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

Abstract

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...

The equivalent of a thallium binding residue from an archeal homolog controls cation interactions in brain glutamate transporters

“…Because the mutants are locked in an exchange mode, we cannot experimentally test this alternative. This is because one or more sodium ions can bind before substrate on the extracellular side (33,34), and all three sodium ions unbind after the substrate on the intracellular side (35). Therefore many labeled substrate molecules can be exchanged for intracellular unlabeled substrate molecules without any accompanying sodium translocation.…”

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

“…TBOA does not bind to EAAC1 in the absence of Na ϩ and may bind, but only weakly, in the presence of extracellular K ϩ . Therefore, TBOA binding was promoted by including a small amount of Na ϩ (2 mM in the presence of extracellular K ϩ and 5 mM in the presence of extracellular NMG ϩ ) in the TBOA-containing solution, as described previously (22). This small amount of Na ϩ did not elicit nonspecific currents.…”

“…Under these ionic conditions, 200 M TBOA is a supersaturating concentration (about 100-fold K m ), so the TBOA binding site should be saturated at all voltages. The K m for TBOA under these conditions has been experimentally determined previously (22).…”

Regulation of Glial Glutamate Transporters by C-terminal Domains

“…ϩ Binding Suggested by Kinetic Modeling-Coupled glutamate transport can be modeled as binding/unbinding transitions between distinct states (32,43). EAAT-associated anion channel properties are included in these models by connecting several states of the transport cycle with open conformations of the EAAT-associated anion channel (Fig.…”