Probing the Mechanism of Substrate Recognition and Translocation in the Mammalian Glutamate Transporters

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Excitatory amino acid transporters (EAATs) located on the plasma membranes of neurons and glia, play a central role in shaping neuronal signaling by removing released glutamate, and maintaining the extracellular concentration below excitotoxic levels. This transporter family is comprised of five subtypes (EAATs1-5), which share high homology and the ability to transport the acidic amino acid substrates, L-Glutamate and L-Aspartate. Despite significant biochemical data and an X-ray structure for a substrate-bound archaeal EAAT ortholog, the mechanism by which the transporters distinguish and translocate potential substrates remains undefined. One interesting observation has been that the subtype EAAT3 displays robust L-Cysteine transport relative to the other EAATs, perhaps accounting for its importance as a primary means for the neuronal import of cysteine for glutathione synthesis. To examine the structural basis for the unique substrate selectivity of EAAT3, we assessed the actions of a cysteine analog, L-Selenocysteine, which substitutes a selenium for sulfur, on the transport kinetics of EAAT3 and EAAT2. L-Selenocysteine has a pKa=5 and thus, at physiological pH the selenol moiety is deprotonated and negatively charged like L-Glu, while the thiol of L-Cys remains protonated and uncharged. We observed that L-Selenocysteine is a potent substrate for both EAAT3 (Km∼8μM) and EAAT2, and is a more potent inhibitor of L-Glu transport in EAAT2 (IC50∼39μM) than L-Cys (IC50>1mM). This result is consistent with the idea that the apparent affinity of a potential substrate should increase as its pKa decreases, and that the arrangement of residues in the substrate binding site may significantly influence this process. These observations are further supported by the mutagenesis of key residues implicated in substrate binding/selectivity and transport, and thus provide insight into the mechanism of substrate selectivity, binding and translocation through this family of transporters.