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The dependence of the Escherichia coli Na+H+ antiporter A (EcNhaA) pH sensor mutant E241C on H+ and Na+ concentrations was tested using a solid supported membrane (SSM) based electrophysiological approach. Proteoliposome preparations with right side out (RSO) oriented carriers were used to investigate the passive downhill uptake mode (physiologically the reverse transport mode) at zero membrane potential. Na+ concentration gradients established with a rapid solution exchange acted as the driving force. When a Na+ concentration gradient was established at symmetrical pH, the transport activity of the E241C EcNhaA variant was similar to that of the wildtype EcNhaA, with no shift of the bell-shaped pH dependence, an increase of the KmNa at acidic pH and a decrease of the KmNa at alkaline pH, supporting the model of a competitive binding of Na+ and H+ to a common binding site.
Background: Amyloid precursor protein (APP) processing is central to Alzheimer’s disease (AD) etiology. As early cognitive alterations in AD are strongly correlated to abnormal information processing due to increasing synaptic impairment, it is crucial to characterize how peptides generated through APP cleavage modulate synapse function. We previously described a novel APP processing pathway producing η-secretase-derived peptides (Aη) and revealed that Aη–α, the longest form of Aη produced by η-secretase and α-secretase cleavage, impaired hippocampal long-term potentiation (LTP) ex vivo and neuronal activity in vivo.
Methods: With the intention of going beyond this initial observation, we performed a comprehensive analysis to further characterize the effects of both Aη-α and the shorter Aη-β peptide on hippocampus function using ex vivo field electrophysiology, in vivo multiphoton calcium imaging, and in vivo electrophysiology.
Results: We demonstrate that both synthetic peptides acutely impair LTP at low nanomolar concentrations ex vivo and reveal the N-terminus to be a primary site of activity. We further show that Aη-β, like Aη–α, inhibits neuronal activity in vivo and provide confirmation of LTP impairment by Aη–α in vivo.
Conclusions: These results provide novel insights into the functional role of the recently discovered η-secretase-derived products and suggest that Aη peptides represent important, pathophysiologically relevant, modulators of hippocampal network activity, with profound implications for APP-targeting therapeutic strategies in AD.
Beta-Barrel-Membranproteine der Omp85-Familie besitzen essentielle Funktionen in der Lipidbiogenese und der Proteinintegration und -assemblierung in der äußeren Membran von Gram-negativen Bakterien. Auch in endosymbiotisch erworbenen Organellen der Eukaryoten übernahmen Vertreter dieser Proteinfamilie wichtige Rollen, wie bei der Translokation von Vorstufenproteinen durch die äußere Hüllmembran von Chloroplasten und der Assemblierung von Proteinkomplexen in der äußeren Hüllmembran von Mitochondrien. Eine phylogenetische Analyse der bekannten Vertreter dieser Proteinfamilie wies auf eine Aufspaltung der Omp85-Familie in zwei Hauptzweige hin. Während der Toc75-Zweig aus plastidären und cyanobakteriellen Omp85-Proteinen besteht, bilden mitochondrielle Proteine sowie die Omp85-Vertreter der Proteobakterien eine zweite Unterfamilie, den Sam50-Zweig. In dieser Arbeit wurden die elektrophysiologischen Eigenschaften von pro- und eukaryotischen Vertretern beider Unterfamilien verglichen. Zu diesem Zweck wurde ein elektrophysiologischer Messstand konstruiert, mit dem diese Kanalproteine mittels der Lipid-Bilayer-Technik untersucht werden konnten. Die Analyse der experimentellen Daten der verwendeten Omp85-Proteine zeigte deutliche Gemeinsamkeiten der gesamten Proteinfamilie hinsichtlich ihrer Kationenselektivität. Hingegen ergab ein Vergleich der Kanalleitwerte signifikante Unterschiede zwischen beiden Unterfamilien. So lagen die aus den Leitwerten berechneten Porendurchmesser bei den Mitgliedern des Toc75-Zweigs um das Zwei- bis Dreifache über den für die Vertreter des Sam50-Zweigs bestimmten Werten. Topologievorhersagen ergaben, dass die Mitglieder der Omp85-Familie aus zwei Domänen aufgebaut sind. Durch die Analyse der elektrophysiologischen Eigenschaften von Teilkonstrukten, die aufgrund dieser Vorhersagen erstellt wurden, konnte gezeigt werden, dass die C-terminale Domäne die Porenregion des beta-Barrel-Proteins bildet. Die elektrophysiologischen Eigenschaften der Omp85-Proteine werden allerdings durch die N-terminale Domäne moduliert. Aufgrund der in dieser Arbeit erzielten elektrophysiologischen Ergebnisse und unter Berücksichtigung der phylogenetischen Analyse der Omp85-Familie konnte eine Hypothese zur evolutionären Entwicklung dieser sehr alten Proteinfamilie aufgestellt werden.
Cross-frequency coupling of sleep oscillations is thought to mediate memory consolidation. While the hippocampus is deemed central to this process, detailed knowledge of which oscillatory rhythms interact in the sleeping human hippocampus is lacking. Combining intracranial hippocampal and non-invasive electroencephalography from twelve neurosurgical patients, we characterized spectral power and coupling during non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Hippocampal coupling was extensive, with the majority of channels expressing spectral interactions. NREM consistently showed delta–ripple coupling, but ripples were also modulated by slow oscillations (SOs) and sleep spindles. SO–delta and SO–theta coupling, as well as interactions between delta/theta and spindle/beta frequencies also occurred. During REM, limited interactions between delta/theta and beta frequencies emerged. Moreover, oscillatory organization differed substantially between i) hippocampus and scalp, ii) sites along the anterior-posterior hippocampal axis, and iii) individuals. Overall, these results extend and refine our understanding of hippocampal sleep oscillations.
Na(+)/H(+) exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na(+)/H(+) exchangers, where a single binding site is alternatively occupied by Na(+) or one or two H(+) ions. The proposed transport mechanism inherently down-regulates Na(+)/H(+) exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na(+)/H(+) exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na(+)/H(+) exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na(+)/H(+) exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na(+)/H(+) exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na(+)/H(+) exchangers.