Pharmacological characterization and second-messenger regulation of the sodium-dependent glutamate transporter subtype, EAAC1

Glutamate and aspartate are the predominant excitatory neurotransmitters in the mammalian central nervous system. Glutamate is thought to be removed from the synapse by a family of sodium-dependent high-affinity transporters. The pharmacology of this transport activity is consistent with at least two subtypes. In the initial part of the present study, the properties of transport in two brain regions were compared and evidence for additional heterogeneity was developed. While these studies were underway, four cDNAs encoding different subtypes of transporters were isolated (GLAST, GLT1, EAAC1, EAAT4). One of these subtypes, EAAC1, is expressed by neurons and is enriched in forebrain regions. This data suggested that EAAC1 would reconstitute the properties of transport observed in forebrain synaptosomes. Although EAAC1 immunoreactivity is present in forebrain synaptosomes, its pharmacological properties do not match those observed in these synaptosomes. However, the pharmacological properties of EAAC1 do parallel those observed in C6 glioma. Western analyses demonstrated that C6 glioma do, indeed, express EAAC1 and do not express GLAST and GLT1. Based on these observations, C6 glioma was identified as a model system for the study of EAAC1.; Because EAAC1 has consensus sites for both protein kinase A (PKA) and protein kinase C (PKC), C6 glioma were pretreated with either forskolin or phorbol ester. Although forskolin did not affect transport activity, phorbol ester caused a rapid (within 2 minutes) and robust (2- to 3-fold) increase in L- ({dollar}sp3{dollar}H) -glutamate transport activity. The increase in transport was due to an increase in V{dollar}sb{lcub}rm max{rcub}{dollar} with no change in K{dollar}sb{lcub}rm m{rcub}{dollar}. The PKC inhibitor, chelerythrine, blocked this increase in activity and the inactive phorbol ester (4{dollar}alpha{dollar}-phorbol) did not alter transport activity. This rapid increase in sodium-dependent L- ({dollar}sp3{dollar}H) -glutamate transport activity may provide a novel mechanism for protection against acute insults to the central nervous system.