January 1, 2011;
Specificity and actions of an arylaspartate inhibitor of glutamate transport at the Schaffer collateral-CA1 pyramidal cell synapse.
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 Mg(2+) concentrations, but in Mg(2+)-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
transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg(2+) 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 Mg(2+) 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 Mg(2+) block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.
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Figure 1. Interaction of L-TBA with EAATs.(A) Docking of L-TBA (green) and L-TBOA (gray) in EAAT3 model showing overlap of functional groups interacting with R447 and D444, with benzyl groups oriented toward extracellular loop HP2 as described in . (B) Surface depiction of the transporter binding site (hydrophobic regions blue) showing L-TBA and alternate docking orientation of L-TBOA with benzyl ring aligned in alternate hydrophobic pocket.
Figure 2. Effects of L-TBA on native and recombinant transporters.(A) Representative recording from voltage-clamped Xenopus oocyte expressing astrocyte transporter subtype EAAT2. 100 µM L-TBA partially blocks equimolar L-Glu uptake current mediated by EAAT2. (B) Summary of L-TBA concentration-dependence of block of 100 µM L-Glu currents in oocytes expressing EAAT1-3, showing approximately four-fold selectivity for EAAT3 by least-squares minimized fits to mean data generating IC50 values of 56, 52, and 13 µM, respectively. (C) Effect of 30 µM L-TBA on synaptically activated transport current (STC) in hippocampal CA1 astrocyte. Currents in the presence or absence of L-TBA were evoked by stimulation in stratum radiatum in the continuous presence of ionotropic receptor antagonists (see methods). 30 µM L-TBA blocked 66.7±10.4 of the peak STC (n = 4).
Figure 3. Representative recordings from outside-out patches excised from CA1 pyramidal neurons illustrating AMPAR and NMDAR responses to rapid application of 100 µM L-glutamate and/or 100 µM L-TBA for durations indicated by solution exchange traces above.Responses at −60 mV showing lack of agonist or antagonist actions of L-TBA on AMPARs (A; with 1.2 mM Mg2+) and NMDARs (B; with 0 mM Mg2+/20 µM glycine/20 µM CNQX). Scale bars are 50/200 ms and 50/100 pA for AMPAR/NMDAR responses respectively. (C) Summary of mean effects of 100 µM L-TBA on 100 µM L-Glu AMPAR and NMDAR responses.
Figure 4. Actions of L-TBA (30 µM) on postsynaptic responses at the CA1 Schaffer collateral-pyramidal neuron synapse of EAAT3 +/+ (A) and EAAT3 −/− (B) mice.Representative whole cell recordings (−60 mV) showing effect of L-TBA on EPSCs evoked by stimulation in stratum radiatum in the presence (A1, B1) and absence (A2, B2) of physiological extracellular Mg2+ (1.2 mM). (C) Summary of data showing EPSC charge transfer increase in slices from EAAT3 (+/+) and (−/−) mice by 30 µM L-TBA in the absence and presence of Mg2+ (n = 5–7 slices; p<0.05). Data normalized to the charge transfer in slices from EAAT3 (+/+) mice. (D) Summary data showing no significant difference in L-TBA concentration-dependence of EPSC charge transfer increase (normalized to control) for EAAT3 +/+ (open squares) and EAAT3 −/− (filled squares) (n = 4).
Figure 5. Actions of L-TBA on field responses at the CA1 Schaffer collateral-pyramidal neuron synapse.(A) Representative field EPSPs elicited in response to three stimuli delivered at 20 Hz in stratum radiatum. 30 µM L-TBA (black trace) prolonged fEPSPs relative to control (gray trace) in an activity-dependent manner (p = 0.02). The TBA prolongation was inhibited by co-application of 50 µM DL-APV (2nd black trace). (B) Summary of effects on fEPSP time-integrals elicited by 1, 2 and 3 stimuli normalized to corresponding fEPSPs in control ACSF (*p<.05 paired t-test; n = 9 slices for one and two stimuli, n = 5 slices for three stimuli).