Rabphilin-3A undergoes phase separation to regulate GluN2A mobility and surface clustering

Area of Science:

• Neuroscience
• Molecular Biology
• Cell Biology

Background:

• N-methyl-D-aspartate receptors (NMDARs) are critical for brain function, mediating excitatory neurotransmission and synaptic plasticity.
• Specific NMDAR subunits, GluN2A and GluN2B, have distinct distributions and mobilities at synapses, but the mechanisms organizing GluN2A remain unclear.

Purpose of the Study:

• To investigate the role of Rabphilin-3A (Rph3A) in the organization and stabilization of GluN2A clusters.
• To elucidate the mechanism by which Rph3A influences GluN2A clustering at neuronal surfaces.

Main Methods:

• Investigated the phase separation properties of Rph3A, focusing on its N-terminal domain and arginine residues.
• Examined the binding interaction between Rph3A and the C-terminal domain of GluN2A.
• Studied the formation of a complex involving Rph3A, GluN2A, and PSD95.
• Disrupted Rph3A phase separation and assessed its impact on GluN2A clustering, localization, stability, and synaptic response in hippocampal neurons.

Main Results:

• Rabphilin-3A (Rph3A) exhibits arginine-dependent phase separation.
• Rph3A phase separation promotes GluN2A clustering by binding to GluN2A's C-terminal domain.
• A ternary complex of Rph3A, GluN2A, and PSD95 enhances Rph3A phase separation.
• Inhibition of Rph3A phase separation impairs GluN2A clustering, synaptic localization, stability, and function.

Conclusions:

• Rph3A phase separation is a key mechanism for organizing and stabilizing GluN2A at synaptic and extrasynaptic sites.
• This process is essential for maintaining normal GluN2A function and synaptic responses in hippocampal neurons.
• The findings reveal a novel role for Rph3A-mediated phase separation in regulating NMDAR organization and synaptic plasticity.