THE NEURONAL AND EPITHELIAL HUMAN HIGH-AFFINITY GLUTAMATE TRANSPORTER- INSIGHTS INTO STRUCTURE AND MECHANISM OF TRANSPORT

High affinity transport of glutamate across plasma membranes of brain neurons and epithelia is mediated by a Na+- and K+-coupled electrogeni c transporter. Here we report the primary structure and functional cha racterization of the human high affinity glutamate transporter (HEAAC1 ). A unique characteristic of HEAAC1-mediated transport is that the af finity for glutamate and the maximal transport rate are strongly depen dent on membrane potential. Our data provide new insights into individ ual steps of high affinity glutamate transport and show that the trans port mechanism is distinct from that of the gamma-aminobutyric acid tr ansporter GAT-1 and the Na+/glucose transporter SGLT1. Under voltage c lamp condition, HEAAC1 mediated large substrate-evoked inward currents (up to 1 mu A). The substrate specificity, stereospecificity, the K-m value (30 +/- 3 mu M at -60 mV) of the L-glutamate-evoked current, an d Northern analysis all agree with previously reported characteristics of high affinity glutamate transport in brain. In contrast to SGLT1 a nd GAT-1, voltage jump studies of HEAAC1 yielded only minor relaxation currents. Classic inhibitors of brain glutamate uptake such as DL-thr eo-beta-hydroxyaspartate, L-trans-pyrrolidine 2,4,-dicarboxylic acid ( PDC), and dihydrokainate were found to be either transport substrates or to have no significant effect on glutamate transport. We also found that the maximal transport rate for PDC was markedly reduced compared to that for L-glutamate. We propose that PDC most likely reduces the turnover rate of the transporter. A search of the sequence data bases revealed weak homology of HEAAC1 to the H+- coupled vesicular monoamin e transporter, suggesting an evolutionary link between plasma membrane and vesicular transporters.