Presynaptic NMDA receptors (preNMDARs) control synaptic release, but it is not well understood how. Rab3-interacting molecules (RIMs) provide scaffolding at presynaptic active zones and are involved in vesicle priming. Moreover, c-Jun N-terminal kinase (JNK) has been implicated in regulation of spontaneous release. We demonstrate that, at connected layer 5 pyramidal cell pairs of developing mouse visual cortex, Mg-sensitive preNMDAR signaling upregulates replenishment of the readily releasable vesicle pool during high-frequency firing. In conditional RIM1αβ deletion mice, preNMDAR upregulation of vesicle replenishment was abolished, yet preNMDAR control of spontaneous release was unaffected. Conversely, JNK2 blockade prevented Mg-insensitive preNMDAR signaling from regulating spontaneous release, but preNMDAR control of evoked release remained intact. We thus discovered that preNMDARs signal differentially to control evoked and spontaneous release by independent and non-overlapping mechanisms. Our findings suggest that preNMDARs may sometimes signal metabotropically and support the emerging principle that evoked and spontaneous release are distinct processes. VIDEO ABSTRACT.
Key points In the hippocampus, calcium‐permeable AMPA receptors have been found in a restricted subset of neuronal types that inhibit other neurons, although their localization in the neocortex is less well understood.In the present study, we looked for calcium‐permeable AMPA receptors in two distinct populations of neocortical inhibitory neurons: basket cells and Martinotti cells. We found them in the former but not in the latter. Furthermore, in basket cells, these receptors were associated with particularly fast responses.Computer modelling predicted (and experiments verified) that fast calcium‐permeable AMPA receptors enable basket cells to respond rapidly, such that they promptly inhibit neighbouring cells and shut down activity.The results obtained in the present study help our understanding of pathologies such as stroke and epilepsy that have been associated with disordered regulation of calcium‐permeable AMPA receptors. AbstractAMPA‐type glutamate receptors (AMPARs) lacking an edited GluA2 subunit are calcium‐permeable (CP) and contribute to synaptic plasticity in several hippocampal interneuron types, although their precise role in the neocortex is not well described. We explored the presence of CP‐AMPARs at pyramidal cell (PC) inputs to Martinotti cells (MCs) and basket cells (BCs) in layer 5 of the developing mouse visual cortex (postnatal days 12–21). GluA2 immunolabelling was stronger in MCs than in BCs. A differential presence of CP‐AMPARs at PC‐BC and PC‐MC synapses was confirmed electrophysiologically, based on measures of spermine‐dependent rectification and CP‐AMPAR blockade by 1‐naphtyl acetyl spermine using recordings from synaptically connected cell pairs, NPEC‐AMPA uncaging and miniature current recordings. In addition, CP‐AMPAR expression in BCs was correlated with rapidly decaying synaptic currents. Computer modelling predicted that this reduces spike latencies and sharpens suprathreshold responses in BCs, which we verified experimentally using the dynamic clamp technique. Thus, the synapse‐specific expression of CP‐AMPARs may critically influence both plasticity and information processing in neocortical microcircuits.
The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
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