Symport and antiport mechanisms of human glutamate transporters

Abstract Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain

The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters.

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation