UC San Diego

AMPA type glutamate receptors are of fundamental importance for brain function, as they mediate the majority of fast excitatory synaptic transmission. They function by opening their transmembrane ion channel upon binding glutamate in the synapse. In addition to their roles in basal synaptic function, AMPA-Rs have also been implicated in synapse formation and stabilization, and their regulation is a primary mechanism underlying synaptic plasticity, a cellular correlate of learning and memory. Here, in 3 separate studies, I investigate different components of AMPA-R function: mechanisms of AMPA-R assembly, the AMPA-R interactome, and mechanisms of AMPA-R modulation by auxiliary subunits. In order to better understand the assembly and trafficking of AMPA-Rs, I investigated early AMPA-R subunit assembly mechanisms. Using a recombinant system, I purified and isolated both mature tetrameric AMPA-Rs as well as the transient AMPA-R dimeric biosynthetic intermediates. I determined the three -dimensional single particle EM structures of both of these AMPA-R forms. This work revealed that efficient subunit assembly requires a preferred conformation of the AMPA-R biosynthetic intermediates in order to efficiently progress into the mature form. This proposed model of assembly complements the x-ray crystallography structure of the full length recombinant AMPA-R solved by Eric Gouaux's group perfectly because this structure shows that there is subunit cross over in the tetrameric AMPA-R complex. In a collaborative proteomics project we utilized mass spectrometry to identify novel proteins that interact with AMPA receptors in the brain. In this way, I came to focus on a predicted protein in the rat genome, GSG1L. Using detailed molecular, cellular, electrophysiological, and biochemical experiments, I validated the interaction between AMPA-Rs and GSG1L and determined that GSG1L enhances AMPA-R surface expression and modulates AMPA-R channel kinetics by slowing desensitization and slowing recovery from desensitization. Thus GSG1L is novel unique modulator of AMPA-R function. In a third project, I investigate the detailed molecular mechanisms of AMPA-R interaction with and modulation by a known class of AMPA-R auxiliary subunits, the cornichon homologues. I have identified specific domains and clusters of residues involved on both sides of the interaction. Most importantly, I show direct evidence for an interaction between the cornichon extracellular loop and both of the extracellular AMPA-R domains, the ligand binding domain and the N-terminal domain. Functional studies had previously hypothesized that such an interaction might occur with the AMPA-R ligand binding domain, however my work confirms and extends it by demonstrating the additional interaction with the N-terminal domain. Overall, this data suggest a completely novel role for the AMPA-R N -terminal domain in which interactions with auxiliary subunits are involved in allosteric modulation of AMPA-R channel function. By elucidating the molecular mechanisms of several aspects of AMPA-R function, this work aids our understanding of synaptic transmission and, and may ultimately useful in efforts to develop therapeutic agents for AMPA-R related disorders