SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions.

SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons.

An Improved Method for Growing Primary Neurons on Electron Microscopy Grids Co-Cultured with Astrocytes.

With the increasing popularity of cryo-electron tomography (cryo-ET) in recent years, the quest to establish a method for growing primary neurons directly on electron microscopy grids (EM grids) has been ongoing. Here we describe a straightforward way to establish a mature neuronal network on EM grids, which includes formation of synaptic contacts. These synapses were thin enough to allow for direct visualization of small filaments such as SNARE proteins tethering the synaptic vesicle (SV) to the active zone plasma membrane on a Titan Krios without prior focused ion-beam milling.

The pathogenic N650K variant in the GluN1 subunit regulates the trafficking, conductance, and pharmacological properties of NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission in the mammalian brain, and their physiological importance is underscored by the large number of pathogenic mutations that have been identified in the receptor's GluN subunits and associated with a wide range of diseases and disorders. Here, we characterized the functional and pharmacological effects of the pathogenic N650K variant in the GluN1 subunit, which is associated with developmental delay and seizures. Our microscopy experiments showed that when expressed in HEK293 cells (from ATCC®), the GluN1-N650K subunit increases the surface expression of both GluN1/GluN2A and GluN1/GluN2B receptors, but not GluN1/GluN3A receptors, consistent with increased surface expression of the GluN1-N650K subunit expressed in hippocampal neurons (from embryonic day 18 of Wistar rats of both sexes).

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials.

The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP ) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP -interaction site (Y1006-Q1031) is involved in TRPA1 regulation.