Functional investigation of GABA and Glutamate re-uptake transporters
EAAT3 and GAT1 using SSM-based electrophysiology
Rocco Zerlotti1,2, Alison Obergrussberger1, Maria Barthmes1, Cecilia George1, Niels Fertig1, Andre Bazzone1
1Nanion Technologies GmbH, Munich, Germany; 2 Regensburg University, Germany

GAT1 transport in CHO membrane vesicles

Introduction
GAT1 (SLC6A1) and EAAT3 (SLC1A1) are secondary active
neurotransmitter transporters coupled to the ion gradients establishedby the NaK-ATPase. Both are expressed in neurons and play a majorrole in the depletion of the neurotransmitters GABA and glutamate
from the synaptic cleft. Therefore, they play a major role intermination of synaptic transmission, maintaining the ambient
extracellular neurotransmitter concentration and neurotransmitter
recycling through reuptake. GAT1 is linked to epilepsy, schizophrenia,
anxiety and ADHD. Subtype specific inhibition of EAAT3 is assumed tobe beneficial during phases of insufficient energy supply by
preventing reversal glutamate transport. Therefore, screening
compounds to find inhibitors of these transporters is of highpharmacological interest.
We used solid-supported membrane (SSM)-based electrophysiology
using the SURFE2R N1 and SURFE2R 96SE to record GAT1 or EAAT3
purified from the plasma membrane of CHO or HEK cells
SURFE2R N1
1 recording channelA
Irec (t)= 1-Cp1* I(t)+CSSM
τ0Valinomycinaddition

D0E

GABA jump
150 mMNaCl

150 mM KCl

150 mM KCl
ΔV < 0150 mM KCl

150 mM KCl

I(t)dtEstablish Na+ ∇100 nM val
150 mM KCl

150 mM KCl

CBt

150 mM KCl
ΔV < 0150 mM KCl
ΔV < 0Incubation
10 min

150 mMNaCl

Figure 1: A: Raw representative current traces upon GABA jumps. B:
Reconstructed steady state currents from A. C: GABA KM obtained byreading reconstructed steady state currents from B. D: Scheme forvalinomycin assay (applying negative voltage to vesicles). E: Effect ofvoltage on hGAT1 currents
GABA jump

Establish Na+ ∇A

Figure 2: A: Scheme for GABA
jump experiments in different
Na+ gradients (lesser gradientare obtained incrementing
internal Na+ concentrations,X). B: Steady state current(Vmax) and peaks (presteady state, PSS) as afunction of Na+ gradient. C:
Representativetraces
obtained in maximum and nullNa+ gradient.

150 mM Na+

SURFE2R 96SE
96 recording channels

150 - X mM K+
X mM Na+B

150 - X mM K+
150 - X mM K+
X mM Na+X mM Na+150 - X mM K+
150 mM Na+

X mM Na+GABA

CSolid supported membrane electrophysiology
explained
SSM (solid supported membrane) based electrophysiology canresolve small currents as they are generated by SLC transporters
like EAAT3 and GAT1.
Plasma membrane of overexpressing
cells is collected – alternativelyreconstituted protein can be used.

EAAT3 activity measurement in overexpressing HEK cells
A sensor is coatedwith the membraneor cells. Changes inthemembranepotential
causedbyelectrogenictransport
eventscan be detectedon the sensor.

ABCD

GlutamateTransport activity istriggered repetitively
by perfusion of themembrane
coatedsensor
witha
substrate containingsolution.
Ionicbackground can beadapted
tothetransporter properties.

Conclusions• The SSM-based electrophysiology technique using the SURFE2R N1or SURFE2R 96SE can be utilized to record GAT1 and EAAT3transporters

Figure 3: Purified plasma membrane of a HEK-EAAT3 cell line shows stable activity induced by glutamate andinhibited by ACBD. A: Activation scheme: Application of glutamate after establishing a Na/K ion gradient. B: EAAT3
activity is dependent on ionic background. C: Apparent glutamate affinity. D: Tool compound ACBD inhibits EAAT3currents.

AB

C

IC50 SURFE2R

IC50 Literature

HIP-B

1.18 µM

10 µM

MPDC

5.1µM

5 µM

ACBD

14 µM

30 µM

Skyblue

8.5 µM

0.8 µM

PDC

0. 92 µM

1.5 µM

L-TBOA

0.29 µM

2 µM

DE

Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, Dubin AE & Patapoutian A(2010). Piezo1 and Piezo2 are essential components of distinct mechanically activated• cation
Smallchannels.
transport
currents
canbe measured which might not beScience(80- ) 330,
55–60.

in whole
cellclamp
electrophysiology
Lou detectable
JY, Laezza F, Gerber
BR, Xiao
M,patchYamadaKA, Hartmann
H, Craig AM, Nerbonne JM &Ornitz DM (2005). Fibroblast growth factor 14 is an intracellular modulator of voltage-gated569, 179–193.on the SURFE2R N1 and the assay was• sodium
EAAT3channels.
activityJ Physiol
was measured
scaled-up
to higher
the SURFE
Rotordam
MG, Fermo
E, Beckerthroughput
N, Barcellini W, using
BrüggemannA, FertigRN, 96SE.
Egée S,Different
Rapedius M,
BianchiP & Kaestner
L (2019). A
novel gain-of-function
mutation ofand
Piezo1
is functionallytransport
and binding
reactioncan be observed,
inhibition
byaffirmed
in redtool
bloodcells by high-throughput
patch clamp. Haematologica 104, e179.
publishedcompounds
was reproduced
2Up to 96 single dose
responses

Van der Valk JBF & Vijverberg HPM (1990). Glutamate-induced inward current in a clonalcell line.
Eur J Pharmacol
99–102. jumps show two distinct• neuroblastoma
GAT1 currents
generatedby185,
GABA

steady state• Application of negative voltages affects the PSS component ofGAT1 currents, enhancing it 2-folds. Na+ gradient affects Vmax andthe PSS component.

Inhibition (10 µM)

10.9
0.8% Inhibition

Vetter
I & Lewis RJ (2010).
of endogenous
calcium
responses
in neuronalcomponents:a Characterization
fast pre-steadystate (PSS)
current
anda slowcelllines. Biochem Pharmacol 79, 908–920.

One to 12 IC50s

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0TBOA

ACBD Skyblue HIP-B

MPCD

PCD

Conclusions

Figure 4: The assay was scaled up
to an HTS format and validated
using 6 known inhibitors. A:
Workflow for the HTS experiment.
Alternative workflows for one or 6
plates are displayed. B: Stability of
the current signal during repetitive
activation of a 96-well sensor
plate C: Comparison of IC50
values measured using SSM
based HTS electrophysiology andpublished values. D & E: Two
different assay modes: Single
dose experiment using multiple
compounds vs. full dose-response
measurement.