No abstract

Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 mm, to 25% of the initial transport activity. This downregulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport.[ 32P] Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. lmmunoprecipitation of [35S] methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. lmmunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site. Key Words: Glutamate transporter-Protein kinase C-Phorbol esterPhosphorylation.

The reuptake of excitatory amino acids, such as glutamate, terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. The L-glutamate/L-aspartate transporter (GLAST-1) is the first member of the recently discovered glutamate transporter family, which includes GLT-1 and EAAC1. The neutral amino acid carrier ASCT1 is structurally closely related to this new family of membrane proteins. Transmembrane transport of neutral amino acids is expected to differ in its binding site from that of the acidic excitatory amino acids glutamate and aspartate. Three positively charged amino acid residues, Arg-122, Arg-280, Arg-479, and one polar Tyr-405 are conserved in all glutamate transporters. They are replaced by apolar amino acid residues in the ASCT1 sequence. We exchanged these residues in the GLAST-1-specific cDNA by site-directed mutagenesis. cRNAs of these mutants were expressed in the Xenopus oocyte system. The functional characterization of the mutants R122I and R280V and the double mutant R122I, R280V revealed that the mutations have no influence on the intrinsic properties and kinetics of glutamate transport but alter the Km-values for L-aspartate and the competitive inhibitor D,L-threo-3-hydroxy aspartate. Substitutions of Tyr-405 by Phe (Y405F) and Arg-479 (R479T) by Thr completely inactivate the glutamate transporter. Immunoprecipitations of [35S]methionine-labeled transporter molecules indicate similar expression levels of wild-type and mutant transporters. Immunostaining of oocyte sections clearly proves the correct targeting to and integration of the mutant GLAST-1 proteins in the plasma membrane. Our results suggest the pivotal function of the hydroxy group of the highly conserved Tyr-405 and the positively charged Arg-479 in the binding of the negatively charged acidic neurotransmitter glutamate.

The L-glutamate transporter GLAST-1 belongs to the newly discovered family of Na(+)-dependent, high-affinity glutamate transporters, which are involved in the regulation of synaptic excitatory neurotransmitter concentration in mammalian brain. The members of this family have a similar topological organisation with at least six transmembrane helices (TMHs) and two putative N-glycosylation sites located in the extracellular loop connecting TMH 3 and TMH 4. Besides these two conserved N-glycosylation motifs at Asn206 and Asn216, GLAST-1 possesses an additional one at Asn35. The putative N-glycosylation consensus motifs (Asn-Xaa-Ser/Thr) were deleted by replacement of Asn206 and/or Asn216 by Thr using site-directed mutagenesis (mutants N206T, N216T and N206,216T). The cDNAs encoding wild-type GLAST-1 and the three glycosylation-defective transport proteins were expressed in the Xenopus laevis oocyte system. Immunoprecipitation of the [35S]methionine-labeled and glycopeptidase-F-treated transporter molecules indicates that GLAST-1 is glycosylated at Asn206 and Asn216, whereas Asn35 remains unglycosylated. To assess a possible functional role of the two glycosylation sites wild-type and glycosylation-deficient GLAST-1 were expressed in Xenopus oocytes and characterized functionally by using the whole-cell voltage-clamp technique. The results prove that N-glycosylation has no impact on the transport activity of GLAST-1.

The rat brain L-glutamate/L-aspartate transporter GLAST-1 is a member of a family of Na(+)-dependent high-affinity L-glutamate transporters proposed to be involved in the termination and modulation of excitatory neurotransmitter signals. Application of electrophysiological and radiotracer techniques on Xenopus oocytes expressing cloned GLAST-1 revealed that the apparent Km value of the transporter for L-glutamate and Na+ ions did not depend on voltage while the maximal transport rate increased with more negative potentials, indicative of a low-field access channel. The apparent Km value of the transporter for L-glutamate depends on the Na+ concentration, suggesting that substrate and ions are transported by GLAST-1 in a simultaneous manner. All of the L-glutamate uptake blockers tested either were substrates or did not affect the current induced by L-glutamate. The changes in the amplitude of the current induced by simultaneous application of two substrates can be interpreted by a competition for one binding site.