Abstract
In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg2+ concentrations, but in Mg2+-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg2+ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg2+ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg2+ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.
Highlights
Five major subtypes of excitatory amino acid transporters exist in the CNS, and three of these (EAAT1-3; known as GLAST, GLT-1, and EAAC1) are expressed in forebrain with distinct distribution patterns on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3) [1]
The most energetically favorable L-TBA complexes corresponded to the benzyl group orientation of L-3Br-TBOA that was determined in the GltPh crystal structure, with interactions between the ring and non-polar residues near the tip of HP2
NMDA receptor (NMDAR) responses to rapid application of 100 mM L-glutamate and/or 100 mM L-TBA for durations indicated by solution exchange traces above
Summary
Five major subtypes of excitatory amino acid transporters exist in the CNS, and three of these (EAAT1-3; known as GLAST, GLT-1, and EAAC1) are expressed in forebrain with distinct distribution patterns on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3) [1]. The widely used glutamate uptake blocker DL-TBOA blocks EAAT2 and EAAT3-mediated [3H]L-Glu uptake with IC50 values approximately seven-fold lower than for EAAT1 [13], and studies utilizing DL-TBOA indicate that it can induce spillover of synaptic glutamate onto NMDARs in hippocampus [7,8,9,10,11]. There is still uncertainty surrounding the relative contributions and detailed roles of neuronal and glial EAAT subtypes in hippocampus Another issue concerns the general role of glutamate transport in restricting NMDAR signaling in the hippocampus under physiological conditions, since this effect has only been reported in conditions permissive for channel activity, i.e. voltage clamp of the postsynaptic neuron at positive potentials or in Mg2+-free ACSF [7,8,9,10,11]
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