Structural Effects of Phosphorylation on C-Terminal Segment of AMPA Receptor

Abstract

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is the primary contributor to neuronal fast excitatory transmission, which is key to learning and building memory. The AMPAR can be divided into four modular domains. The structure of the outermost three has been shown in detailed crystal structures, but very little is known about the structure of the cytoplasmic domain. Although it is thought that this segment is disordered, it is unknown whether local order may exist in the cytoplasmic terminus, or whether structure changes may occur as conformational shifts due to functional modifications. Previous studies have established protein kinase C (PKC) phosphorylation sites at residues S818, S831, and T840 in the GluA1 subtype receptor, and confirmed their effect to enhance receptor function. Our studies examined a membrane-proximal section of the GluA1 cytoplasmic terminus in order to consider local structural changes brought about by these phosphorylation events. Peptides were examined using Fourier transform infrared technology, which showed a conversion to lesser helix content, more extension, in the phospho-protein. Studies of the peptides in a solution of small unilamellar vesicles were conducted and showed that the change in secondary structure is enhanced in the context of close proximity to a charged lipid membrane. To confirm, single molecule fluorescence resonance energy transfer was used to examine the peptide in both the unphosphorylated and phosphomimetic states. Phosphorylation yielded a narrower breadth of FRET efficiencies between labeled residues bookending all three PKC-phosphorylated sites, indicative of the reduced helix allowing the peptide flexibility to primarily probe a larger distance between flors. Again this effect was dependent on charged lipid environment. Ongoing electrophysiological studies are being conducted to assess the effects of phosphorylation on receptor function in the context of a physiological lipid membrane environment.