Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses

Tanaka, Jun; Ichikawa, Ryoichi; Watanabe, Masahiko; Tanaka, Kohichi; Inoue, Yoshiro

doi:10.1097/00001756-199707280-00010

Cited by 59 publications

(38 citation statements)

References 9 publications

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“…The other component, termed ''residual current,'' has several features inconsistent with glutamate transport. First, we have confirmed that the residual current is not blocked by high concentrations of TBOA, a competitive antagonist for EAAC1 and EAAT4 (36,37), the two EAATs expressed by PCs (12,14,24,38). Second, I-V analysis of the residual synaptic and photolysis-evoked currents shows that they uniformly reverse polarity at potentials near 0 mV, which would not be expected of stoichiometric EAAT currents or of responses consisting of mixed stoichiometric and anion currents.…”

Section: Discussionsupporting

confidence: 57%

Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber–Purkinje cell synapse

Brasnjo

Otis²

2004

Proc. Natl. Acad. Sci. U.S.A.

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Excitatory amino acid transporters (EAATs) located on neurons and glia are responsible for limiting extracellular glutamate concentrations, but specific contributions made by neuronal and glial EAATs have not been determined. At climbing fiber to Purkinje cell (PC) synapses in cerebellum, a fraction of released glutamate is rapidly bound and inactivated by neuronal EAATs located on postsynaptic PCs. Because transport involves a stoichiometric movement of ions and is electrogenic, postsynaptic currents mediated by EAATs should permit precise calculation of the amount of postsynaptic glutamate uptake. However, this is possible only if a stoichiometric EAAT current can be isolated from all other contaminating signals. We used synaptic stimulation and photolysis of caged glutamate to characterize the current in PCs that is resistant to high concentrations of glutamate receptor antagonists. Some of this response is inhibited by the high-affinity EAAT antagonist TBOA (DL-threo-␤-benzyloxyaspartic acid), whereas the remaining current shows properties inconsistent with glutamate transport. By subtracting this residual non-EAAT current from the response recorded in glutamate receptor antagonists, we have obtained an estimate of postsynaptic uptake near physiological temperature. Analysis of such synaptic EAAT currents suggests that, on average, postsynaptic EAATs take up Ϸ1,300,000 glutamate molecules in response to a single climbing fiber action potential. E xtracellular neurotransmitter concentrations in the central nervous system are regulated by highly specific systems of reuptake that rapidly bind and quickly move neurotransmitters across membranes. Neurotransmitter reuptake facilitates chemical synaptic transmission by limiting receptor activation and by recycling released neurotransmitter molecules. In the case of glutamate, the primary excitatory neurotransmitter of the central nervous system, uniquely localized excitatory amino acid transporters (EAATs), influence both ionotropic (1-5) and metabotropic glutamate receptor (GluR) (6 -9) activation. EAATs on glial (10-12) and neuronal (12-14) processes accomplish this task by relying on the Na ϩ and K ϩ electrochemical gradients to move glutamate against its own large concentration gradient.A single cycle of transport is estimated to take tens of milliseconds (15)(16)(17)(18)(19)(20) and is accompanied by the cotransport of 3 Na ϩ , 1 H ϩ and 1 glutamate Ϫ and the countertransport of 1 K ϩ across the membrane (21, 22). Such stoichiometry results in a net inward movement of two charges per cycle, meaning that glutamate transport is electrogenic. In addition to these stoichiometric fluxes, EAATs become permeable to anions at specific points during the transport cycle (16-19, 23, 24). Under conditions in which anion flux through EAATs is minimized, stoichiometric currents can be used to estimate glutamate flux by taking advantage of the fact that the number of glutamate molecules transported is equal to half the net movement of elementary charges.In an effort to deter...

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Section: Discussionsupporting

confidence: 57%

Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber–Purkinje cell synapse

Brasnjo

Otis²

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…In the cerebellar cortex, both GLAST and EAAT4 are abundantly expressed as predominant glial and neuronal transporters, respectively (Danbolt, 2001). GLAST is located on the plasma membranes of Bergmann glial processes surrounding the excitatory synapses in Purkinje cells (PCs) (Rothstein et al, 1994;Chaudhry et al, 1995), whereas EAAT4 is concentrated in extrasynaptic regions at excitatory synapses in PCs (Tanaka et al, 1997;Dehnes et al, 1998). This different distribution of GLAST and EAAT4 at the electron microscopic level suggests that these transporters serve distinct functions in excitatory synapses on PCs.…”

Section: Introductionmentioning

confidence: 99%

Glial Glutamate Transporters Maintain One-to-One Relationship at the Climbing Fiber-Purkinje Cell Synapse by Preventing Glutamate Spillover

Takayasu

Iino

Shimamoto

et al. 2006

Journal of Neuroscience

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A glial glutamate transporter, GLAST, is expressed abundantly in Bergmann glia and plays a major role in glutamate uptake at the excitatory synapses in cerebellar Purkinje cells (PCs). It has been reported that a higher percentage of PCs in GLAST-deficient mice are multiply innervated by climbing fibers (CFs) than in the wild-type (WT) mice, and that CF-mediated EPSCs with small amplitude and slow rise time, designated as atypical slow CF-EPSCs, are observed in these mice. To clarify the mechanism(s) underlying the generation of these atypical CF-EPSCs, we used (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA), an inhibitor of glial glutamate transporters. After the application of PMB-TBOA, slow-rising CF-EPSCs were newly detected in WT mice, and their rise and decay kinetics were different from those of conventional fast-rising CF-EPSCs but similar to those of atypical CF-EPSCs in GLAST-deficient mice. Furthermore, both slow-rising CF-EPSCs in the presence of PMB-TBOA in WT mice and atypical CF-EPSCs in GLAST-deficient mice showed much greater paired-pulse depression compared with fast-rising CF-EPSCs. In addition, both of them were more markedly inhibited by ␥-D-glutamyl-glycine, a low-affinity competitive antagonist of AMPA receptors. These results indicated that both of these types of EPSCs were mediated by a low concentration of glutamate released from neighboring CFs. Based on all of these findings, we suggest that glial transporters prevent glutamate released from a single CF from spilling over to neighboring PCs other than the synaptically connected PC, and play an essential role in the maintenance of the functional one-to-one relationship between CFs and PCs.

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“…Two of the five characterized glutamate transporters, excitatory amino acid carrier 1 (EAAC1) (Kanai and Hediger, 1992) and excitatory amino acid transporter 4 (EAAT4), have been reported to be expressed in Purkinje neurons (Kanai et al, 1995;Furuta et al, 1997;Dehnes et al, 1998) (but see Tanaka et al, 1997a). G TA can be detected upon activation of climbing fiber synaptic inputs and elicited in membrane patches by glutamate application (Otis et al, 1997).…”

mentioning

confidence: 99%

Anion Currents and Predicted Glutamate Flux through a Neuronal Glutamate Transporter

1998

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Kinetic properties of a native, neuronal glutamate transporter were studied by using rapid applications of glutamate to outside-out patches excised from Purkinje neurons. Pulses of glutamate activated anion currents associated with the transporter that were weakly antagonized by the transporter antagonist kainate. In addition, kainate blocked a resting anion conductance observed in the absence of glutamate. Transporter currents in response to glutamate concentration jumps under a variety of conditions were used to construct a cyclic kinetic model of the transporter. The model simulates both the anion conductance and the glutamate flux through the transporter, thereby permitting several predictions regarding the dynamics of glutamate transport at the synapse. For example, the concentration-dependent binding rate of glutamate to the transporter is high, similar to binding rates suggested for ligand-gated glutamate receptors. At saturating glutamate concentrations, transporters cycle at a steady-state rate of 13/sec. Transporters are predicted to have a high efficiency; once bound, a glutamate molecule is more likely to be transported than to unbind. Physiological concentrations of internal sodium and glutamate significantly slow net transport. Finally, a fixed proportion of anion and glutamate flux is expected over a wide range of circumstances, providing theoretical support for using net charge flux to estimate the amount and time course of glutamate transport.

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Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses

Cited by 59 publications

References 9 publications

Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber–Purkinje cell synapse

Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber–Purkinje cell synapse

Glial Glutamate Transporters Maintain One-to-One Relationship at the Climbing Fiber-Purkinje Cell Synapse by Preventing Glutamate Spillover

Anion Currents and Predicted Glutamate Flux through a Neuronal Glutamate Transporter

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