Ion transport and regulation in a synaptic vesicle glutamate transporter

Abstract

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo-electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.

Fig. 1.. Structure of VGLUT2.

(A) Cryo-EM map of the VGLUT2-Fab complex. The two domains are colored blue (N-domain) and red (C-domain), and the Fab is colored yellow. (B) Schematic representation of the structural arrangement of VGLUT2. Three-helix bundles are related to each other by a twofold pseudosymmetry, and each bundle is colored using shades of the same color group. (C) Structure of VGLUT2. Helices are colored according to the representation in (B), with connecting strands shown in gray. The VGLUT2 structure includes residues 59 to 508 except for the disordered loop 1 between TM1 and TM2 (residues 98 to 123) and 10 residues between ICH1 and ICH2 (residues 288 to 299). (D) Electrostatic surface of VGLUT2 shown at the plane parallel to the membrane through the central substrate binding site of the protein. The central cavity is indicated by a black dashed circle.

Fig. 2.. R88 and R322 are critical for glutamate transport.

(A) Alignment of human SLC17 family proteins, presented alongside the Escherichia coli homolog DgoT, with binding site residues indicated by magenta triangles. Residues are colored according to sequence conservation in descending order of red, orange, yellow, green, and no color. Putative substrate binding residues conserved in VGLUTs alone are highlighted by red boxes. (B) Structure of the substrate binding site in VGLUT2. Glutamate was manually placed into the binding site to mimic D-galactonate in DgoT. Both R88 and R322 are located at distances suitable for interacting with the carboxyl groups. (C) Structure of the substrate binding site in DgoT with substrate D-galactonate bound in the outward occluded conformation [PDB: 6E9O (22)]. Structures of VGLUT2 (B) and DgoT (C) are colored following the same pattern as (A), and substrates are colored cyan. (D) mEPSCs recorded from hippocampal neurons of VGLUT1 and VGLUT2 double knockout (DKO) mice rescued with WT, R88A, and R322A VGLUT2. Synaptic transmission is impaired by the R88A mutation and eliminated by the R322A mutation. mEPSC frequency (left) and amplitude (right) are normalized to those of VGLUT2-WT (n = 10 to 15 cells per condition). Data indicate means and SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. *P < 0.05; ***P < 0.001; ns, not significant. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 3.. Two functional clusters of charged and polar residues embedded within the transmembrane domains.

(A) Electrostatic surface of VGLUT2. Two internal charged and polar cavities (referred to as R88 and R322 clusters) are colored according to the potential scale (bottom right). The N-domain is colored light green and the C-domain is tan. Cyan and magenta boxes in (A) match the insets in (B) (top view), (C) (side view), and (D) (top view), showing key residues and distances between polar groups (A). (B and C) R88 cluster with proposed Cl⁻ (R184) and H⁺ binding sites (E191 and H128) highlighted in orange. (D) R322 cluster.

Fig. 4.. Putative Cl⁻ channels and proposed transport mechanism.

(A) Channels consistent with Cl⁻ conductance (surface). (B) Top view of the central channel. (C) Top view of the cytoplasmic two-His gate. Distances (Å) between polar groups are shown with dotted lines. (D) Proposed mechanism by which VGLUT integrates the dynamic change of ionic conditions during synaptic vesicle recycling to regulate its activity.