Mechanisms regulating lateral diffusion and positioning of glutamate receptors within the postsynaptic density (PSD) determine excitatory synaptic strength. Scaffold proteins in the PSD are abundant receptor binding partners, yet electron microscopy suggests that the PSD is highly crowded, potentially restricting the diffusion of receptors regardless of binding. However, the contribution of macromolecular crowding to receptor retention remains poorly understood. We combined experimental and computational approaches to test the effect of synaptic crowding on receptor movement and positioning in Sprague Dawley rat hippocampal neurons. We modeled AMPA receptor diffusion in synapses where the distribution of scaffold proteins was determined from photoactivated localization microscopy experiments, and receptor-scaffold association and dissociation rates were adjusted to fit single-molecule tracking and fluorescence recovery measurements. Simulations predicted that variation of receptor size strongly influences the fractional synaptic area the receptor may traverse, and the proportion that may exchange in and out of the synapse. To test the model experimentally, we designed a set of novel transmembrane (TM) probes. A single-pass TM protein with one PDZ binding motif concentrated in the synapse as do AMPARs yet was more mobile there than the much larger AMPAR. Furthermore, either the single binding motif or an increase in cytoplasmic bulk through addition of a single GFP slowed synaptic movement of a small TM protein. These results suggest that both crowding and binding limit escape of AMPARs from the synapse. Moreover, tight protein packing within the PSD may modulate the synaptic dwell time of many TM proteins important for synaptic function.
Significance statement: Small alterations to the distribution within synapses of key transmembrane proteins, such as receptors, can dramatically change synaptic strength. Indeed, many diseases are thought to unbalance neural circuit function in this manner. Processes that regulate this in healthy synapses are unclear, however. By combining computer simulations with imaging methods that examined protein dynamics at multiple scales in space and time, we showed that both steric effects and protein-protein binding each regulate the mobility of receptors in the synapse. Our findings extend our knowledge of the synapse as a crowded environment that counteracts molecular diffusion, and support the idea that both molecular collisions and biochemical binding can be involved in the regulation of neural circuit performance.
Keywords: FRAP; GFP imaging; inducible dimerization; single-molecule tracking; stochastic modeling; uPAINT.
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