Abstract 1368 Poster Session II, Sunday, 5/2 (poster 58)

Glutamate has been implicated in the pathophysiology of HIE. The glutamate in the neuro-synaptic junction is removed via the action of the amino acid transport family known as the excitatory amino acid transport family (EAAT). ASCT1 is an amino acid transporter, which bears a strong homology to the EAAT family and is highly expressed in human brain. ASCT1 undergoes an interesting pH-dependent change in its substrate specificity. At pH 7.4 it transports zwitterionic amino acids. However, at a lower pH, it undergoes a shift in its substrate specificity to include anionic amino acids. Therefore, ASCT1 may contribute to glutamate export during periods of hypoxia-induced acidosis. Therefore, a more thorough understanding of the ASCT1 transporter mechanism may contribute to our understanding of HIE. We sought to determine the mechanism of the pH-dependent shift by mutation analysis of specific amino acid residues, which are conserved within the cloned ASCT1 family members. We have demonstrated using cysteate as a competitive anionic inhibitor that anionic amino acid transport increases as the pH is lowered. These data suggested titration of a group with a pK value around 6.5-7.0. The most likely residue to account for this response is a histidine, for which the imidazole sidechain has a pK value around 6. Therefore, at the lower pH the imidazole group become positively charged and therefore, binds anionic substrates. An analysis was performed which revealed several histidine residues that were conserved in the ASCT1 family. One residue, histidine 305, isolated in the putative membrane domain. To study the importance of this residue on the pH shift of ASCT1, site-directed mutagenesis was performed to convert histidine 305 to alanine. The mutant cDNA (H305A) was then inserted into the pDR2 plasmid and stably transfect into 293c18 cells. Serine uptake was measured at extracellular pH value of 5.5 or 7.5. Northern analysis revealed equal mRNA expression for both the mutant and the wild-type ASCT1. The H305A mutant transport activity was decreased compared with the wild-type both at a pH of 7.5 (133±113 and 3070±430, p<0.05) and 5.5 (1981±364 and 4447±568, p<0.05). However, preliminary experiments suggest that cysteate inhibition of H305A still occurs at an assay pH of 5.5. Therefore the conclusion was drawn that the mutation decreased transport activity in a non-pH dependant manner, but did not alter the cysteate-inhibitable serine transport. Experiments in progress will mutate H305 position to a larger, sterically similar amino acid, glutamine, followed by similar transport analysis. Localization of the transporter in human fetal tissue using immunohistochemistry will also be documented.