Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines

Abstract

Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.

Fig. 1. Overall structures of human VMAT2 at different ligand-bound states.

a Cryo-EM density map (top) and structural model (bottom) of WT VMAT2 in the absence of ligand (VMAT2A), with N- and C-domain colored differently. The lumen-facing state is depicted by black dashed lines on the model. b WT VMAT2 in complex with substrate serotonin (serotonin, green) captured in a lumen-facing state (VMAT2S). The 2D chemical structure of serotonin is shown on the bottom right. c Cryo-EM density (top) and structure (bottom) of WT VMAT2 with non-competitive inhibitor TBZ (cyan) (VMAT2T). d Competitive inhibitor RES (pink) locks the VMAT2 Y422C mutant at a cytosol-facing state (VMAT2_YCR).

Fig. 2. Central binding site for 5-HT in the lumen-facing state.

a Structural superimposition of the lumen-facing serotonin-bound VMAT2S (N-domain in green and C-domain in purple) with apo VMAT2A (gray). The expanded view (right) shows density fitting of 5-HT (lime green). b Binding affinity for the WT and the F433F VMAT2 mutant with 5-HT measured using microscale thermophoresis (MST) assay (mean ± SEM, n = 3–4 independent experiments). c Residues lining the central substrate binding cavity that accommodates 5-HT. TMs are indicated with numbers. d Cutaway side-view of the electrostatic surface potential (negative in red, positive in blue) surface of the 5-HT binding pocket. e FFN uptake activity of VMAT2 variants. Activity values (mean ± SEM, n = 3 biologically independent experiments with 3 technical replicates each) are normalized to that of the WT.

Fig. 3. Non-competitive inhibitor TBZ locks VMAT2 in occluded state.

a TBZ-bound VMAT2T structure (N-domain in light green and C-domain in salmon) is overlaid onto VMAT2A (gray). The expanded view (right) shows density fitting of TBZ (cyan). b Conformational changes induced by TBZ binding viewed from lumen. Green and red arrows indicate movement in the N- and C-domain, respectively. Luminal loops connecting TM1/TM2 (LuL1–2) and TM7/TM8 (LuL7–8) exhibiting the largest movement are labeled. c TBZ–VMAT2 interactions viewed from lumen. d Electrostatic potential surface of the hydrophobic/electronegative TBZ-binding pocket. e Residues sealing the luminal exit viewed from the cytosol. f Binding affinity for VMAT2 mutants with TBZ measured using MST assay. The table (right) summarizes Kd values (mean ± SEM, n = 3–4 independent experiments).

Fig. 4. Cytosol-facing VMAT2_YCR captured by the competitive inhibitor RES.

a Binding affinity of VMAT2 variants with RES measured by MST assay, with results summarized below (mean ± SEM, n = 3–4 independent experiments). b RES-bound VMAT2_YCR structure (N-domain in cyan and C-domain in yellow-green) captured at cytosol-facing state. An expanded view of the elongated density of RES (purple) is shown on the left. c Cutaway side-view for the overall electronegative vestibule hosting RES molecule. d Residues in close vicinity of RES lining the translocation funnel are detailed, viewed from membrane plane.

Fig. 5. Cytosolic and luminal gates.

a The cytosolic gate. Gate residues from TMs 4, 5, 10 and 11 from the VMAT2A structure (semi-transparent gray cartoon) are shown in sticks. Left, expanded view from membrane plane; right, expanded view from cytosol. The hydrophobic Met-layer and hydrophilic Arg-layer are highlighted by dashed rhomboids. b The luminal gate. Gate residues from TMs 1, 2, 7 and 8 from the cytosol-facing RES-bound VMAT2_YCR structure are shown. Left, expanded view from membrane plane; right, expanded view from lumen. The Phe-layer and Pro-layer are highlighted.

Fig. 6. Proposed mechanism for VMAT2-mediated transport of monoamines.

Schematic representation of the alternating access transport cycle. The 7 states are derived from direct experimental structures (States 4 and 6), docking poses (States 1, 2 and 5), and AlphaFold2 prediction (States 3 and 7). For clarity, only TMs 1, 2, 7 and 8 are shown as empty tubes in color. States that are not experimentally determined are shown in faint shades. Two negative residues D399 and E312 along the translocation pathway (empty circles, non-protonated; solid circles filled with green, protonated) facilitate substrate movement by alternating protonation states. Significant conformational shifts, particularly in TM2 and TM7, triggered by TBZ (green) entrance at the luminal side induce a dead-end occluded state.