Abstract
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.DOI: http://dx.doi.org/10.7554/eLife.02283.001.
Highlights
Glutamate transporters, or excitatory amino acid transporters (EAATs), reside in the plasma membranes of glial cells and neurons, where they catalyze the re-uptake of the neurotransmitters glutamate and aspartate (L-asp) (Danbolt, 2001)
We propose that the closed translocationcompetent conformation of the transport domain free of Na+ and substrate is intrinsically stable in glutamate transporter homologue (GltPh) but not in EAATs, in which K+ binding at the newly identified site is required, coupling transport cycle completion to K+ counter-transport
GltPh-R397A crystallizes in the outward-facing state, like wild type GltPh, except that L-asp coordination is slightly altered because the mutant is missing the key coordinating side chain of Arg397 (Figure 1B, Figure 1—figure supplement 2; Bendahan et al, 2000; Boudker et al, 2007)
Summary
Excitatory amino acid transporters (EAATs), reside in the plasma membranes of glial cells and neurons, where they catalyze the re-uptake of the neurotransmitters glutamate and aspartate (L-asp) (Danbolt, 2001). To maintain steep trans-membrane glutamate gradients, EAATs transport one substrate molecule together with three sodium ions (Na+) and one proton. After their release into the cytoplasm, countertransport of one potassium ion (K+) resets the transporter for the cycle (Zerangue and Kavanaugh, 1996; Levy et al, 1998; Owe et al, 2006). When bound to Na+ and L-asp (‘fully bound’ from here on), each transport domain moves by ∼15 Å across the membrane from an outwardto an inward-facing position, in which the substrate binding site is near the extracellular solution and the cytoplasm, respectively (Reyes et al, 2009).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
