Copine-6 integrates calcium, phosphoinositide and Rab11 signals to coordinate glutamate receptor recycling

Endosomal trafficking is a major pathway that delivers cell-surface proteins, including glutamate receptors, to support neurotransmission and normal brain functions. Activity-dependent insertion of glutamate receptors is essential for synaptic plasticity, learning and memory. Copine-6 is a neuronal-specific calcium (Ca ²⁺ ) binding protein that mediates activity-induced exocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors. The activation of N -methyl- D -aspartate (NMDA) receptors triggers Ca ²⁺ -dependent translocation of Copine-6 to intracellular endosomal compartments. However, the mechanisms underlying the activity-dependent accumulation of Copine-6 in endosomes remain unknown. Here, we show that Copine-6 exhibits Ca ²⁺ -dependent binding to phosphatidylinositol-3-phosphate (PI(3)P) through the C2B domain and displays enhanced interaction with active Rab11a in a Ca ²⁺ -independent manner via the vWA domain. Mutations in the C2B that inhibit binding to PI(3)P not only block the activity-induced translocation of Copine-6 to early endosomes, but it also causes an aberrant accumulation of Copine-6 in recycling endosomes. Consequently, loss of Copine-6 expression impairs the efficient coupling of early and recycling endosomes and blocks activity-dependent delivery of both AMPA and NMDA receptors onto the neuronal plasma membrane. These defects can be restored by re-expressing wild-type Copine-6, but not the C2B phospholipid-binding mutant. Together, our findings establish Copine-6 as a molecular bridge that enhances coupling between the early and recycling endosomal membranes, thereby facilitating the activity-dependent forward trafficking of glutamate receptors to the neuronal plasma membrane to maintain synaptic potentiation.