Abstract
Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homolog, sodium, and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.
Highlights
Sodium and aspartate symporter GltPh is an archaeal homologue of human glutamate transporters, which clear the neurotransmitter glutamate from the synaptic cleft following rounds of neurotransmission (Danbolt, 2001)
To picture gating in the inward-facing state (IFS), we imaged the GltPh reconstituted into MSP1E3 nanodiscs in the presence of various ligands by single-particle Cryo-EM
One remarkable aspect of the observed structural transitions is the conformational plasticity of HP2 and the interface between the transport domain and scaffold, which differ in each functional intermediate of the transporter
Summary
Sodium and aspartate symporter GltPh is an archaeal homologue of human glutamate transporters, which clear the neurotransmitter glutamate from the synaptic cleft following rounds of neurotransmission (Danbolt, 2001). Structural studies on other members of the family, including human variants, have enriched the field and have been mostly consistent with earlier findings on GltPh (Canul-Tec et al, 2017; Garaeva et al, 2018; Yu et al, 2019). These studies provide what appears to be a nearly complete picture of the structural changes that underlie transport. The crucial conformational transition from the outward-facing state (OFS), in which L-asp binding site is near the extracellular solution, into the inward-facing state (IFS), from which the substrate is released into the cytoplasm, involves a rigid-body “elevator-like” movement of the transport domain by ca 15 Å across the lipid membrane (Reyes et al, 2009; Akyuz et al, 2013; Erkens et al, 2013; Ruan et al, 2017). The structures of the apo transporters in the OFS and IFS showed similar positions of the transport domains that have undergone local structural rearrangements associated with the release of the bound L-asp and Na+ ions (Jensen et al, 2013; Verdon et al, 2014)
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