The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport

Watzke, Natalie; Grewer, Christof

doi:10.1016/s0014-5793(01)02715-6

Cited by 46 publications

(42 citation statements)

References 21 publications

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“…7), indicating that the apparent dissociation constant of glutamate from its intracellular binding site, K m i , also depends on [Na i ϩ ]. As expected, K m i values are 20-to 40-fold larger for reverse transport than the K m o for forward transport (7). In contrast to the K m values, the maximum glutamateinduced anion current, I max , determined at saturating [glutamate i ] was independent of [Na i ϩ ] (SI Fig.…”

Section: Resultssupporting

confidence: 57%

Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1

Zhang

Tao

Gameiro

et al. 2007

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

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Glutamate transport by the excitatory amino acid carrier EAAC1 is known to be reversible. Thus, glutamate can either be taken up into cells, or it can be released from cells through reverse transport, depending on the electrochemical gradient of the co-and countertransported ions. However, it is unknown how fast and by which reverse transport mechanism glutamate can be released from cells. Here, we determined the steady-and pre-steady-state kinetics of reverse glutamate transport with submillisecond time resolution. First, our results suggest that glutamate and Na ؉ dissociate from their cytoplasmic binding sites sequentially, with glutamate dissociating first, followed by the three cotransported Na ؉ ions. Second, the kinetics of glutamate transport depend strongly on transport direction, with reverse transport being faster but less voltage-dependent than forward transport. Third, electrogenicity is distributed over several reverse transport steps, including intracellular Na ؉ binding, reverse translocation, and reverse relocation of the K ؉ -bound EAAC1. We propose a kinetic model, which is based on a ''first-in-first-out'' mechanism, suggesting that glutamate association, with its extracellular binding site as well as dissociation from its intracellular binding site, precedes association and dissociation of at least one Na ؉ ion. Our model can be used to predict rates of glutamate release from neurons under physiological and pathophysiological conditions. excitatory amino acid transporter ͉ electrophysiology ͉ reverse transport ͉ patch-clamp ͉ caged compounds G lutamate transporters belong to the class of Na ϩ -driven secondary-active transporters. They couple the uphill uptake of glutamate into cells to the movement of three Na ϩ ions down their ion concentration gradient (1). Neurons, like many other cells, express glutamate transporters, allowing them to keep a 10 6 -fold glutamate concentration gradient across their cell membranes (2). This steep concentration gradient is essential for neuronal signaling, because it ensures submicromolar resting concentrations of extracellular glutamate.Glutamate transporters are not strictly unidirectional and are able to change the direction of glutamate transport (3). Under physiological conditions, forward transport from the extracellular side to the cytosol is favored. However, if the driving force for glutamate uptake decreases, glutamate can be released from cells through reverse glutamate transport (3, 4). This situation may arise in oxygen-deprived cells when the Na ϩ concentration gradient across the membrane runs down, and/or when cells depolarize. In ischemic neurons, the majority of glutamate release upon oxygen/glucose deprivation was shown to be caused by reverse glutamate transport and not by vesicular release (5, 6). Considering the severe neurotoxic effects of elevated extracellular glutamate concentrations, it is of major importance to understand the mechanism of how glutamate is released through reverse transport.Here, we investigated with high time re...

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Section: Resultssupporting

confidence: 57%

Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1

Zhang

Tao

Gameiro

et al. 2007

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

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“…ϩ /H ϩ /K ϩ , and the reverse transport-activated chloride conductance (5,22,25). The lack of any conductance blocked by TBOA after MTSET treatment of V452C suggests that both the reverse transport and reverse transport-activated chloride conductance are not activated by high extracellular K ϩ .…”

Section: Resultsmentioning

confidence: 98%

Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1

Ryan¹,

Vandenberg

2002

Journal of Biological Chemistry

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Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na ؉ , 1 H ؉ , and the counter-transport of 1 K ؉ ion. In addition to coupled ion fluxes, glutamate and Na ؉ binding to the transporter activates a thermodynamically uncoupled anion conductance through the transporter. In this study, we have distinguished between these two conductance states of the EAAT-1 transporter using a [2-(trimethylammonium)ethyl]methanethiosulfonatemodified V452C mutant transporter. Glutamate binds to the modified mutant transporter and activates the uncoupled anion conductance but is not transported. The selective alteration of the transport function without altering the anion channel function of the V452C mutant transporter suggests that the two functions are generated by distinct conformational states of the transporter.Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system, and the role of the excitatory amino acid transporters (EAATs) 1 is to regulate synaptic glutamate concentrations to maintain dynamic signaling between the presynaptic and postsynaptic membranes and also to prevent a build-up of toxic levels of glutamate. The energy used to drive the uphill transport of glutamate into the cell against a significant concentration gradient is derived from the coupling of transport to the co-transport of 3 Na ϩ and 1 H ϩ and the counter-transport of 1 K ϩ for each glutamate molecule (1, 2). In addition to these coupled ion fluxes, glutamate bound to the transporter activates a thermodynamically uncoupled anion conductance through the transporter (3-5).Five different glutamate transporters have been identified, and their amino acid sequences were determined from the cDNA sequences. The human glutamate transporters are termed EAAT-1 to 6,7), whereas the rat homologs of EAAT-1 and EAAT-2 are termed glutamate/aspartate transporter-1 (8) and glutamate transporter-1 (9) and the rabbit homolog of EAAT-3 is termed excitatory amino-acid carrier-1 (10). The five different transporter subtypes show a high degree of similarity and are likely to form similar structures in the membrane. A number of models for the membrane topology of glutamate transporters have been proposed. Initial predictions of the topology from hydrophobicity analysis of the amino acid sequence lead to three different models with 8, 10, and 12 transmembrane domains (8 -10), but more direct structural information has been obtained from method studies of substituted cysteine accessibility (11, 12). The models proposed from these studies both predict six ␣-helical transmembrane domains in the amino-terminal portion of the protein followed by a series of reentrant loops and membrane-associated structures followed by a final ␣-helical transmembrane domain and an intracellular carboxyl-terminal domain.The reentrant loop and membrane-associated structures are highly conserved in their amino acid sequences, and from mutagenesis studies, it has been proposed that this region forms the por...

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“…3 and 4). The existence of a glutamate-independent anion conductance has been reported (15,31,32); however, previous estimates of the relative amplitude of this current amplitude for EAAT1 and EAAT3 were much lower (between 0.05 and 0.2 (15,32)). This difference likely arises from the methods used to measure the glutamateindependent anion currents.…”

Section: Discussionmentioning

confidence: 94%

Glutamate Modifies Ion Conduction and Voltage-dependent Gating of Excitatory Amino Acid Transporter-associated Anion Channels

Melzer

Biela

Fahlke

2003

Journal of Biological Chemistry

108

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Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na ؉ , K ؉ , and H ؉ , and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na ؉ /H ؉ /K ؉ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na ؉ -or K ؉ -based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels. Excitatory amino acid transporters (EAATs)1 mediate the removal of glutamate from the synaptic cleft in the central nervous system and the uptake of glutamate in kidney and intestine (1-3). Five structurally distinct subtypes of mammalian glutamate transporters, EAAT1-EAAT5, have been identified in recent years (4 -9). Each of these isoforms exhibits two separate transport processes: a stoichiometrically coupled cotransport of one glutamate with three Na ϩ ions and one H ϩ , in countertransport to one K ϩ ion (10, 11); and an anion conductance that appears to be pore-mediated. The EAAT-associated anion channel is currently thought to function as a glutamategated channel with a tight coupling of channel opening and closing to conformational changes of the corresponding carrier domain. In this model, only certain carrier conformations are associated with conducting anion pores, and the anion channel cycles between conducting and non-conducting states during transitions through various conformational states of the glutamate carrier (8,9,(12)(13)(14)(15).We investigated anion conduction properties of two EAAT isoforms, human EAAT2 and rat EAAT4, using patch clamp recordings of transfected tsA201 cells under conditions that eliminate the Glu/Na ϩ /H ϩ /K ϩ current component. Our results demonstrated that opening and closing of the EAAT-associated anion channels as well as the interaction with the glutamate uptake process are more complex than previously assumed. External glutamate modifies intrinsic properties of EAAT2-and EAAT4-associated anion channels such as anion selectivity and the rate constants of anion permeation. Moreover, EAAT4 anion channels exhibit voltage-dependent opening and closing transitions that are modified by glutamate. These results provide novel insights into the function of neurotransmitter transporters and illustrate similarities as well as differences between transporter-associated pores and ion channe...

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The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport

Cited by 46 publications

References 21 publications

Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1

Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1

Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1

Glutamate Modifies Ion Conduction and Voltage-dependent Gating of Excitatory Amino Acid Transporter-associated Anion Channels

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