Trafficking mechanisms that regulate AMPA receptor Ca2+ permeability

AMPA receptors (AMPARs) mediate the majority of synaptic excitation in the brain, so the precise regulation of AMPAR trafficking is crucial to brain function. AMPARs are tetrameric assemblies of subunits GluA1-4, and most AMPARs contain GluA2, which is important because this subunit renders AMPARs calcium-impermeable. The small population of GluA2-lacking, calcium-permeable AMPARs is not expressed at synapses under basal conditions. Precise regulation of their synaptic expression therefore provides intriguing possibilities for synaptic calcium signaling events.

PICK1 binds GluA2, and is required for AMPAR trafficking events that modulate AMPAR Ca2+-permeability. PICK1 restricts GluA2 traffic from recycling endosomes back to the plasma membrane, but has been suggested to function in endocytosis, although this has not been tested. Data from our lab indicate that PICK1 binds core components of the endocytic machinery.

The objective of this proposal is to apply novel techniques to investigate the role of PICK1 as a regulator of GluA2 endocytosis, and examine how the actin cytoskeleton is regulated during this process by PICK1 and also by cortactin. Since endocytosis is a very dynamic process, live imaging using pHluorin-tagged subunits to analyse AMPAR trafficking, and TIRF microscopy to visualise protein recruitment to endocytic sites will be key techniques. A detailed view can only be obtained through EM. We will combine these techniques using Correlative Light and Electron Microscopy to acquire an ultrastructural view of neuronal endocytosis events observed live. A mathematical model will be developed based on cytoskeletal and membrane dynamics for direct comparison to imaging data.