The AMPA type glutamate receptors are heteromultimeric proteins consisting of 4 or 5 subunits (GluR1Ð4). We, and others, using yeast two-hybrid and biochemical analyses have identified a number of proteins that interact with specific motifs on the intracellular c-terminus of the GluR2 AMPA receptor subunit. These proteins, some of which are novel, are thought to be involved in the targeting to and regulation of AMPARs at excitatory synapses in neurons. However, the mechanisms by which these interactors achieve this are only starting to be investigated.

To study the functional roles of these interacting proteins we have developed approaches to investigate these interactions in hippocampal neurons in acute brain slices. To block the interactions of specific proteins we used short peptides corresponding to binding motifs for these proteins and introduced them into hippocampal neurons during whole-cell patch-clamp recordings. The effects of the peptides on AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) during basal transmission and during long-term synaptic plasticity were investigated. These acute studies show that interaction proteins such as NSF, PICK1 and GRIP regulate AMPA receptors over a time scale of minutes at synapses, and that these interactions are required for the expression of long-term synaptic plasticity.

To investigate the more long-term effects of these interactions, we used Sindbis virus to express blocking peptides, full length interacting proteins, or mutant interacting proteins in acute hippocampal slices maintained overnight in culture. These studies show that the interactors can regulate surface expression and subunit composition of synaptic AMPA receptors but that distinct interactors have different roles in this regulation. Using this approach to study mutant forms of the interactors, this approach has also allowed us to investigate the downstream signalling mechanisms of these proteins.

These novel approaches have allowed us to directly investigate the functions of these poorly characterised proteins in neurons in acute brain slices. When combined with other techniques such as biochemical analyses and immunocytochemistry, these studies have enabled much progress to be made in understanding the role of proteinÐprotein interactions in regulating AMPA receptors at synapses in neurons.

All procedures accord with current local guidelines.Research Symposium Ð Functional Genomics in the Nervous SystemResearch Symposium Ð Functional Genomics in the Nervous SystemResearch Symposium Ð Functional Genomics in the Nervous Systemnding the role of proteinÐprotein interactions in regulating AMPA receptors at synapses in neurons.All procedures accord with current local guidelines.