8 research outputs found
Transport Mechanisms of the Vesicular Glutamate Transporter 1
In der neuronalen Signaltransmission spielen die vesikulären Neurotransmittertransporter eine Schlüsselrolle in der Modifikation der Quantengröße. Sie sind verantwortlich für die Befüllung der synaptischen Vesikel (SVs) mit Neurotransmittern und werden vom elektrochemischen Gradienten (ΔµH+) angetrieben, welcher durch die vakuole ATPase (V-ATPase) generiert wird. Die Aufklärung der Regulationsmechanismen der SV-Beladung kann entscheidend zur Beantwortung fundamenteller Fragen zu synaptischen Funktionen beitragen. Insbesondere der Mechanismus des vesikulären Glutamattransporters (VGLUT), der die SVs mit L-Glutamat befüllt, konnte bisweilen nicht zufriedenstellend aufgeklärt werden. Beispielsweise ist bisweilen nicht klar welcher Mechanismus der biphasischen Abhängigkeit des Transporters von Chlorid-Ionen unterliegt. Desweiteren wurden widersprüchliche Daten zur Chlorid-Leitfähigkeit des Transporters veröffentlicht. Kürzlich wurde ein Regulationsmodus vorgeschlagen, der die Glutamataufnahme durch den Austausch eines luminalen Protons mit einem cytosolischen Kalium-Ion stimuliert.
Der Fokus der vorliegenden Studie liegt hauptsächlich auf dem Chlorid-Transport durch VGLUT1 und der Regulierung von VGLUT1 durch den vermeintlichen Kalium-Ion/Proton Austausch mit dem Ziel den ionischen Mechnismus des Glutamattransportes aufzuklären. Die Fragen um den ionischen Kupplungsmechanismus wurden unter Verwendung eines minimalen Systems adressiert, das aus dem rekombinanten Maus-VGLUT1 und einer Protonenpumpe aus dem Bacillus thermophilus (TF0F1) rekonstituiert in künstliche Membranen besteht und in der vorliegenden Arbeit etabliert wurde. Die VGLUT1/TF0F1 Liposomen wiesen keine Chlorid-Leitfähigkeit auf -im Gegensatz zu früheren Studien. Zudem haben hohe luminale Chlorid-Konzentrationen keinen stimulierenden Effekt auf die Glutamataufnahme gezeigt, wie zuvor berichtet. Dieses Ergebnis bestätigt zusätzlich eine fehlende Chlorid-Leitfähigkeit durch VGLUT1. Bei Untersuchungen an SVs konnte der kürzlich beschriebene Kalium-Ion/Proton Austausch beobachtet werden. Bemerkenswerterweise haben erste Ergebnisse in der vorliegenden Arbeit gezeigt, dass VGLUT1 selbst für den Kalium-Ion/Proton Austausch in SVs verantwortlich ist. Desweiteren wurde in dieser Arbeit ein neuartiges „Hybrid“-System entwickelt, bestehend aus SVs und Proteoliposomen mit TF0F1 und fusionsmediierenden Proteinen, die miteinander fusioniert werden. Die Einzigartigkeit dieser „fusionierten SVs“ liegt in der erstmals möglichen Modifikation des SV-Lumens, eine bisweilen schwer umsetzbare Herausfoderung. Die Glutamataufnahme kann in „fusionierten SVs“ ohne Aktivitätsverlust erhalten werden. Die „fusionierten SVs“ bieten u.a. eine einzigartige Möglichkeit den luminalen Effekt hoher Chlorid-Konzentrationen auf die Glutamataufnahme in SVs zu untersuchen
Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts
Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of
synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such
as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report
robust elevation in the probability of release of readily releasable vesicles with both high and low
release probabilities at a variety of synapse types from knockout mice missing all four family
members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the
readily releasable pool was affected. The results suggest that family members serve as negative
regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection
strength selectively when presynaptic action potentials fire at low frequency. The widespread
expression suggests that chemical synapses may play a frequency filtering role in biological
computation that is more elemental than presently envisioned
Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts
Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report robust elevation in the probability of release of readily releasable vesicles with both high and low release probabilities at a variety of synapse types from knockout mice missing all four family members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the readily releasable pool was affected. The results suggest that family members serve as negative regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection strength selectively when presynaptic action potentials fire at low frequency. The widespread expression suggests that chemical synapses may play a frequency filtering role in biological computation that is more elemental than presently envisioned.Peer reviewe
Isolation of large dense-core vesicles from bovine adrenal medulla for functional studies
Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.Other Information Published in: Scientific Reports License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41598-020-64486-3</p
Pump, Rest and Repeat: Single Molecule Measurements Reveal Mode-Switching in the Mammalian Brain V-ATPase
Elevated synaptic vesicle release probability in synaptophysin/gyrin family quadruple knockouts
Synaptophysins 1 and 2 and synaptogyrins 1 and 3 constitute a major family of
synaptic vesicle membrane proteins. Unlike other widely expressed synaptic vesicle proteins such
as vSNAREs and synaptotagmins, the primary function has not been resolved. Here, we report
robust elevation in the probability of release of readily releasable vesicles with both high and low
release probabilities at a variety of synapse types from knockout mice missing all four family
members. Neither the number of readily releasable vesicles, nor the timing of recruitment to the
readily releasable pool was affected. The results suggest that family members serve as negative
regulators of neurotransmission, acting directly at the level of exocytosis to dampen connection
strength selectively when presynaptic action potentials fire at low frequency. The widespread
expression suggests that chemical synapses may play a frequency filtering role in biological
computation that is more elemental than presently envisioned
The Na+/H+ Exchanger Nhe1 Modulates Network Excitability via GABA Release
Brain functions are extremely sensitive to pH changes because of the pH-dependence of proteins involved in neuronal excitability and synaptic transmission. Here, we show that the Na+/H+ exchanger Nhe1, which uses the Na+ gradient to extrude H+, is expressed at both inhibitory and excitatory presynapses. We disrupted Nhe1 specifically in mice either in Emx1- positive glutamatergic neurons or in parvalbumin-positive cells, mainly GABAergic interneurons. While Nhe1 disruption in excitatory neurons had no effect on overall network excitability, mice with disruption of Nhe1 in parvalbumin-positive neurons displayed epileptic activity. From our electrophysiological analyses in the CA1 of the hippocampus, we conclude that the disruption in parvalbumin-positive neurons impairs the release of GABA-loaded vesicles, but increases the size of GABA quanta. The latter is most likely an indirect pH-dependent effect, as Nhe1 was not expressed in purified synaptic vesicles itself. Conclusively, our data provide first evidence that Nhe1 affects network excitability via modulation of inhibitory interneurons