SUMMARY AMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1–4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ubiquitination of all subunits occurs exclusively on AMPARs located on the plasma membrane post-endocytosis. The sites of ubiquitination were mapped to Lys-868 in GluA1 and Lys-870/Lys-882 in GluA2 C-terminals. Mutation of these lysines did not affect basal surface expression or AMPA-induced internalization of GluA1 and GluA2 subunits. Instead, it reduced the intracellular trafficking of AMPARs to the late endosomes and thus protein degradation. These data indicate that ubiquitination is an important regulatory signal for controlling AMPAR function, which may be crucial for synaptic plasticity.

Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.

Kasula et al. use single-molecule imaging to reveal the diffusional signature for the SNARE proteins Munc18-1 and syntaxin-1A during secretory vesicle priming. The authors show that a conformational change in the Munc18-1 domain 3a hinge-loop regulates engagement of syntaxin-1A in the SNARE complex.