# Structural Basis of Vesicular Neurotransmitter Transport

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2023 · $627,108

## Abstract

The synaptic vesicle uptake of classical transmitters depends on a H+ electrochemical driving force
(ΔµH+), and generally involves the exchange of cytosolic transmitter for lumenal H+. However, vesicular
glutamate transport relies almost entirely on the electrical component of this gradient (Δψ) rather than
the pH gradient (ΔpH), and undergoes unusual, allosteric regulation by H+ and Cl-. The vesicular
glutamate transporters (VGLUTs) also exhibit an associated Cl- conductance, and the physiological role
of these properties remains unknown. Further, the VGLUTs belong to the solute carrier 17 (SLC17)
family which includes other members that rely on ΔpH rather than Δψ for transport in the opposite
direction from VGLUTs. The long-term objective of this proposal is to understand how the properties of
vesicular glutamate transport contribute to synaptic transmission. The strategy uses structure to
identify the mechanisms common to all family members and understand how their adaptation confers
the specific properties of vesicular glutamate transport. We have determined the first structures of an SLC17 family member, E. coli D-galactonate transporter DgoT, which is closely related in sequence to the VGLUTs. DgoT contains a polar pocket within the N-terminal lobe connected to the periplasm through a putative H+ tunnel evident in the
inwardly oriented structure. An outwardly oriented structure contains galactonate occluded in the
substrate recognition site. The structures predict that delivery of periplasmic H+ to a glutamate in
transmembrane domain (TM) 4 liberates an interacting arginine in TM1 to bind substrate. In contrast to
the VGLUTs but like other SLC17 proteins, DgoT catalyzes H+ cotransport. Although the critical
residues are conserved to the VGLUTs, they thus serve a different function in DgoT. We will now
1) Elucidate the mechanism that couples transport of galactonate to H+ in DgoT.
Using assays for exchange and binding as well as net uptake, we will determine how protonation of
DgoT contributes to substrate recognition. 2) Determine the structural basis for vesicular glutamate transport.
We will use a combination of crystallography and cryo-electron microscopy to determine the structure of
a VGLUT. 3) Elucidate the mechanisms responsible for allosteric regulation of the VGLUTs.
We will leverage the structures as well as the available assays for both DgoT and the mammalian
proteins to understand the allosteric regulation of VGLUTs by H+ and Cl-. We will also use
electrophysiology to assess a channel suggested by the structure, and determine its relationship to
glutamate flux.

## Key facts

- **NIH application ID:** 10614384
- **Project number:** 5R01NS089713-09
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** ROBERT H EDWARDS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $627,108
- **Award type:** 5
- **Project period:** 2015-08-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10614384

## Citation

> US National Institutes of Health, RePORTER application 10614384, Structural Basis of Vesicular Neurotransmitter Transport (5R01NS089713-09). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10614384. Licensed CC0.

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