Dancing with the ions: symport and antiport mechanisms of human glutamate transporters

Abstract

Excitatory amino acid transporters (EAATs) pump glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton and counter-transporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms remain unclear. We report high-resolution Cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or in the absence of ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the counter-transported 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.

TheSLC1A1/EAAT3 Dicarboxylic Amino Acid Transporter is an Epigenetically Dysregulated Nutrient Carrier that Sustains Oncogenic Metabolic Programs

“…3C). Our model closely resembles the recently published Cryo-EM structure of SLC1A1 (26). We compared our model with SLC1A1 loss-of-function mutations, which have been previously described in a human pathology called Dicarboxylic Aminoaciduria, such as Ile395Δ (I395Δ) and Arg445->Trp [R445W (RW)] (27).…”

Evolutionary analysis reveals the origin of sodium coupling in glutamate transporters

“…The two apo Glt Ph structural classes differ in the position of the transport domain. One corresponds to the outward-facing state ( Figure 3a,e ) and the other to a previously observed intermediate ( 16, 21, 30, 56 ), where the transport domain moves toward the inward-facing state by about a quarter of the way ( Supplementary Figure 12, 13 ). The HP2 gate is open in the outward-facing states, propped by M311, and closed in in the intermediate, with M311 pointing out into the lipid from underneath ( Supplementary Figure 13 ).…”

“…In both classes, the NMD motif is in a “flipped-out” configuration, which distorts substrate- and Na + -binding sites ( Figure 3a,e, Supplementary Figure 13 ). We call these conformations, characteristic of apo Na + -coupled transporters, “low-affinity” states ( 11, 15, 16, 30, 55 ). The two apo Glt Ph structural classes differ in the position of the transport domain.…”

“…Previously determined Glt Ph and Glt Tk structures show that upon Na + -binding to Na1 and Na3, Na + -coupled transporters restructure from the low-affinity apo state to a “high-affinity” state through allosterically coupled changes ( 6, 7, 11, 28 ) ( Figure 3b,f ). Their NMD motifs go from the ‘flipped-out’ to the ‘flipped-in’ configuration, repositioning M311 proximal to the substrate ( Figure 3b ) and altering the tilt of TM7b ( 30 ). R397 moves from occupying the substrate-binding site to forming a cation-ν interaction with Y317 in TM7b ( Figure 3b ), its guanidium group in place to salt-bridge with L-Asp.…”

“…R397 moves from occupying the substrate-binding site to forming a cation-ν interaction with Y317 in TM7b ( Figure 3b ), its guanidium group in place to salt-bridge with L-Asp. Also, N401 repositions slightly to coordinate L-Asp optimally ( 30 ). Together, these changes underlie high-affinity binding by optimally positioning residues coordinating the substrate and the additional Na2 ( Figure 3c-d,g ).…”