The influence of binding and crowding on the mobility and organization of transmembrane proteins within the synapse

Abstract

Postsynaptic transmembrane proteins are important elements of synapses. Positioning and mobility of each member of this large class of proteins dictate their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. However, factors that control receptor mobility and spatial organization within the synapse are not well understood. Scaffold proteins in the PSD are abundant receptor binding partners, and electron microscopy suggests that the PSD is highly crowded, potentially restricting the diffusion of receptors regardless of binding. I complemented computational approaches with empirical data to test the effect of synaptic crowding on receptor movement and positioning in rat hippocampal neurons. Simulation of receptor diffusion in synapses containing previously measured distribution of scaffold proteins predicted that the variation of receptor size and the organization of scaffold proteins each strongly influences the positioning and mobility of receptors within the synapse. Using high-resolution and super-resolution imaging of custom-designed transmembrane (TM) probes, I found that a single-pass TM protein with a single PDZ binding motif was more mobile than the much larger AMPAR. Moreover, either the single binding motif or an increase in bulk slowed the synaptic movement of the small TM probe, suggesting that both crowding and binding can limit the escape of AMPARs from the synapse. By measuring synaptic architecture and TM protein movement simultaneously, I found that the TM probe that does not bind PSD-95 could be as stabilized as the binding variant in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize receptors even in the absence of binding. Interestingly, single-molecule mapping showed that excitatory neurotransmitter release preferentially occurs over these regions. Using a deterministic computer modeling approach, I found that this type of release-receptor alignment can modulate the synapse potency by 20-30%. Altogether these results demonstrate that tight protein packing within the PSD may organize TM proteins within the synapse by modulating their dwell times, thereby tuning synaptic strength.