Application NoteTransporter:
EAAT3
Sample:Plasma membraneOrigin:
CHO cellsTools:
SURFE2R N1Electrophysiological recordings of EAAT3(SLC1A1) activity on Nanion’s SURFE2R N1The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

The excitatory amino acid transporter 3 (EAAT3; also

To activate transport on the SURFE2R N1, a sensor withknown as EAAC1) is a sodium-dependent neuronal uptake

attached EAAT3-containing membrane fragments wastransporter encoded by the slc1a1 gene1. It plays ainserted into the device and perfused with a buffer

major role in the reuptake of glutamate from thecontaining NaCl and glutamate. When the substrate issynaptic

cleft,

therebymaintaining

alow

extra-​ present, Na+ and K+ movement across the membrane

cellular concentration of glutamate and regulating thecan be observed until an electrochemical equilibrium isexcitatory neurotransmission. EAAT3 is also involved inreached. To generate Na+ and K+ gradients, necessary

the uptake of aspartate and cysteine into the cells . The

as a driving force, the sensor was flushed with KCl before

transporter is highly expressed in mature neurons, where it

and after the EAAT3 activation (Figure 1).

1,2*

is distributed in somata and dendrites .

1nA

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 oftransport is 1 glutamate with 3 Na+ and 1 H+ moving into
the cell to 1 K+ moving out of the cell1,2. Therefore EAAT3
is an electrogenic transporter, generating a net charge
flow.
Dysfunction of glutamate transporters leads to increased

200 msAPerfusion*GlutamateNaClKClA

extracellular glutamate levels, thereby causing neurotoxicity

andmechanisms

neurodegeneration2.
facilitating

EAAT3

Regulatory

functionare,therefore, interesting as targets for the treatment ofneurodegenerative diseases2.
Here we present EAAT3 activity measurements on theSURFE2R N1 instrument using purified plasma membraneof CHO cells expressing EAAT3.

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Figure 1: Typical EAAT3 current response on the SURFE2R N1. Glutamate wasused as the substrate for EAAT3. When applying glutamate to the sensor, atransient current occurs (top).

Application NoteA

consecutively, showing a stable signal height after theNormalized Peak Current [%]second application (n = 5; Figure 2A).
A
100
80
60
40
2000Normalized Peak Current [%]B
120

510Time [min]152010 mM NaCl
20 mM NaCl
50 mM NaCl
140 mM NaCl

100
80
60
40
200110100
1000
Sodium Concentration [mM]

500 pA

activation cycles on the same sensor were performed

EC50 = 42.2 ± 2.4 mM (n = 4)
Normalized Peak Current [%]The stability of the current response was tested. Ten

B

50 ms
Figure 3:
EAAT3 is sodium-dependent, with an EC50 of 42.2 ± 2.4 mM (n = 4).

The apparent affinity of EAAT3 for the substrates

Glutamate, EC50 = 80.5 ± 18.7 µM (n = 4)
Aspartate, EC50 = 70.7 ± 18.3 µM (n = 10)
Cysteine, EC50 = 5.3 ± 0.6 mM (n = 6)

glutamate, aspartate and cysteine was investigated.
EC50 values of 80.5 ± 18.7 µM (n = 4) for glutamate, 70.7 ±18.3 µM (n = 10) for aspartate and 5.3 ± 0.6 mM (n = 6) for100

cysteine were determined (Figure 2B). The Na+ sensitivity

8060of EAAT3 was investigated using buffers with different

40

concentrations of Na+, revealing a Na+ EC50 of 42.2 ± 2.4
mM (n = 4; Figure 3).

2000.001

0.01

0.1

1

10

Substrate Concentration [mM]

Figure 2: A Repeated measurements on sensors with 1 mM glutamate as
the substrate (n = 5). B Concentration response curves for 3 different substrates shown overlaid. Increasing concentrations of the substrates weretested cumulatively on the same population of membrane fragments.

References

sure EAAT3 activity. This has important implications for drug
discovery targeting EAAT3 for neurodegenerative diseases.
Control buffer contained: 140 mM NaCl, 2 mM MgCl2 30
mM HEPES, pH 7.4 with NMG, activating buffer identical

1. Zhou, Y & Danbolt, N.C. 2013. Front. Endocrinol. 4:165.
2. Aoyama, K & Nakaki, T. 2013. Amino acids. 45(1): 133–142

to control plus x mM glutamate.
SURFE2R sensor preparation
According to the Nanion standard procedure “SURFE2RMethodsSensor Preparation”. Sensors are prepared in resting
buffer, membrane is diluted 1:10 with resting buffer.

Plasma membrane preparation
According

In conclusion, the SURFE2R N1 can be used to reliably mea-totheNanion’s

standardprocedure(“Quickguide Membrane Preparation from CHO cells”).
Total protein concentration was between 5 - 10 µg/µl.

SURFE2R N1 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.

BuffersTherefore, any 3-buffer Nanion standard protocol is

EAAT3 experiments were performed by the exchange ofsuitable.

a sodium and glutamate free (“resting”) buffer for a
sodium containing (“control”) buffer and afterwards a
substrate and sodium containing (“activating”) buffer.
Resting buffer contained: 140 mM KCl, 2 mM MgCl2 30
mM HEPES, pH 7.4 with NMG.
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