Control of AMPA receptor function by phosphorylation

Emory University, Atlanta GA

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Abstract

DESCRIPTION (provided by applicant): The AMPA-type glutamate receptors are ligand-gated cation channels that mediate fast excitatory neurotransmission in the brain, and thus are critically involved in all aspects of brain function including cognition, movement, learning, and memory. The function and number of postsynaptic AMPA receptors are dynamically regulated to control the strength of synaptic connections, and this plasticity is a key feature of cellular models of learning and memory. Signals that trigger synaptic plasticity lead to phosphorylation of AMPA receptors by protein kinases, and this phosphorylation controls AMPA receptor trafficking and function. Phosphorylation by protein kinase C (PKC) or Ca2+/calmodulin dependent kinase II (CamKII) of an intracellular serine residue (Ser831) located on the GluR1 subunit enhances AMPA receptor function to increase synaptic strength during expression of long-term potentiation (LTP), one model of synaptic plasticity. Although previous studies observed that CamKII phosphorylation of GluR1 enhances the single channel conductance, no conceptual or structural mechanism has been described for this unique form of ion channel regulation. The goal of the experiments proposed here is to understand functionally, structurally, and conceptually how phosphorylation of GluR1 Ser831 potentiates AMPA receptor function. We will focus on Ser831 in GluR1 because of the unique mechanism of potentiation (increased unitary conductance), and will expand the study to evaluate for the first time how three nearby phosphorylation sites (Ser818, Thr840, Ser845) might functionally interact with phospho-Ser831. Furthermore, we will test whether the effects of phospho-Ser831 reflect either intra-protein interactions between the phospho-Ser831 and intracellular portions of the receptor, or inter-protein interactions between phospho-Ser831 and GluR1 binding partners. Completion of these studies will provide a comprehensive functional and structural understanding of an under-studied feature of synaptic plasticity-phosphorylation mediated changes in postsynaptic AMPA channel function. The proposed experiments address three questions: 1. What is the mechanism by which phosphorylation regulates AMPA receptor function? Single channel currents will be recorded to determine how phosphorylation of Ser831 controls GluR1 function. We will also evaluate the interactions of Ser831 with nearby phosphorylation sites, and validate our conclusions in neurons. 2. What is the structural basis for phospho-serine regulation of AMPA receptor function? We will identify intracellular GluR1 residues as phospho-Ser831 hydrogen bonding partners. We will additionally search for inter-protein interactions involving GluR1 that depend on the phosphorylation of Ser831. 3. Can models of independent subunit gating describe AMPA receptor regulation by phosphorylation? We will analyze the response of patches with one active GluR1 channel (plus stargazin) to the rapid application of a maximally effective concentration of glutamate. These data will be used to develop a novel model of subunit gating that can account for the potentiation of GluR1 channel function by phosphorylation of Ser831.