Beleuchten der Funktion von AMPA-Rezeptoren mit fluoreszierenden Sonden

The family of ionotropic glutamate receptors (iGluRs) mediates the majority of fast excitatory synaptic transmission in the central nervous system. Their implication in higher brain function and life-threatening neurological disorders makes this protein family the target of numerous investigations. Despite immense progress in unraveling the structure and function of iGluRs, central questions remain unanswered. What conformational changes accompany and enable the sub- millisecond activation rates of the receptors? Which sites of the receptor are central for function and regulation? Although structural details are available for all iGluR subtypes through crystallographic and electron microscopy studies, little evidence exists to support the theories of the dynamic structural rearrangements within the receptor during the gating cycle. Furthermore, as the intracellular C-terminal tail and loops are unresolved in all structures available to date, structural and dynamic information about this region of the receptor is limited. In the present study AMPA receptor subunits were labelled with variants of green fluorescent protein (GFP) through genetic incorporation into various insertion sites in both the extracellular and the intracellular regions of the receptor. Fluorescence resonance energy transfer (FRET) signals between these strategically positioned fluorophores were combined with simultaneous electrophysiological measurements to record conformational rearrangements during receptor gating in real time. On the level of the intracellular domains, insertion of yellow and cyan fluorescent proteins (YFP and CFP, respectively) in the loop connecting the M1 and M2 transmembrane helices and the C- terminal tail enabled us to measure conformational rearrangements in response to an extracellularly- binding allosteric modulator cyclothiazide (CTZ), which blocks receptor desensitisation. Following the lead of the conformational changes of this region, FRET between single YFP insertions in the two intracellular positions and a membrane bound quencher showed state- dependent changes during receptor gating, and allowed us to map the positions of the insertion sites relative to the membrane in the receptor resting, desensitised and active states. Fluorescent insertions within the extracellular domains of the AMPA receptor allowed us to probe both lateral and orthogonal movements of the ligand binding and amino terminal domains. Additionally, genetically labelling a transmembrane AMPA receptor regulatory protein (TARP) with an acceptor fluorophore enabled direct visualisation of complex association and dynamics, which could be functionally confirmed through simultaneous electrophysiological recordings. In summary, these experiments gave – for the first time – a concurrent visualisation of structure- function correlation of AMPA receptors, and could ultimately lead to optically active glutamate receptors capable of reporting their own activity.Die Familie von ionotropen Glutamatrezeptoren (iGluRs) vermittelt die Mehrzahl der schnellen exzitatorischen synaptischen Transmission im Zentralnervensystem. Ihre Verwicklung in höheren Gehirnfunktion und lebensbedrohlichen neurologischen Störungen macht diese Proteinfamilie das Ziel zahlreicher Untersuchungen. Trotz immensen Fortschritte in der Struktur und Funktion von iGluRs entwirren, zentrale Fragen bleiben unbeantwortet. Welche Konformationsänderungen begleiten und ermöglichen die Untermillisekunde Aktivierungsraten der Rezeptoren? Welche Websites des Rezeptors sind für Funktion und Regulation zentraler? Obwohl strukturelle Details für alle iGluR Subtypen durch kristallographische und Elektronenmikroskopie-Studien verfügbar sind, gibt es wenig Beweise für die Theorien der dynamischen Strukturänderungen innerhalb des Rezeptors während des Gating-Zyklus zu unterstützen. Ferner ist, wie die intrazelluläre C-terminale Schwanz und Schleifen in allen Strukturen verfügbar bisher ungelöst sind, strukturelle und dynamische Informationen über diese Region des Rezeptors ist begrenzt. In der vorliegenden Studie AMPA-Rezeptor-Untereinheiten wurden mit Varianten des Green Fluorescent Protein (GFP), durch genetische Einarbeitung in unterschiedliche Insertionsstellen markierten sowohl in der extrazellulären und intrazellulären Regionen des Rezeptors. Fluoreszenz- Resonanz-Energie- Transfer (FRET) Signale zwischen diesen strategisch positionierte Fluorophore wurden bei gleichzeitiger elektrophysiologischen Messungen kombiniert, um Konformationsänderungen während Rezeptor-Gating in Echtzeit aufnehmen. Auf der Ebene der intrazellulären Domänen, Insertion von Gelb und Cyan fluoreszierende Proteine (YFP und CFP sind) in der Schleife, die M1 und M2 Transmembran- Helices und den C-terminalen Schwanz verbindet es uns ermöglicht, Konformationsänderungen in Reaktion auf eine extracellularly- zu messen Bindung allosterischer Modulator Cyclothiazid (CTZ), die Blöcke Rezeptor Desensibilisierung. Nach dem Vorbild der Konformationsänderungen dieser Region, FRET zwischen einzelnen YFP Einfügungen in den beiden intrazellulären Positionen und eine Membran gebundenen Quencher zeigte zustandsabhängigen Veränderungen während der Rezeptor- Gating und erlaubt es uns, die Positionen der Insertionsstellen in Bezug auf die Membran zu kartieren in dem Rezeptor ruhende, desensibilisiert und aktive Zustände. Fluorescent Einfügungen innerhalb der extrazellulären Domänen des Rezeptors AMPA erlaubt uns sowohl lateral als auch orthogonale Bewegungen der Ligandenbindung und aminoterminalen Domänen zu untersuchen. Zusätzlich Kennzeichnung genetisch ein Trans AMPA-Rezeptor- Regulatorprotein (TARP) mit einem Akzeptor-Fluorophor direkte Visualisierung komplexer Assoziation und Dynamik ermöglicht, die funktionell durch gleichzeitige elektrophysiologischen Ableitungen bestätigt werden konnte. Zusammenfassend ergaben diese Experimente - zum ersten Mal - eine gleichzeitige Visualisierung von Struktur-Funktions-Beziehung von AMPA- Rezeptoren und letztendlich zu optisch aktiven Glutamat-Rezeptoren fähig berichten ihre eigene Aktivität führen könnte

Structural rearrangement of the intracellular domains during AMPA receptor activation

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact

Relocation of an Extrasynaptic GABAA Receptor to Inhibitory Synapses Freezes Excitatory Synaptic Strength and Preserves Memory

The excitatory synapse between hippocampal CA3 and CA1 pyramidal neurons exhibits long-term potentiation (LTP), a positive feedback process implicated in learning and memory in which postsynaptic depolarization strengthens synapses, promoting further depolarization. Without mechanisms for interrupting positive feedback, excitatory synapses could strengthen inexorably, corrupting memory storage. Here, we reveal a hidden form of inhibitory synaptic plasticity that prevents accumulation of excitatory LTP. We developed a knockin mouse that allows optical control of endogenous α5-subunit-containing γ-aminobutyric acid (GABA)A receptors (α5-GABARs). Induction of excitatory LTP relocates α5-GABARs, which are ordinarily extrasynaptic, to inhibitory synapses, quashing further NMDA receptor activation necessary for inducing more excitatory LTP. Blockade of α5-GABARs accelerates reversal learning, a behavioral test for cognitive flexibility dependent on repeated LTP. Hence, inhibitory synaptic plasticity occurs in parallel with excitatory synaptic plasticity, with the ensuing interruption of the positive feedback cycle of LTP serving as a possible critical early step in preserving memory

Topical reinforcement of the cervical mucus barrier to sperm

International audienceClose to half of the world’s pregnancies are still unplanned, reflecting a clear unmet need in contraception. Ideally, a contraceptive would provide the high efficacy of hormonal treatments, without systemic side effects. Here, we studied topical reinforcement of the cervical mucus by chitosan mucoadhesive polymers as a form of female contraceptive. Chitosans larger than 7 kDa effectively cross-linked human ovulatory cervical mucus to prevent sperm penetration in vitro. We then demonstrated in vivo using the ewe as a model that vaginal gels containing chitosan could stop ram sperm at the entrance of the cervical canal and prevent them from reaching the uterus, whereas the same gels without chitosan did not substantially limit sperm migration. Chitosan did not affect sperm motility in vitro or in vivo, suggesting reinforcement of the mucus physical barrier as the primary mechanism of action. The chitosan formulations did not damage or irritate the ewe vaginal epithelium, in contrast to nonoxynol-9 spermicide. The demonstration that cervical mucus can be reinforced topically to create an effective barrier to sperm may therefore form the technological basis for muco-cervical barrier contraceptives with the potential to become an alternative to hormonal contraceptives