THE GLUTAMATE TRANSPORTER EAAT4 IN RAT CEREBELLAR PURKINJE-CELLS - A GLUTAMATE-GATED CHLORIDE CHANNEL CONCENTRATED NEAR THE SYNAPSE IN PARTS OF THE DENDRITIC MEMBRANE FACING ASTROGLIA

Antibodies to an excitatory amino acid transporter (EAAT4) label a gly coprotein of similar to 65 kDa strongly in the cerebellum and weakly i n the forebrain. Cross-linking of cerebellar proteins with bis(sulfosu ccinimidyl) suberate before solubilization causes dimer bands of EAAT4 and both dimer and trimer bands of the other glutamate transporters G LAST (EAAT1) and GLT (EAAT2) to appear on immunoblots. In contrast to GLAST, GLT, and EAAC (EAAT3), EAAT4 is unevenly distributed in the cer ebellar molecular layer being strongly expressed in parasagittal zones ; it is located in cerebellar Purkinje cells, and the highest con; cen trations are seen in ones expressing high levels of zebrin II (aldolas e C). The labeling of Purkinje cell spines and thin dendrites is stron ger than that of large diameter dendrites and cell bodies. EAAT4 is pr esent at low concentrations in the synaptic membrane, but is highly en riched in the parts of the dendritic and spine membranes facing astroc ytes (which express GLAST and GLT) compared with parts facing neuronal membranes, suggesting a functional relationship with glial glutamate transporters. The presence of EAAT4 in intracellular cisterns and mult ivesicular organelles may reflect turnover of transporter in the plasm a membrane. The total Purkinje cell spine surface and the EAAT4 concen tration were found to be 1.1 m(2)/cm(3) and 0.2 mg/cm(3), respectively , in the molecular layer, corresponding to 1800 molecules/mu m(2). The juxtasynaptic location of EAAT4 may explain electrophysiological obse rvations predicting the presence of a neuronal glutamate transporter n ear the release site at a Purkinje cell spine synapse. EAAT4 may funct ion as a combined transporter and inhibitory glutamate receptor.