The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft, and thus, is crucial for proper excitatory signaling. Initial steps in the process of glutamate transport take place within a time scale of microseconds to milliseconds. Here we compare the steady-state and pre-steady-state kinetics of the neuronal heterologously expressed glutamate transporter EAAC1, cloned from the mammalian retina. Rapid transporter dynamics, as measured by using whole-cell current recordings, were resolved by applying the laserpulse photolysis technique of caged glutamate with a time resolution of 100 s. EAAC1-mediated pre-steady-state currents are composed of two components: A transport current generated by substratecoupled charge translocation across the membrane and an anion current that is not stoichiometrically coupled to glutamate transport. The two currents were temporally resolved and studied independently. Our results indicate a rapid glutamate-binding step occurring on a submillisecond time scale that precedes subsequent slower electrogenic glutamate translocation across the membrane within a few milliseconds. The voltage-dependent steady-state turnover time constant of the transporter is about 1/10 as fast, indicating that glutamate translocation is not rate limiting. A third process, the transition to an anion-conducting state, is delayed with respect to the onset of glutamate transport. These rapid transporter reaction steps are summarized in a sequential shuttle model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotransmission in the central nervous system.

Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. The reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed the presence of three different high-affinity glutamate transporters in the rat retina, viz. GLAST-1, GLT-1 and EAAC-1. No message has been found in the retina for EAAT-4, a transporter recently cloned from human brain. By using membrane vesicle preparations of total rat retina, we show that glutamate uptake in the retina is a high-affinity electrogenic sodium-dependent transport process driven by the transmembrane sodium ion gradient. Autoradiography of intact and dissociated rat retinae indicates that glutamate uptake by Müller glial cells dominates total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. RT-PCR, immunoblotting and immunohistochemistry have revealed that Müller cells express only GLAST-1. The Km for glutamate of GLAST-1 is 2.1+/-0.4 microM. This study suggests a major role for the Müller cell glutamate transporter GLAST-1 in retinal transmitter clearance. By regulating the extracellular glutamate concentration, the action of GLAST-1 in Müller cells may extend beyond the protection of neurons from excitotoxicity; we suggest a mechanism by which Müller cell glutamate transport might play an active role in shaping the time course of excitatory transmission in the retina.

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At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

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We investigated the localization and possible function of EAAC1 in the rat retina. Immunocytochemical localization of EAAC1 at the light-microscopic level revealed a fine dust-like labelling pattern across the two synaptic layers. Horizontal cell and subpopulations of amacrine cell somata were labelled, as were some somata within the ganglion cell layer. Some immunoreactive puncta were observed within the cytoplasm of amacrine cells, in regions well away from synaptic sites. At the ultrastructural level, EAAC1 immunolabelled one postsynaptic element at synapses and also processes well away from the synaptic release site. Since EAAC1 was localized away from synaptic sites, we evaluated the role EAAC1 plays in GABA formation by measuring GABA concentrations via reversed-phase high-performance liquid chromatography following incubation of retinae in enzyme and glutamate uptake inhibitors. Incubation of retinae in D-threo-beta-hydroxyaspartate or D/ L-threo-beta-benzyloxyaspartate, which are known to inhibit the glutamate transporters GLAST1, GLT1, and EAAC1, caused a decrease in GABA synthesis by around 50%. Incubation in 6-diazo-5-oxo- L-norleucine, a phosphate-activated glutaminase inhibitor, decreased GABA formation by 40%. Taken together with the anatomical data, the results of this study suggest that EAAC1 plays very little role in GABA synthesis - indeed GABA formation occurs predominantly from glutamine. By virtue of its location both near and well away from synaptic release sites, EAAC1 may regulate glutamate uptake differentially.