Posterior cerebellar Purkinje cells in an SCA5/SPARCA1 mouse model are especially vulnerable to the synergistic effect of loss of ß-III spectrin and GLAST.

Clinical phenotypes of spinocerebellar ataxia type-5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type-1 (SPARCA1) are mirrored in mice lacking ß-III spectrin (ß-III-/-). One function of ß-III spectrin is the stabilization of the Purkinje cell-specific glutamate transporter EAAT4 at the plasma membrane. In ß-III-/- mice EAAT4 levels are reduced from an early age. In contrast levels of the predominant cerebellar glutamate transporter GLAST, expressed in Bergmann glia, only fall progressively from 3 months onwards. Here we elucidated the roles of these two glutamate transporters in cerebellar pathogenesis mediated through loss of ß-III spectrin function by studying EAAT4 and GLAST knockout mice as well as crosses of both with ß-III-/- mice. Our data demonstrate that EAAT4 loss, but not abnormal AMPA receptor composition, in young ß-III-/- mice underlies early Purkinje cell hyper-excitability and that subsequent loss of GLAST, superimposed on the earlier deficiency of EAAT4, is responsible for Purkinje cell loss and progression of motor deficits. Yet the loss of GLAST appears to be independent of EAAT4 loss, highlighting that other aspects of Purkinje cell dysfunction underpin the pathogenic loss of GLAST. Finally, our results demonstrate that Purkinje cells in the posterior cerebellum of ß-III-/- mice are most susceptible to the combined loss of EAAT4 and GLAST, with degeneration of proximal dendrites, the site of climbing fibre innervation, most pronounced. This highlights the necessity for efficient glutamate clearance from these regions and identifies dysregulation of glutamatergic neurotransmission particularly within the posterior cerebellum as a key mechanism in SCA5 and SPARCA1 pathogenesis.

Neuronal glutamate transporters regulate glial excitatory transmission.

In the CNS, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter-driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG, although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuroglial signaling.

Mapping of glutathione and its precursor amino acids reveals a role for GLYT2 in glycine uptake in the lens core.

PURPOSE: To correlate the distribution of glutathione (GSH) and its precursor amino acids (cysteine, glycine, and glutamate) with the expression of their respective amino acid transporters in the rat lens. METHODS: Whole rat lenses were fixed, cryoprotected, and cryosectioned in either an equatorial or axial orientation. Sections were double labeled with cystine, glycine, glutamate, GSH, GLYT1, or GLYT2 antibodies, and the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal laser scanning microscopy. Cystine, glycine, glutamate, and GSH labeling were quantified by using image-analysis software and intensity profiles plotted as a function of distance from the lens periphery. Western blot analysis was used to verify regional differences in amino acid transporter expression. RESULTS: Cystine and glycine labeling in equatorial sections was most intense in the outer cortex, was diminished in the inner cortex, but was increased again in the core relative to the inner cortex. Glutamate and GSH labeling was most intense in the outer cortex and was diminished in the inner cortex to a minimum that was sustained throughout the core. The distribution of cystine and glutamate levels correlated well with the expression patterns observed previously for the cystine/glutamate exchanger (Xc-) and the glutamate transporter (EAAT4/5), respectively. Although high levels of glycine labeling in the outer cortex correlated well with the expression of the glycine transporter GLYT1, the absence of GLYT1 in the core, despite an increase of glycine in this region, suggests an alternative glycine uptake system such as GLYT2 exists in the core. Equatorial sections labeled with GLYT2 antibodies, showed that labeling in the outer cortex was predominantly cytoplasmic, but progressively became more membranous with distance into the lens. In the inner cortex and core, GLYT2 labeling was localized around the entire membrane of fiber cells. Western blot analysis confirmed GLYT2 to be expressed in the outer cortex, inner cortex, and core of the lens. Axial sections labeled for glycine revealed a track of high-intensity glycine labeling that extended from the anterior pole through to the core that was associated with the sutures. CONCLUSIONS: The mapping of GSH and its precursor amino acids has shown that an alternative glycine uptake pathway exists in mature fiber cells. Although GLYT1 and -2 are likely to mediate glycine uptake in cortical fiber cells, GLYT2 alone appears responsible for the accumulation of glycine in the center of the lens. Enhancing the delivery of glycine to the core via the sutures may represent a pathway to protect the lens against the protein modifications associated with age-related nuclear cataract.

Neuronal glutamate transporters vary in substrate transport rate but not in unitary anion channel conductance.

Excitatory amino acid transporters (EAATs) not only sustain a secondary active glutamate transport but also function as anion-selective ion channels. The relative proportion of currents generated by glutamate transport or by the chloride conductance varies for each cloned EAAT subtype. For EAAT1, EAAT2, and EAAT3, the anion current is only a small component of the total transporter-associated current amplitude, whereas EAAT4 and EAAT5 transporters mediate predominantly anion currents. We here demonstrate that the distinct current proportions are entirely due to differences in glutamate transport rates. EAAT3 and EAAT4 differ in unitary glutamate transport rates as well as in the voltage and substrate dependence of anion channel opening, but ion conduction properties are very similar. Noise analysis revealed identical unitary current amplitudes and similar absolute open probabilities for the two anion channels. The low glutamate transport rate of EAAT4 allows regulation of cellular excitability without interfering with extracellular glutamate homeostasis and makes this EAAT isoform ideally suited to regulate excitability in dendritic spines of Purkinje neurons.

Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1.

Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.

The glutamate-activated anion conductance in excitatory amino acid transporters is gated independently by the individual subunits.

Excitatory amino acid transporters (EAATs) use sodium and potassium gradients to remove glutamate from the synapse and surrounding extracellular space, thereby sustaining efficient synaptic transmission and maintaining extracellular glutamate concentrations at subneurotoxic levels. In addition to sodium-driven glutamate uptake, EAATs also mediate a glutamate-activated chloride conductance via a channel-like mechanism. EAATs are trimeric proteins and are thought to comprise three identical subunits. Previous studies have shown that the sodium-driven uptake of glutamate occurs independently in each of the three subunits. In contrast, a recent study reports high Hill coefficients for the activation of EAAT anion currents by glutamate and suggests that the subunits function cooperatively in gating the chloride conductance. In the present work, we find that the Hill coefficient for the activation of the anion current by glutamate is approximately 1 in both EAAT3 and EAAT4. Furthermore, we also used fluorescent labeling and inactivation correlation on EAAT3 and EAAT4 to determine whether the glutamate-activated chloride conductance is gated independently or cooperatively by the transporters. We found that both glutamate uptake currents and glutamate-activated chloride currents are mediated independently by each subunit of an EAAT multimer. It has been suggested that EAAT subtypes with particularly large anion conductances can directly influence the excitability of presynaptic terminals in certain neurons. Thus, the finding that the anion conductance is gated independently, rather than cooperatively, is important because it significantly alters predictions of the influence that EAAT-mediated anion currents will have on synaptic transmission at low glutamate concentrations.

Intersubunit interactions in EAAT4 glutamate transporters.

Excitatory amino acid transporters (EAATs) play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian CNS. A functional transporter is assembled as oligomer consisting of three subunits, each of which appears to transport glutamate independently from the neighboring subunits. EAATs do not only sustain a secondary-active glutamate transport but also function as anion channel. We here address the question whether intersubunit interactions play a role in pore-mediated anion conduction. We expressed a neuronal isoform, EAAT4, heterologously in Xenopus oocytes and mammalian cells and measured glutamate flux and anion currents under various concentrations of Na+ and glutamate. EAAT4 anion channels are active in the absence of both substrates, and increasing concentrations activate EAAT4 anion currents with a sigmoidal concentration dependence. Because only one glutamate molecule is cotransported per uptake cycle, the cooperativity between glutamate binding sites most likely arises from an interaction between different carrier domains. This interaction is modified by two point mutations close to the putative glutamate binding site, G464S and Q467S. Both mutations alter the dissociation constants and Hill coefficient of the substrate dependence of anion currents, leaving the concentration dependence of glutamate uptake unaffected. Our results demonstrate that glutamate carriers cooperatively interact during anion channel activation.

Roles of glial glutamate transporters in shaping EPSCs at the climbing fiber-Purkinje cell synapses.

Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). Here we aimed to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporter, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors, DHK prolonged the decay time constant (tau(w)) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by approximately 80%, caused no change in tau(w) in WT in the absence of CTZ, whereas it prolonged tau(w) in the presence of CTZ. This prolonged value of tau(w) was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion ( approximately 20%) of functional transporters is preserved.

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism.

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.

Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses.

Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate-aspartate transporter (GLAST) and excitatory amino acid transporter 4 (EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.