Glutamate transporters play a crucial role in the central nervous system, helping to terminate synaptic excitatory transmission by lowering the extracellular glutamate concentration, keeping the extracellular glutamate concentration below neurotoxic levels, and being involved in the pathology of ischaemia. Little is known about the proteins that target and anchor these transporters to the plasma membrane or modulate their activity. Using electrophysiological, molecular, biochemical and cell biological techniques, I have studied the role of such interacting proteins. I dialysed into retinal Muller cells peptides homologous to the amino- or carboxy- terminals of the GLAST glutamate transporter to competitively disrupt interactions of the terminals with endogenous proteins. By studying their effects on the membrane current generated by the transporter, using whole-cell patch-clamping, I have shown that protein interactions at the carboxy-terminal of GLAST modulate the apparent glutamate affinity of the transporter. Using the yeast two-hybrid system, I identified two proteins, the I1 imidazoline receptor candidate protein and the ajuba protein, that interact with the amino-terminal of the glutamate transporter GLT-1. To further characterise the interaction between GLT-1 and ajuba, I generated polyclonal antibodies to ajuba. Ajuba and GLT-1 interact directly in biochemical assays, and they co-localise and interact in a heterologous mammalian system. Furthermore, they are both expressed in brain and retina and they may interact and co-localise in retinal bipolar cells and cerebellar structures in vivo. Electrophysiological studies suggest that ajuba does not modulate the apparent affinity of GLT-1 for glutamate. Previous work has shown that ajuba activates MAP kinase and c-jun kinase, that it shuttles between the nucleus and the cell membrane and that it is likely to interact with the cytoskeleton. I demonstrate here that ajuba is a component of adherens junctions and binds to the epithelial-cadherin complex in biochemical assays, implying that ajuba might also have a role in the formation or regulation of adherens junctions
