Several subtypes of
sodium-dependent high affinity (SDHA)
glutamate transporters have been pharmacologically differentiated in brain tissue. Recently, four distinct cDNAs (EAAC1, GLT1, GLAST, and EAAT4) encoding Na+-dependent
glutamate transporters have been isolated, but the properties of some of these transporters do not fully match the properties of transport observed in brain tissue or astrocyte-enriched cultures. The purpose of the current investigation was to determine whether the pharmacological properties of EAAC1 parallel those observed in cortical or cerebellar synaptosomes, C6
glioma, or primary astrocyte-enriched cultures. EAAC1
cRNA was expressed in Xenopus oocytes, an expression system with no detectable endogenous Na+-dependent
glutamate transport activity. EAAC1-mediated
glutamate transport was >98% Na+ dependent, and the transport was saturable and consistent with a single site.
Glutamate transport activates in EAAC1-injected oocytes and C6
glioma have similar Km values for
glutamate (Km = 15-24 microM) and Na+ (apparent Km = 35-50mM), and these values markedly differ from those observed in rat synaptosomes (
glutamate, Km = 1-5 microM; Na+,
Km = 13-20 mM). Several
excitatory amino acid analogues were tested as inhibitors of L-[3H]
glutamate transport in oocytes expressing EAAC1
cRNA. The potencies of several compounds for inhibition of EAAC1-mediated transport differed from those previously observed in cerebellar synaptosomes and astrocyte-enriched cultures. Although EAAC1-mediated transport and cortical synaptosomal transport have similar pharmacological profiles, five
excitatory amino acid analogues were > or= 3-fold more potent as inhibitors of transport into cortical synaptosomes than of transport into EAAC1-injected oocytes. For example, L-trans-pyrrolidine-2,4-dicarboxylate was approximately 5-fold more potent in cortical synaptosomes, and
dihydrokainate was approximately 10-fold more potent in cortical synaptosomes than in EAAC1-injected oocytes. In contrast, all of the compounds examined inhibit transport observed in C6
glioma wtih potencies similar to that observed in oocytes injected with EAAC1
cRNA. Consistent with these data, C6
glioma expressed EAAC1- but not GLT1- and GLAST-like immunoreactivity. Although this immunoreactivity migrated as
proteins of slightly different molecular masses in each system, treatment with
N-glycosidase F shifted all
proteins to a molecular mass consistent with that predicted from the
cDNA sequence. In cortical synaptosomes, EAAC1-, GLT1-, and GLAST-like immunoreactives were apparent. These results indicate that (i) EAAC1 but not GLAST or GLT1 transporters are expressed in C6
glioma, (ii) synaptosomes contain a heterogeneous population of transporters, (iii) EAAC1 does not account for the pharmacology previously observed in cortical synaptosomes, and (iv) based on the pharmacology and tissue distribution of EAAC1, GLT1, GLAST, and EAAT4, it appears that there are additional
glutamate transporter subtypes.