RESUMO
The extracellular concentration of glutamate and other related excitatory amino acids (EAA) is regulated by the action of transporter proteins located on either presynaptic terminals or adjacent astroglial processes. Recent molecular advances have led to the cloning of three separate cDNAs encoding for Na(+)-dependent glutamate transporters; two are thought to be primarily glial in origin (GLAST and GLT-1) and the third (EAAC1) is localized to neurons in the brain and other nonneural tissues. An EAAC1 cDNA was initially cloned from rabbit small intestine (13). In this study, we report isolation and characterization of the homologous clone from rat brain. Northern blot hybridization revealed high levels of EAAC1 mRNA in rat brain and kidney and low levels in heart, lung, and skeletal muscle. Transient expression of EAAC1 in HeLa cells resulted in an increase in Na(+)-dependent high-affinity L-[3H]glutamate and D-[3H]aspartate transport. The pharmacological profile of EAAC1 was very similar to that reported for the rabbit and human EAAC1 homologues. Transport activity was potently inhibited by D- and L-threo-beta-hydroxyaspartate and L-trans-pyrrolodine-2,4-dicarboxylate. Dihydrokainate and L-alpha-aminoadipate did not inhibit transport at concentrations below 1 mM. Oligonucleotide cDNA probes (45-mer) were constructed and labeled with 35S-ATP for film- and emulsion-based in situ hybridization of rat brain. EAAC1 mRNA had the highest density in the cerebellar granule cell layer, hippocampus, superior colliculus, and neocortex. Sections that were emulsion-dipped and counterstained with cresyl violet revealed EAAC1 labeling localized exclusively over neuronal cell bodies, including some nonglutamatergic neurons such as spinal cord ventral horn cells.