Application Note

Transporter:
EAAT3Sample:
Plasma membraneOrigin:
CHO cellsTools:
SURFE2R 96SE

High throughput SSM recordings of EAAT3(SLC1A1) activity on the SURFE2R 96SE
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

The excitatory amino acid transporter 3 (EAAT3; also

To activate transport on the SURFE2R 96SE, the sensor

known as EAAC1) is a sodium-dependent neuronal uptake

plate containing 96 individual sensors with attachedtransporter encoded by the SLC1A1 gene1. It plays aEAAT3-containing membrane fragments was perfused

major role in the reuptake of glutamate from thewith a buffer containing NaCl and glutamate. When thesynaptic

cleft,

therebymaintaining

alow

extra-​ substrate is present, Na+ and K+ movement across thecellular concentration of glutamate and regulating themembrane can be observed until an electrochemical

excitatory neurotransmission. EAAT3 is also involved inequilibrium is reached. To generate Na+ and K+ gradients,

the uptake of aspartate and cysteine into the cells . The

necessary as a driving force, the sensor was flushed withtransporter is highly expressed in mature neurons, where it

KCl before and after the EAAT3 activation (Figure 1).

1,2

is distributed in somata and dendrites .
1,2*

EAAT3 functions as a co-transporter, coupling the uphill
substrate transport into the cells to the electrochemical
gradients of sodium and potassium. The stoichiometry ofAtransport is 1 glutamate with 3 Na+ and 1 H+ moving intoPerfusion

the cell to 1 K+ moving out of the cell1,2. Therefore EAAT3
is an electrogenic transporter, generating a net chargeflow.

200 pA
200 ms*Glutamate
NaClDysfunction of glutamate transporters leads to increased
extracellular glutamate levels, thereby causing neurotoxicity

andmechanisms

neurodegeneration2.
facilitating

EAAT3

Regulatory

functionKClA

are,

therefore, interesting as targets for the treatment ofneurodegenerative diseases2.
Here we present EAAT3 activity measurements on
the SURFE2R 96SE instrument using purified plasma
membraneofCHO

cells

expressing

EAAT3.

The

transporter was activated by glutamate and blockedby six different known inhibitors with IC50 values similar to
those found in the literature.

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Figure 1: Typical EAAT3 current response on an individual sensor of theSURFE2R 96SE. Glutamate was used as the substrate for EAAT3. When
applying glutamate to the sensor, a transient current occurs (top; actualrecording trace from an experiment on the SURFE2R 96SE).

Success rate and signal stability
An investigation into reproducibility revealed that in a
typical experiment 97 ± 3% of the sensors in a 96 well plate(average of 10 plates) met the quality criteria and generated current signals >150 pA. A screenshot showing atypical plate with color-coding for quality of the SSM isNormalized IPeak

Application Note
1.2
0.8
0.4
0.020Normalized IPeak

shown in Figure 2.

46Time (hr)81012

1.20.8

Typical Experiment
0.4 duration (~50 min)
0.00

20

4060
Time (min)

80

100

Once prepared, sensors are very stable and signals arehighly reproducible. Figure 3 shows normalized currentsof EAAT3 activated by glutamate in the presence ofsodium. EAAT3 can be repetitively activated for over
12 hours with similar amplitudes. A typical experiment
duration is approximately 50 minutes (shown in the inset).
In this time, EAAT3 was activated 9 times with similar peak
amplitudes proving the suitability of the technique forNormalized Peak Current

Figure 3: Stability of EAAT3 signals on the SURFE2R 96SE. EAAT3 can bereproducibly and repetitively activated for 12 hours. Over the courseof a typical experiment (approx 50 mins; bottom panel), EAAT3 can be
repetitively stimulated and reproducible currents are recorded. Datashown is average normalized peak current for a 96 well plate (n = 94 wells) ±2
Figure 2: Success rate of experiments on the SURFE R 96SE. Shown is astandard deviation.
2screenshot of one experiment on the SURFE R 96SE. Wells are color-coded
based sensor quality (green: high quality; blue: intermediate quality or grey:Glutamate
Failed well). Shown is signal generated upon perfusion with activating buffer
EC50 = 99 µM+1.0(Na and glutamate).

0.6
0.4
0.2
0.0

the concentration response curve (CRC) for glutamate
was calculated across the whole plate with n = 8 wellsper concentration. In each experiment, negative control (baseline) and maximum current are also recorded

Normalized block

centration of glutamate was applied to each well and10-10

1.4
1.2
1.0
0.8
0.6
0.4
0.20.0revealing an EC50 for glutamate of 99 µM, similar to thatrecorded using the SURFE R N1 (see Application Note).
210-6
10-4
[Glutamate]

PDC
IC50 = 1.9 ± 0.4 µM (n = 15)

10-2

Control (100 µM Glu)
1 µM PDC
10 µM PDC
100 µM PDC

50 pA
50 ms

10-9in each well and data was normalized to the maximum
current in each well. The CRC was fit with a Hill Equation

10-8

Figure 4: Concentration response curve for activation of EAAT3 by glutamate.
Shown are average ± S.E.M of 8 wells per concentration of glutamate. The
curve was fit using a Hill equation revealing an EC50 for glutamate of 99 µM.

pharmacological investigation.
Glutamate was applied to the sensors, a single con-Max = 1.0

0.8

10-8

10-7

10-6 10-5[PDC (M)]

10-4

10-3

10-2

Figure 5: Concentration response curve for PDC. Shown is the concentration
response curve for PDC for an average of 15 wells. Traces for an exemplar
well are shown on the right.

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fax

80339 Munich, Germany

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Application Note

Figure 6: Graphical user interface of the screening and data analysis software used on the SURFE2R 96SE. Screenshot of depiction of online analysis data ofEAAT3 activated by glutamate and blocked by different compounds. Six different compounds were used on one plate, cumulative concentrations wereadded to each well and full concentration response curves in each well were calculated. Two columns of the plate were exposed to each compound
giving n = 16 wells per compound. All wells were successful in terms of quality of sensor (green wells). Average IC50 values for each of the 6 compounds aregiven in Table 1, an example of a concentration curve for one of the compounds (PDC) is shown in Figure 5.
Instrument
Sensor Preparation
Initialization
Start-up ofinsrument

SensorCoatingCentrifugation

10 min10 min1 Plate30 minMeasurements
ExperimentSensor loadand QC

60 min6 Plates

30 min

5 min~50 min

Dataanalysis

5 min

Experiment: 2 x negative control
3 x full signal (glutamate)
2-6 compound applications
washout + 2 x full signal

Start new experiment withpre-prepared sensor plate
Figure 7: Workflow for experiments on the SURFE2R 96SE. Sensors (96 well plate format) are prepared on the robot and subsequently centrifuged. If multiple
sensors are used on one day, they are prepared sequentially and centrifuged together. One sensor plate containing 96 sensors is then loaded onto theSURFE2R 96SE and experiments conducted. In these experiments, total time was approximately 50 mins. Once the experiment is finished, a new sensor platecontaining 96 sensors is loaded onto the SURFE2R 96SE and a new experiment started.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70fax

80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note
Compound

IC50 (µM)

Literature value(µM)

HIP-B

3.3 ± 1.4 (16)17-183 (IC50)UMPDC

7.2 ± 1.5 (15)54 - 75(Ki) U

ACBD

1.5 ± 0.2 (16)305(Ki) U

SkyBlue

0.6 ± 0.1 (16)0.86 (IC50) B

PDC

1.9 ± 0.4 (15)1.55(Ki) U

L-TBOA

0.4 ± 0.05 (15)

7 - 157,8 (IC50) U

concentrations of the compound were added in
duplicate (1, 10 and 100 µM) via the activating buffer.
At the end of the experiment, the compounds were
washed out and control activating buffer was added.
Two columns per compound were used giving n = 16
wells per compound. An example of a concentration
curve for one of the drugs (PDC) with the correspond-Table 1: IC50 values for 6 different blockers of EAAT3 calculated using theSURFE2R 96SE. Shown are average IC50 values ± S.E.M obtained on theSURFE2R 96SE (number of wells shown in brackets) and the expected
literature IC50 or Ki values. B = binding assay; U = uptake assay.

ing traces from one example well is shown in Figure 5.
The IC50 values calculated on the SURFE2R 96SE aresummarized in Table 1. Shown are average IC50s calculated from the individual IC50 values from each well ± S.E.M.

The IC50 values calculated on the SURFE2R 96SE agreed
well with the literature3-8, some being a little lower, e.g.

Pharmacology experiments on the SURFE R 96SE canHIP-B, ACBD and L-TBOA presumably due to differences

be performed in different ways. Single point CRCs canin the technique used (binding versus electrophysiology).

2

be performed calculated across the whole plate or cumulative CRCs can be performed on individual wells,

In conclusion, EAAT3 can be reliably measured on theas shown in Figure 6. This increases the number of dataSURFE2R 96SE with expected EC50 values for glutamate

points that can be obtained from 1 sensor plate. In theexperiment shown in Figure 6, six different compounds

and IC50 values for a number of known blockers. TheSURFE2R 96SE is a highly sensitive SSM-based electrophysi-

were investigated in one experiment. The experiment

ology device for higher throughput recordings of trans-

started with 2 applications of resting buffer (negativeporters and ion pumps.

control) followed by 3 applications of glutamate (100µM). Once a stable signal was established, increasing

Referencesa sodium and glutamate free (“resting”) buffer for a

1. Zhou, Y & Danbolt, N.C. 2013. Front. Endocrinol. 4:165.

sodium containing (“control”) buffer and afterwards a

2. Aoyama, K & Nakaki, T. 2013. Amino acids. 45(1): 133–142

substrate and sodium containing (“activating”) buffer.

3. Funicello, M., et al. 2004. Mol. Pharmacol. 66(3): 522-529

Resting buffer contained: 140 mM KCl, 2 mM MgCl2 30

4. Bridges, R.J., et al. 1994. Neurosci. Lett. 174: 193-197

mM HEPES, pH 7.4 with NMG.

5. Koch, H. P., et al. 1999. Mol. Pharmacol. 56: 1095–1104

Control buffer contained: 140 mM NaCl, 2 mM MgCl2 30

6. Bai, F., et al. 2012. J. Biol. Chem. 287(36): 30653–30663

mM HEPES, pH 7.4 with NMG, activating buffer identical

7. Shimamoto, K., et al. 2000. Bioorg. Med. Chem. Lett. 10: 2407-2410

to control plus x mM glutamate.

8. Shimamoto, K. The Chemical record. 8: 182–199

SURFE2R sensor preparation

Methods

According to the Nanion standard procedure “SURFE2RSensor Preparation”. Sensors are prepared in resting

Plasma membrane preparation
AccordingtotheNanion’s

standardprocedure(“Quickguide Membrane Preparation from CHO cells”).
Total protein concentration was between 5 - 10 µg/µl of
the undiluted protein preparation which is about 0.1 µg
per well.

buffer, membrane is diluted 1:10 with resting buffer.
SURFE2R 96SE measurement workflow
EAAT3 can be activated by providing glutamate,aspartate or cysteine as a substrate. A sodium gradientneeds to be established in advance of substrate addition.
Therefore, any double solution exchange protocol isBuffers
EAAT3 experiments were performed by the exchange ofsuitable. All sensors are prepared at the start of the day
and then used sequentially.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70

fax

80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960