Application NoteTransporter:
hGAT1
Sample:Plasma membraneOrigin:
HEK cellsTools:
SURFE2R N1Electrophysiological recordings of hGAT1(SLC6A1) activity on Nanion’s SURFE2R N1The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Axxam S.p.A

SummaryResults

GABA is the major inhibitory neurotransmitter in the brain

To activate transport on the SURFE2R N1, a sensor withand is important in controlling excitability. After release,attached hGAT1-containing plasma membranes wasGABA is removed from the extracellular space by GABAinserted into the device and perfused with a buffertransporters (GATs), thus terminating inhibitory synaptic

containing NaCl and GABA. When the substrate istransmission. The GABA transporters belong to the family

present, Na+ and Cl- movement across the membraneof neurotransmitter:sodium symporters referred to as thecan be observed until an electrochemical equilibrium issolute carrier 6 (SLC6) family in humans1. GATs co-trans-reached. To generate Na+ and Cl- gradients, necessary as

port GABA, Na+ and Cl- with the proposed stiochiometrya driving force, the sensor was flushed with Kgluconate to

1 GABA: 2 Na : 1 Cl , resulting in a net influx of 1 positive

create a Na+ and Cl- gradient simultaneously before andcharge per cycle . So far, 4 GATs have been identified,after the hGAT1 activation (Figure 1). To ensure stable

GAT1, GAT2, GAT3 and BGT1 .

GAT1 current response for multiple activation cycles,+-2

3a waiting time and thorough rinsing of the sensor wasnecessary between measurements.

ergic and non-GABAergic neurons, and is expressedin particularly high levels in the olfactory bulb, basal*ganglia, cerebellum and retina4. The physiological
function of GAT1 is primarily to terminate synaptictransmission but also to ensure the fidelity of synaptictransmission by preventing the spread of neurotransmitter to neighbouring synapses5. GABA transporters also
play an important role in neurotransmitter reutilization .
5

In certain circumstances, e.g. when the sodium gradient

1 nA

GAT1 is expressed throughout the brain in both GABA-

200 msAPerfusion*GABANa+Cl-increases during ischemia or following seizures, GATs canact in reverse which may have a protective effect during

Kgluconate

Aseizures, by inhibiting electrical excitability5. There is someevidence that GATs may play a role in neurodegenerative diseases such as Parkinson’s3 and Alzheimer’s6 andmay provide a novel target for treating these conditions.
Here we present human GAT1 activity measurementson the SURFE R N1 instrument using purified plasma2

membranes from HEK cells. GABA affinity and effect ofinhibitors were investigated.
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Figure 1: Typical GAT1 current response on the SURFE2R N1. GABA was usedas the substrate for GAT1. When applying GABA to the sensor, a transientcurrent occurs (top) corresponding to the co-transport of GABA, Na+ and
Cl- (bottom).

Application Note
Aapparent affinity of 0.95 ± 0.10 mM (Figure 2).

0.6

5 mM GABA (GAT1 HEK)
100 µM GABA (GAT1 HEK)
5 mM GABA (parental HEK)

1.0

0.4
0.2
0.01100.001 0.01 0.1[Nipecotic acid (mM)]

0.8Tiagabine
IC50 = 22.9± 5 µM
(n = 3)1.00.80.60.40.21000.010-410-2100102104[Tiagabine (µM)]

Figure 3:
GAT1 is blocked by nipecotic acid (A) and Tiagabine (B). Experiments
performed in the presence of 1 mM GABA.

0.6500 pA

Normalized IPeak

0.8
Normalized IPeak

was applied in increasing concentrations giving anNipecotic acidB
IC50 = 0.18 ± 0.03 mM(n = 3)

Normalized IPeak

The affinity of GAT1 for GABA was investigated. GABA

1.00.4

GAT1 was blocked by nipecotic acid and tiagabine withan IC50 of 0.18 ± 0.03 mM (n = 3) and 22.9 ± 5 µM (n = 3),

200 msrespectively. These values are a little higher than thoseGABA
Km = 0.95 ± 0.10 mM(n = 3)

0.2

found in literature5,7, presumably due to differences intechnique used.
In conclusion, the SURFE2R N1 can be used to reliably

0.01

10.1
[GABA (mM)]

10

Figure 2: Affinity of GAT1 for GABA. GABA was applied in increasing concentrations revealing a Km = 0.95 ± 0.10 mM (n = 3). In cells not expressing
GAT1 (parental HEK), no transport was observed (grey).

measure GAT1 activity using whole cells attached to thesensors. The assay could be scaled up to the SURFE2R 96SE
for higher throughput screening and hit identification for
GAT1 inhibitors for the treatment of neurodegenerative
diseases such as Parkinson’s and Alzheimer’s.

References

Buffers

1. Scimemi, A. 2014. Front. Cell. Neurosci. 8:161.

GAT1 experiments were performed by the exchange of a

2. Loo, D.D.F., et al. 2000. J. Biol. Chem. 275(48): 37414–37422

sodium and GABA-free (“resting”) buffer for a sodium

3. Jin, X-T. et al. 2011. Front. Sys. Neurosci. 5: 63

containing (“control”) buffer and afterwards a substrate

4. Durkin, M.M., et al. 1995. Mol. Brain Res. 33: 7-21

and sodium containing (“activating”) buffer. Resting

5. Borden, L.A. Neurochem. Int. 1996. 29(4): 335–356

buffer contained: 140 mM Kgluconate, 2 mM MgCl2 30

6. Fuhrer, T.E., et al. Neurosci. 2017. 351:108-118

mM HEPES, pH 7.4 with NMG. Control buffer contained:

7. Soudijn, W. & van Wijngaarden, I. 2000. Cur. Med. Chem. 7:

140 mM NaCl, 2 mM MgCl2 30 mM HEPES, pH 7.4 with

NMG, activating buffer identical to control plus x mM

1063-1079

GABA.

Methods

SURFE2R sensor preparation

Plasma membrane preparation
HEK cells expressing hGAT1 were kindly provided by
Axxam S.p.A and membranes were prepared according
to the Nanion’s standard procedure (“QuickguideMembrane Preparation from CHO cells”). Total protein
concentration was between 5 - 10 µg/µl.

According to the Nanion standard procedure “SURFE2R
Sensor Preparation”. Sensors are prepared in resting
buffer, membrane is diluted 1:10 with resting buffer.
SURFE2R N1 measurement workflow
GAT1 can be activated by providing GABA as thesubstrate. A sodium gradient needs to be established inadvance of substrate addition. Therefore, any 3-buffer
Nanion standard protocol is suitable.

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