# Functional dynamics of glutamate transporters probed by high-speed atomic force microscopy with micro- to millisecond time resolution

> **NIH NIH R01** · WEILL MEDICAL COLL OF CORNELL UNIV · 2020 · $351,162

## Abstract

Glutamate or excitatory amino acid transporters (EAATs) are members of the Solute Carrier 1 family of
transmembrane proteins. EAATs are key in the function of neuronal synapses responsible for the removal of
excitatory neurotransmitters from the synaptic cleft after each neurotransmission. Malfunction of EAATs is
involved in cerebral stroke, epilepsy, Alzheimer's disease, dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma. Thus, a profound understanding of the molecular determinants of EAAT
function is crucial to understand these diseases. In the last decade, the structure and function of a prokaryotic
glutamate transporter homolog, the sodium/aspartate symporter from archaebacterium Pyrococcus horikoshii,
GltPh, has been extensively studied and made a perfect model system for EAAT studies. More recently a first
structure of the human EAAT1 has been solved, but little is known about its transport dynamics and kinetics. In
this project we will study both GltPh and hEAAT1 proteins, with the aim to characterize the so far elusive
transport sub-states and kinetics in the ms and µs range. While the transport kinetics of the prokaryotic GltPh
are slow (seconds to tens of milliseconds), EAATs are expected to work at faster rates (~100 to ~1000
transport cycles per second). Here, we will reconstitute GltPh and hEAAT1 in lipid membranes and employ
high-speed atomic force microscopy (HS-AFM) to directly image transport cycles of individual unlabeled
glutamate transporters with the aim to resolve transport-related conformational sub-states and fast kinetics. To
achieve this goal, in addition to HS-AFM (HS-AFM) imaging, we develop and employ HS-AFM line scanning
(HS-AFM-LS) and HS-AFM height spectroscopy (HS-AFM-HS). In these two novel sub-modes, we reach
millisecond and microsecond time resolution, respectively. This makes our approach unique in three ways:
i) We analyze the dynamics of membrane transporters in membrane. ii) We analyze unlabeled single
transporter molecules. iii) We reach so far inaccessible temporal resolution. These novel approaches allow to
unveil the occluded state in GltPh (only the outward and inward facing states in the transport cycle were
assigned to date), its lifetime, the frequency with which it is being visited, and if it is visited on passage of a
complete cycle or if returns from the occluded state occur regularly. In addition, given the fast transport rates of
the eukaryotic homologues, we will pioneer single molecule dynamics measurements on human EAAT1
and provide first insights into their transport state probabilities and kinetics. The overall goal of the project is to
establish a new experimental tool to study millisecond and microsecond dynamics in transporters and
to apply this tool towards uncovering intermediates states in the transport cycles and measure currently
inaccessible fast transport kinetics on the single molecule level.

## Key facts

- **NIH application ID:** 10023957
- **Project number:** 5R01NS110790-02
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Simon Scheuring
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $351,162
- **Award type:** 5
- **Project period:** 2019-09-30 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10023957

## Citation

> US National Institutes of Health, RePORTER application 10023957, Functional dynamics of glutamate transporters probed by high-speed atomic force microscopy with micro- to millisecond time resolution (5R01NS110790-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10023957. Licensed CC0.

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