EAAT3 INVESTIGATED USING
SSM-BASED ELECTROPHYSIOLOGYMaria Barthmes1, Andre Bazzone1, Stephan Holzhauser1, Michael George1, Niels Fertig1, Andrea Brügeemann1
1Nanion Technologies GmbH, Ganghoferstr. 70A, 80339 Munich, Germany, contact: Info@nanion.de

EAAT3

SSM-based electrophysiology

The excitatory amino acid transporter 3 (EAAT3, EAAC1) is an electrogenic secondary
active cotransporter of the SLC-family (SLC1A1). Structure and function of this transporterare comparatively well characterized.
A crystal structure and other data from the glutamate transporter homologue GltPhrevealed, that the protein is a homo trimer, with each monomer consisting of 7 TMDs and2 hairpins. Driven by the concentration gradients of sodium and potassium it transports
glutamate, aspartate and cysteine into the cell lumen. It is expressed throughout the CNS,
but also in many organs such as intestines, kidney and liver, as well as in cardiac andskeletal muscle.
EAAT3 is involved in the neuronal re-uptake and the homeostasis of glutamate. It plays acentral role in the regulation of excitatory neurotransmission and synaptic plasticity. It alsotransports cysteine, necessary for the synthesis of glutathione and GABA, and seems toprovide the main pathway for aspartate in some tissues.
Linked to this physiological functions several connections of EAAT3 to severe neuronal
disorders like Huntington’s and Alzheimer’s disease, epilepsy and schizophrenia have been
described, as well as metabolic disturbances concerning the maintenance of aspartateand cysteine levels.
Given all this implications of EAAT3, pharmacologicalmodulation of this transporter is of interest. The
unusually
detailedcharacterizationofthe
physiological and biophysical properties qualify thisprotein as a potential drug target. However, noselective inhibitors of EAAT3 are known today. To
facilitate the search for inhibitors, we developed anfunctional assay using SSM-based electrophysiology
with high throughput and robustness.

“Solid Supported Membrane (SSM) based
electrophysiology” is a method, whichallows to resolve low amplitude electric
events in biological membranes. This is why
it enables real-time activity measurement ofelectrogenic membrane proteins for whichconventional electrophysiology is unsuitable,
like many membrane transporters andpumps, or targets localized in intracellular orbacterial membranes. Unlike in cell-based
electrophysiology a large sensor coatedwith purified membrane vesicles
orproteoliposomes is used, leading to a highsum current signal. The method is establishedsince the 90ies and was up scaled to 96-wellformat recently.

The Importance of the Excitatory Amino Acid
Transporter 3 (EAAT3), W Bjørn-Yoshimoto and S M
Underhill, Neurochem. Int. 2016 Sep; 98: 4–18.

The method in detail. Top: Structure of a SSM sensor. The 3 mmdiameter, thin layer gold electrode is coated with a SSM consisting of athiol and a lipid monolayer. On top liposomes or membrane vesicles
containing the target protein are adsorbed. Bottom: Protocol of one
activation cycle. The sensor is perfused with a passive non activating
solution followed by an activating solution, containing a transported
substrate or ATP. During this step a transient current occurs, since theactive transporter moves charge into the vesicles what generates amembrane potential.
Up scaling: Right: The SURFE2R 96SE is a platform for automatedrecording of 96 SSM sensors in parallel. Upper left: 96 sensors in a wellplate. Lower left: interface view of 96 measurements.

intermediate sensor quality

Failed well(no contact, no signal or SSM not sufficient)

Representative view of one 96 well plate withcurrent traces. The color coding indicates thequality of the sensor

Time course of the mean transporter peak currentof 94 wells normalized to initial amplitude over 12hours. The average assay time is marked in red.
Errors indicate the standard deviation, N=94.

Signalstability:The
activation cycle describedabove was repeated every
5 minutes for 45 minutes,
followed by a reduced
repetition frequency (30min, later 2 hours). After 12hours
thesensor
stillgeneratedacurrent
response of the initial
amplitude, demonstratinga remarkable stability ofthe assay setup. A typicalinhibitor screening assay
takes about 15-25 min.

ACBDSkyBlue

HIP-B

MPDC

PCD

ACBD
SkyBlue
HIP-B
MPDC
PCD

2x negativecontrol

2x 1µM

3x wash out

2x 10µM

Screening mode:
A reduced number of concentrations is used,instead the number of compounds per plate isincreased. It is possible to test up to 96
compounds, each corresponding to one well. In
addition of the compound injection at least one
negative control and two positive controls (fullactivation with glutamate) should be performed.
This experiment takes 20 minutes.

2x 100µM

1

Example of an experiment setup to test several
compounds in one plate. Here 16 wells are used
per compound. The figure displays the userinterface during the measurement. The points in
the graphs reflect the peak amplitude during thetime course of the experiment. One well is enlarged
at the bottom and 12 wells at the right side.

KD=99 µM

10 nM
100 nM
1 µM

IC50 SURFE2R

IC50 Literature

HIP-B

1.18 µM

10 µM (Voltage Clamp)

3 µM

MPDC

5.1µM

5 µM (uptake, radio)

10 µM

ACBD

14 µM

30µSkyblue

8.5 µM

0.8µM

0. 92 µm

1.5 µM

L-TBOA

0.29 µm

2µM

30 µM

30 µM
100 µM

Example of an experiment setup to determine an
IC50. 8 wells/one column is used per concentration.
The figure displays the user interface during themeasurement, showing the current response ineach well and several enlarged views.

Inhibition (10 µM)

0,80,60,40,20

control

PDCGlutamate

Assay modes
TBOA

3x
positive
control

Control experiment: If the vesicles are perfused with sodium a big
transient current occurs, indicating the net inward current. Are thevesicles perfused with potassium, glutamate causes only a small peakindicating the binding of glutamate. In absence of glutamate nocurrent signal occurs.

The SURFE2R 96SE instrument:
Current recording and pipetting% Inhibition

High sensor quality

96-well format
Sensor plateTBOA

Comparison with other assay techniques:
EAAT3 has been investigated in the past using two-electrodevoltage clamp with oocytes, patch clamp and uptake assays
with radiolabeled substrates. To confirm validity of the SSM-based
assay, IC50 values of 6 compounds were determined andcompared to published values. Although the inhibitor potency isinfluenced by the glutamate concentration and the assay
conditions, the IC50s correlate well.

EAAT3 was overexpressed in CHO cells.
The cells were grown, harvested, lysed andthe plasma membrane isolated by partial
centrifugation and a density gradientcentrifugation. The resulting membrane
vesicles were frozen.
At the day of the experiment an 10 µl
aliquot of the membrane vesicles wasunfrozen, diluted and adsorbed to a 96well SSM-sensor. During this process thevesicles were loaded with potassium.
During the experiment the sensor isperfused with a sodium buffer, leading to
Rinsing of sensor an inward directing sodium gradient.
When adding glutamate a transientcurrent occurs. This current is caused bysodium
potassium
sodium and proton inward current whileGlu
potassium moves out of the vesicles. Thesensor is rinsed in potassium containing
Top leftEAAT3 is expressed in CHO cells and the membrane is purified
and stored. Top right: The measurement principle: EAAT3 transports
buffer to restore the ion gradients before
glutamate into the cells driven by the physiological gradients of sodium
the next activation cycle.
and potassium. To simulate this, the membrane vesicles were loadedwith potassium. After immobilization of the vesicles on the SSM theexternal buffer was exchanged to a sodium based solution. Under
these conditions glutamate was added to the vesicles triggering EAAT3activity. Bottom: The resulting current response and the perfusion
protocol.

96-well view inuser interface

Validation
Success rate: The conductance, capacitance andcurrent response in eachwell were analyzed. A
conductance below 200
MΩ or a capacitance
below 15 nF indicates aleaky
SSMandtherespective wellswererejected. This resulted in a
total success rate of 97±3 %(meanvalueof960
individual sensors).

Assay development

Characterization mode:
A high number of concentrations of one or twocompounds is tested to determine an IC50 or EC50.
In the beginning a negative control and a full
activation is performed. In total this experimenttakes 15 minutes.

Conclusion
 We developed an assay to measure theactivity of EAAT3 direct and in real time using
SSM-based electrophysiology.
 We tested for the affinity of glutamate,determined IC50s for several known inhibitorsand compared them with values derived
from other techniques (TEVC, uptake), whichshowed good correlation.
 The automated SSM-based assay has athroughput of about 2000 data points during
an 8 hour day (negative and positive controlexcluded), which is sufficient for an
secondary screening assay.
 The method has some relevant advantages:
In contrast to other assaying techniques norunning cell culture is required. The read-out
techniques is direct and does not requireradioactive labelling.
 We expect, that the developed EAAT3 assay
can be transferred to other glutamate
transporters without modification.
Literature:
The Importance of the Excitatory Amino Acid Transporter 3 (EAAT3), W BjørnYoshimoto and S M Underhill, Neurochem. Int. 2016 Sep; 98: 4–18.
An automatic electrophysiological assay for the neuronal glutamate transporter
mEAAC1, R Krause, Journal of Neuroscience Methods 177 (2009) 131–141
The substrate specificity of a neuronal glutamate transporter is determined by thenature of the coupling ion, D Menaker, Journal of Neurochemistry, 2006, 99, 20–28
Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate
transporter blockers, K Shimamoto, Bioorg. Med. Chem. Lett., 10 (21): 2407-10.