation pathway, located between the 4th, 5th and 6th helices, we have targeted this study on the 2nd and the 3rd extracellular loops linking respectively the transmembrane segment (TMS) 3 and 4, and the TMS 5 and 6. And on the other side, we have tried to understand the functional effects of mutations linked with the Familial Hemiplegic Migraine Type 2 (FHM2). In contrast with the transmembrane segments and the cytoplasmic domains, the extracellular loops show lots of difference between Na,K-ATPase and SERCA, the model is not a good approach to know the structure and the function of the extracellular loops. Thus, we have used a functional approach consisting in directed mutagenesis and the study of the functional activity of the Na,K-ATPase by electrophysiological techniques with Xenopus oocytes. In conclusion, we have demonstrated that the third extracellular loop could participate in the structure of the entry of the cations pathway and that the second extracellular loop could control the K+ accessibility to their binding sites. Concerning the mutations associated with the FHM2, our results showed a strong decrease in the functional activity of the Na,K-pump under physiological conditions and for one of mutations, induce a decrease in the apparent external K+ affinity. We could make the hypothesis that the pathogenesis of migraine is related to the decrease in Na,K-pump activity. Résumé au large publique De la même manière que l'assemblage des mots forme des phrases et que l'assemblage des phrases forme des histoires, l'assemblage des cellules forme des organes et l'ensemble des organes constitue les êtres vivants. La fonction d'une cellule dans le corps humain peut se rapprocher de celle d'une usine hydroélectrique. La matière première apportée est l'eau, l'usine électrique va ensuite convertir l'eau en énergie hydraulique pour fournir de l'électricité. Le fonctionnement de base d'une cellule suit le même processus. La cellule a besoin de matières premières (oxygène, nutriments, eau...) pour produire une énergie sous forme chimique, l'ATP. Cette énergie est utilisée par exemple pour contracter les muscles et permet donc à l'individu de se déplacer. Morphologiquement la cellule est une sorte de petit sac rempli de liquide (milieu intracellulaire) baignant elle-même dans le liquide (milieu extracellulaire) composant le corps humain (un adulte est constitué environ de 65 % d'eau). La composition du milieu intracellulaire est différente de celle du milieu extracellulaire. Cette différence doit être maintenue pour que l'organisme fonctionne correctement. Une des différences majeures est la quantité de sodium. En effet il y a beaucoup plus de sodium à l'extérieur qu'à l'intérieur de la cellule. Bien que l'intérieur de la cellule soit isolé de l'extérieur par une membrane, le sodium arrive à passer à travers cette membrane, ce qui a tendance à augmenter la quantité de sodium dans la cellule et donc à diminuer sa différence de concentration entre le milieu extracellulaire et le milieu intracellulaire. Mais dans les membranes, il existe des pompes qui tournent et dont le rôle est de rejeter le sodium de la cellule. Ces pompes sont des protéines connues sous le nom de pompe à sodium ou Na,K-ATPase. On lui attribue le nom de Na,K-ATPase car en réalité elle rejette du sodium (Na) et en échange elle fait entrer dans la cellule du potassium (K), et pour fonctionner elle a besoin d'énergie (ATP). Lorsque les pompes à sodium ne fonctionnent pas bien, cela peut conduire à des maladies. En effet la Migraine Familiale Hémiplégique de type 2, est une migraine très rare qui se caractérise par l'apparition de la paralysie de la moitié d'un corps avant l'apparition du mal de tête. C'est une maladie génétique (altération qui modifie la fonction d'une protéine) qui touche la pompe à sodium située dans le cerveau. On a découvert que certaines altérations (mutations) empêchent les pompes à sodium de fonctionner correctement. On pense alors que le développement des migraines est en partie dû au fait que ces pompes fonctionnent moins bien. Il est important de bien connaître la fonction de ces pompes car cela permet de comprendre des mécanismes pouvant conduire à certaines maladies, comme les migraines. En biologie, la fonction d'une protéine est étudiée à travers sa structure. C'est pourquoi l'objectif de cette thèse a été d'étudier la structure de la Na,K-ATPase afin de mieux comprendre son mécanisme d'action.

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium.

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.

Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices.

BACKGROUND: The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. RESULTS: In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. CONCLUSION: On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence that structural genomic changes, such as exonic indel mutations and gene duplications are less rare than previously thought and that these also occur within fungal populations.

alphaENaC-mediated lithium absorption promotes nephrogenic diabetes insipidus.

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.