COMPARISON OF NA-DEPENDENT GLUTAMATE TRANSPORT ACTIVITY IN SYNAPTOSOMES, C6 GLIOMA, AND XENOPUS OOCYTES EXPRESSING EXCITATORY AMINO-ACID CARRIER-1 (EAAC1)()

Several subtypes of sodium-dependent high affinity (SDHA) glutamate tr ansporters have been pharmacologically differentiated in brain tissue. Recently, four distinct cDNAs (EAAC1, GLT1, GLAST, and EAAT4) encodin g Na+-dependent glutamate transporters have been isolated, but the pro perties of some of these transporters do not fully match the propertie s of transport observed in brain tissue or astrocyte-enriched cultures . The purpose of the current investigation was to determine whether th e pharmacological properties of EAAC1 parallel those observed in corti cal or cerebellar synaptosomes, C6 glioma, or primary astrocyte-enrich ed cultures. EAAC1 cRNA was expressed in Xenopus oocytes, an expressio n system with no detectable endogenous Na+-dependent glutamate transpo rt activity. EAAC1-mediated glutamate transport was >98% Na+ dependent , and the transport was saturable and consistent with a single site. G lutamate transport activates in EAAC1-injected oocytes and C6 glioma h ave similar K-m values for glutamate (K-m = 15-24 mu M) and Na+ (appar ent K-m = 35-50 mM), and these values markedly differ from those obser ved in rat synaptosomes (glutamate, K-m = 1-5 mu M; Na+, K-m = 13-20 m M). Several excitatory amino acid analogues were tested as inhibitors of L-[H-3]glutamate transport in oocytes expressing EAAC1 cRNA. The po tencies of several compounds for inhibition of EAAC1-mediated transpor t differed from those previously observed in cerebellar synaptosomes a nd astrocyte-enriched cultures. Although EAAC1-mediated transport and cortical synaptosomal transport have similar pharmacological profiles, five excitatory amino acid analogues were greater than or equal to 3- fold more potent as inhibitors of transport into cortical synaptosomes than of transport into EAAC1-injected oocytes. For example, L-trans-p yrrolidine-2,4-dicarboxylate was similar to 5-fold more potent in cort ical synaptosomes, and dihydrokainate was similar to 10-fold more pote nt in cortical synaptosomes than in EAAC1-injected oocytes. In contras t, all of the compounds examined inhibit transport observed in C6 glio ma with potencies similar to that observed in oocytes injected with EA AC1 cRNA. Consistent with these data, C6 glioma expressed EAAC1- but n ot GLT1- and GLAST-like immunoreactivity. Although this immunoreactivi ty migrated as proteins of slightly different molecular masses in each system, treatment with N-glycosidase F shifted all proteins to a mole cular mass consistent with that predicted from the cDNA sequence. In c ortical synaptosomes, EAAC1-, GLT1-, and GLAST-like immunoreactivities were apparent. These results indicate that (i) EAAC1 but not GLAST or GLT1 transporters are expressed in C6 glioma, (ii) synaptosomes conta in a heterogeneous population of transporters, (iii) EAAC1 does not ac count for the pharmacology previously observed in cortical synaptosome s, and (iv) based on the pharmacology and tissue distribution of EAAC1 , GLT1, GLAST, and EAAT4, it appears that there are additional glutama te transporter subtypes.