Effect of Phosphorylation on Structure of C-Terminal Segment of AMPA Receptor

Abstract

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is the primary contributor to fast excitatory transmission in neurons. The AMPA receptor can be divided into four domains. Extracellularly, there are the amino-terminal and ligand-binding domains. The transmembrane domain serves as the actual ion-channel pore and, of course, links the extracellular domains to the cytoplasmic domain. Of these four domains, the structure of the outermost three has been shown in detailed crystal structures of the tetramer. However, very little is known about the structure of the cytoplasmic domain. Although it is widely thought that this segment is highly disordered, it is unknown whether local order (higher levels of secondary and/or tertiary structure) exists in the cytoplasmic terminus, or whether structural changes may occur as conformational shifts in the terminal due to functional modifications. Previous studies have established phosphorylation sites at residues S818, S831, and T840 in the GluA1 subtype receptor. These studies examined a representative membrane-proximal section of the GluA1 c-terminus comprising residues 809-841 in order to consider structural changes brought about by these phosphorylation events. The peptide was examined using circular dichroism (CD) investigation, which showed a conversion to greater helix content in the phosphomimetic sample. CD studies of the peptide in a solution of small unilamellar vesicles were conducted and showed that the increase in helical content is also present in the context of close proximity to a lipid membrane. To confirm, single molecule fluorescence resonance energy transfer (smFRET) was used to examine the peptide in both the unphosphorylated state and in the PKCα-phosphorylated state, in order to gauge the distance between two native cysteines in the peptide. Phosphorylation yielded a reduced distance between these cysteines, indicative of a shift to more compressed secondary structure, that is, coil to helix.