Glutamate transport by retinal Mueller cells in glutamate/aspartate transporter-knockout mice

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Mueller cells in GLAST super(-/-) mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the K sub(m) value for glutamate uptake was similar in wild-type and GLAST super(-/-) mouse retinas, but the V sub(max) was [sim]50% lower in the mutant. In Na super(+)-free medium, the V sub(max) was further reduced by 40%. In patch-clamp recordings of dissociated Mueller cells from GLAST super(-/-) mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Mueller cells. [ super(3)H]D-Aspartate uptake autoradiography, however, showed that Na super(+)-dependent, high-affinity transporters account for most of the glutamate uptake by Mueller cells, and that Na super(+)-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Mueller cells in the GLAST super(-/-) mouse retina is not due to known glutamate transporters - cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Mueller cells, new Na super(+)-dependent, high-affinity glutamate transporters remain to be identified.