Neurophysiologie Excitatory amino acid transporters (EAATs) are crucial for the termination of glutamatergic synaptic transmission and for the prevention of glutamate excitotoxicity and neuronal cell death. Five EAAT isoforms have been cloned and shown to be expressed in diverse parts of the central nervous system. EAATs form an evolutionarily conserved trimeric structure (Gendreau et al. (2004), JBC 279: 39505 - 39512), but it is not clear whether different isoforms can assemble into hetero-oligomers with novel functional properties. EAAT3 and EAAT4 are two neuronal glutamate transporters. To test whether these two transporters can associate, we co-expressed human EAAT3 and rat EAAT4 in tsA201 cells. EAAT3 was labeled with a high affinity protein purification tag (His- or strep-tag), and EAAT4 was expressed as YFP-fusion protein. YFP-EAAT4 could be purified by His affinity chromatography form cells co-expressing His-EAAT3 and YFP-EAAT4, but not from cells only expressing YFP- EAAT4 alone. Using Blue Native PAGE and fluorescence imaging, we could demonstrate that EAAT oligomers purified from co-transfected cells exhibit a lower molecular mass than YFP-EAAT4 trimers, and a higher mass than dimers, in full agreement with the notion that they represent His-EAAT3-YFP- EAAT4 heterotrimers. We are currently developing concatameric constructs to study the functional features of these heterotrimeric transporters.