Gating of the Hv1 proton channel

Hv1 is a voltage-gated proton channel, which is involved in the regulation of the intracellular pH (pHi) in immune cells. Among the voltage-gated ion channels, Hv1 is unique because it lacks a classical pore domain and its voltage-sensor domain (VSD) also forms the pore. Thus, its pore structure, as well as the opening and closing of the pore (gating), differs from classical voltage-gated ion channels. Molecular mechanisms underlying gating, in particular the coupling of voltage sensing and pore opening, are not well understood. In this thesis I used targeted crosslinking of engineered cysteine side chains to measure interactions, distances, and distance changes between amino-acid side chains. 1,1-methanediyl bismethanethiosulfonate (MTS-1-MTS) was suitable for crosslinking specific cysteine side chains in transmembrane segments S1 (151C) and S4 (262C) in the closed state of the channel, which showed that the cysteine side chains approached 3-4 Å during gating. By combining crosslinking with patch-clamp fluorometry (PCF), I was able to show that crosslinking blocks the S1 gating movement. In further experiments, this combinatorial approach will contribute to our understanding of structure-function relationships in Hv1. In Hv1, gating is controlled not only by membrane voltage but also by the difference between extracellular and intracellular pH (ΔpH), being coupled such that Hv1 opens only when the electrochemical proton gradient is outwardly directed. The coupling between voltage- and ΔpH-sensing is not understood at the molecular level. Here, I investigated the underlying molecular mechanism in PCF experiments by altering the ΔpH by means of perfusion or light- controlled proton release. I could show that a ΔpH change can induce conformational changes of S4 but not of S1. The results are consistent with the idea that S4 can sense both voltage and ΔpH. Furthermore, I could show that S4 of another voltage-sensitive protein (voltage-gated phosphatase, VSP) is not sensitive to ΔpH, indicating Hv1-specific structure-function relationships for ΔpH sensing.Hv1 ist ein spannungsgesteuerter protonenselektiver Ionenkanal, der unter anderem in Immunzellen an der Regulation des intrazellulären pH (pHi) beteiligt ist. Unter den spannungsgesteuerten Ionenkanälen ist Hv1 einzigartig, da der Kanal keine klassische Porendomäne aufweist und seine Spannungssensordomäne (VSD) auch die Pore bildet. Damit unterscheidet sich seine Porenstruktur, sowie das Öffnen und Schließen der Pore (Gating) von klassischen, spannungsgesteuerten Ionenkanälen. Molekulare Mechanismen, die dem Gating zugrunde liegen, insbesondere die Kopplung von Spannungsdetektion und Porenöffnung, sind nicht gut verstanden. Im Rahmen dieser Arbeit benutzte ich gezieltes Quervernetzen (Crosslinking) von künstlich eingeführten Cystein-Seitenketten, um Interaktionen sowie Abstände und Abstandsänderungen zwischen einzelnen Aminosäureseitenketten zu messen. 1,1-Methanediyl Bismethanethiosulfonate (MTS-1-MTS) eignete sich zum Crosslinking von bestimmten Seitenketten in Transmembransegmenten S1 (151C) und S4 (262C) im geschlossenen Zustand des Kanals, was zeigte, dass sich die Cystein-Seitenketten während des Gatings auf 3-4 Å annähern. Durch Kombination von Crosslinking mit Patch-Clamp-Fluorometrie (PCF) konnte ich zeigen, dass die Quervernetzung die S1-Gatingbewegung blockiert. Weitere Experimente mit diesem kombinatorischen Ansatz werden zu unserem Verständnis von Struktur-Funktionsbeziehungen in Hv1 beitragen. Das Gating von Hv1 wird neben der Membranspannung auch durch die Differenz zwischen extrazellulärem und intrazellulärem pH (ΔpH) gesteuert. Dabei sind ΔpH- und Spannungssteuerung derart gekoppelt, dass Hv1 nur öffnet, wenn der elektrochemische Protonengradient nach außen gerichtet ist. ΔpH-Detektion, sowie die Kopplung von ΔpH- und Spannungsdetektion sind auf molekularer Ebene nicht verstanden. Hier untersuchte ich den zugrunde liegenden Mechanismus in PCF-Experimenten durch ΔpH-Änderungen mittels Perfusion oder lichtgesteuerter Protonenfreisetzung. ΔpH-Änderungen führten zu Konformationsänderungen von S4, nicht jedoch von S1. Die Ergebnisse erlauben die Schlussfolgerung, dass S4 neben der Membranspannung auch ΔpH detektiert. Darüber hinaus konnte ich zeigen, dass S4 eines anderen spannungssensitiven Proteins (spannungsgesteuerte Phosphatase, VSP) nicht ΔpH gesteuert ist, was auf Hv1-spezifische Struktur-Funktionsbeziehungen zur Detektion von ΔpH hindeutet

Corrole-silica hybrid particles: synthesis and effects on singlet oxygen generation

The present study describes the first example of hybrid particles composed of amorphous silica and corrole. The hybrid particles were obtained by covalent linking of the gallium(III)(pyridine) complex of 5,10,15-tris(pentafluorophenyl) corrole (GaPFC) at the surface of functionalized silica spheres. The functionalization step was achieved by a nucleophilic substitution reaction between corrole and 3-aminopropyltriethoxysilane previously grafted at the silica surfaces. The hybrids were morphologically and chemically characterized and the results have confirmed covalent linkages between corrole molecules and the silica particles. Preliminary studies on the capacity of corrole and hybrid particles to generate singlet oxygen was evaluated by a chemical method in which 1,3-diphenylisobenzofuran was used to trap singlet oxygen. The new corrole-silica hybrid particles have shown lower efficiency to generate singlet oxygen as compared to the pure corrole precursor. This effect was interpreted as a consequence of interparticle interactions mediated by the corrole molecules grafted at the silica surfaces that result in their clustering. Taken together, these findings demonstrate that despite lower efficiency in terms of singlet oxygen generation, the hybrid materials offer an alternative route to develop new platforms with potential for photodynamic therapy

The impact of donor and recipient common clinical and genetic variation on estimated glomerular filtration rate in a European renal transplant population

Genetic variation across the HLA is known to influence renal‐transplant outcome. However, the impact of genetic variation beyond the HLA is less clear. We tested the association of common genetic variation and clinical characteristics, from both the donor and recipient, with post‐transplant eGFR at different time‐points, out to 5‐years post‐transplantation. We conducted GWAS meta‐analyses across 10,844 donors and recipients from five European ancestry cohorts. We also analysed the impact of polygenic risk scores (PRS), calculated using genetic variants associated with non‐transplant eGFR, on post‐transplant eGFR. PRS calculated using the recipient genotype alone, as well as combined donor and recipient genotypes were significantly associated with eGFR at 1‐year post‐transplant. 32% of the variability in eGFR at 1‐year post‐transplant was explained by our model containing clinical covariates (including weights for death/graft‐failure), principal components and combined donor‐recipient PRS, with 0.3% contributed by the PRS. No individual genetic variant was significantly associated with eGFR post‐transplant in the GWAS. This is the first study to examine PRS, composed of variants that impact kidney function in the general population, in a post‐transplant context. Despite PRS being a significant predictor of eGFR post‐transplant, the effect size of common genetic factors is limited compared to clinical variables

Schmitt Trigger Using a Self-Healing Ionic Liquid Gated Transistor

Electrical double layer transistors using ionic liquids as the gate and ZnO as the semiconductor exhibit stable operation in the presence of redox active additives. The characteristics of the device enable single components with the response of a Schmitt trigger

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM

Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

Background: In addition to HLA genetic incompatibility, non-HLA difference between donor and recipients of transplantation leading to allograft rejection are now becoming evident. We aimed to create a unique genome-wide platform to facilitate genomic research studies in transplant-related studies. We designed a genome-wide genotyping tool based on the most recent human genomic reference datasets, and included customization for known and potentially relevant metabolic and pharmacological loci relevant to transplantation. Methods: We describe here the design and implementation of a customized genome-wide genotyping array, the ‘TxArray’, comprising approximately 782,000 markers with tailored content for deeper capture of variants across HLA, KIR, pharmacogenomic, and metabolic loci important in transplantation. To test concordance and genotyping quality, we genotyped 85 HapMap samples on the array, including eight trios. Results: We show low Mendelian error rates and high concordance rates for HapMap samples (average parent-parent-child heritability of 0.997, and concordance of 0.996). We performed genotype imputation across autosomal regions, masking directly genotyped SNPs to assess imputation accuracy and report an accuracy of >0.962 for directly genotyped SNPs. We demonstrate much higher capture of the natural killer cell immunoglobulin-like receptor (KIR) region versus comparable platforms. Overall, we show that the genotyping quality and coverage of the TxArray is very high when compared to reference samples and to other genome-wide genotyping platforms. Conclusions: We have designed a comprehensive genome-wide genotyping tool which enables accurate association testing and imputation of ungenotyped SNPs, facilitating powerful and cost-effective large-scale genotyping of transplant-related studies. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0211-x) contains supplementary material, which is available to authorized users