Functional characterization of human GAT-1 using solid supported membraneelectrophysiology and automated patch clamp
Rocco Zerlotti1, Andre Bazzone1, Maria Barthmes1, George Okeyo2, Irene Lu2, Nadine Becker1, Alison Obergrussberger1, Elena Dragicevic1
1 Nanion Technologies GmbH, Germany
2 Nanion Technologies Inc., Livingston, NJ, USAA

ON signal
Baseline
Mechanical artifact

OFF signal

Non-activating solution

-80
-100

80

510time [s]

155A

20

1,31,4

1,51,61,71,81,1

1,21,3B∆𝑉 = 0ΔQ = 0 → ΔG = 01.5[Na ]in[Na+]out [GABA]in [GABA]out

1.0

20 mM
20 mM
20 mM
20 mM
20 mM

60 mM 50 µM 40 µM
60 mM 10 µM 20 µM
60 mM
5 µM 50 µM
40 mM 100 µM
2 µM
50 mM
1 µM 100 µM

0.50.0140
120
100
80

Transport (time point)
KM = (19 ± 6) µM

6040n = 1.0 ± 0.2
R2 > 0.95

2010100log10(GABA [µM])A) Traces obtained with CHO cells over-expressing hGAT1, using a Patchliner in whole cell modeand at membrane potential of -70 mV. Solutions containing GABA and NaCl have beenperfused for 10 seconds. B) GABA KM obtained with the Patchliner. The result is in perfectagreement with literature data (Bicho, 2005; Gonzales, 2007) and in very good agreement withSSME data.

1.25

1.50time [s]1,51,61,71,81,92,01,01,11,21,3

1,4

1,5

1,61,71,81,92,0time [s]140120100

Transport (reco)
KM = (27 ± 3) µM80

n = 0.77 ± 0.08
R2 > 0.98

60
4001

10

100

1000log10(GABA [µM])

10100

1000

10000log10(GABA [µM])A) Above: GABA-induced currents in high Na+ gradient; Below: GABA KM in high Na+ gradientB) Above: GABA-induced currents in low Na+ gradient; Below: GABA KM in low Na+ gradientC) Above: GABA-induced currents at low GABA; Below: peak deconvolution

6∆µ𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
𝜕𝑄
𝑄= 𝑄0 +∆µ𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
∆µ𝑖𝑜𝑛
𝜕
∆µ

0,06

∆µ𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
∆µ𝑖𝑜𝑛

𝑖𝑜𝑛

Summary• SSME is a suitable technique for studying hGAT1,revealing multiple electrogenic events upon
GABA binding and transport.

0,04

Na+:GABA=2.05:1

0,02

0,00

1.75

• The cells from which the vesicles for SSME havebeen generated have been tested with patch
clamp, validating the functionality of the sample.
• The stoichiometry of Na+ and GABA has beensuccessfully assessed with SSME, confirming themost accepted one of GABA:Na+=1:2.

-0,04
1.00

τtransport

160-0,02

𝐺𝐴𝐵𝐴 𝑖𝑛𝑙𝑛∆𝜇𝐺𝐴𝐵𝐴𝑛𝐺𝐴𝐵𝐴 𝑜𝑢𝑡==−𝑁𝑎+ 𝑜𝑢𝑡∆𝜇𝑁𝑎+𝑚𝑙𝑛
𝑁𝑎+ 𝑖𝑛0,20,01,4time [s]160C+

τPSS,slow

180-0.5

12,0SSME

40

0,11,9+
GABA:Na stoichiometry with

∆𝐺 = 𝑚∆𝜇𝐺𝐴𝐵𝐴 + 𝑛∆𝜇𝑁𝑎+-20
01,21,00,30,11.12 s-0,21800-140

1,1

τtransport

time [s]20-1200,40,01.15 s
1,0n = 1.2 ± 0.1
R2 > 0.99

60

τPSS,fast

0,6

τtransport-0,2charge [nC]-60

0,40,20,0Transport (steady-state)
KM = (11.76 ± 1.26) µM

1000,80,2IGAT1 - control [nA]

current [pA]-40

τPSS,slow

1,00,4160

120

τPSS,fastcurrent [nA]

Activating solution

Lipid
ThiolGoldB-20

0,50,81,20,6

GABA-induced currents andGABA KM with patch clamp
140150 mM
KCl1,0current [nA]

Non-activating solutionThe protein of interest (expressed in membrane vesicles or reconstituted in proteoliposomes) isadsorbed to an artificial bilayer that functionalize a gold-coated sensor. The measurement iscarried out at 0 mV and the addition of substrate generates a concentration gradient that drives
the transport reaction. In a general SSME workflow a non-activating solution is perfused to obtain
the electrical signal baseline and – if needed, to generate a co-substrate gradient to energize
transport. Then, the activating solution containing the main substrate is perfused and transport isactivated: because of the capacitive-coupled nature of the system, only transient currents arerecorded. Finally, a second flow of non-activating solution brings the system back to the initialstate.

0135 mM
NaCl + 15mM KCl

1,2150 mM
NaCl +30 µM GABA

C150 mM
NaCl +5 mM GABA

1,4Here, CHO plasma membrane vesicles stably
overexpressing hGAT1 were used to measure GABA
induced currents and GABA:Na+ stoichiometry withsolid supported membrane-based electrophysiology(SSME) using a SURFE2R N1 system. We also measured
GABA transport through automated patch clamp
using a Patchliner, confronting the GABA
concentration dependence with literature results.

-70 mV holding potential, whole cell mode

150 mM
KCl

1,6Schematic of the measurement chamber of a SURFE2R N1 and measurement principle of SSME.

AB

150 mM
NaCl +
5 mM GABA

1,8

The apparent affinity (KM) for GABA re-uptake isknown to be in the low µM range and the currently
most accepted stoichiometry of GABA transport is
Na+:Cl-:GABA = 2:1:1.

4current [pA]

3GABA-induced currents andGABA KM with SSMEreconstructed current [pA]

The human γ-aminobutyric acid (GABA) transporter
1 (hGAT1) is the main regulator of GABA homeostasis
in the central nervous system. It is a secondary
active transporter that exploits the inward-directed
Na+ chemical gradient to energize the uphill reuptake of GABA from the synaptic cleft to thepresynaptic neuron.

2SSME – an overview

current [nA]1Introduction

current [pA]

Contact: Rocco.Zerlotti@nanion.de-5-4-3-2-10Dµ(GABA)/Dµ(Na+)A) Physical principle of stoichiometric assay with SSME. B) Traces obtained at all different
conditions of Na+ and GABA inside and outside the vesicles. The resulting traces from CHO
vesicles expressing GAT1 have been compared with traces from empty CHO vesicles used ascontrol, which have then been subtracted from the sample traces. C) Resulting linear fitting ofthe data. When the total charge is null, then the ratio between the chemical potential ofGABA and Na+ reflects the stoichiometry of the two substrates in the transport reaction(Thomas, 2021). At 0 charge, the stoichiometric ratio reflects a stoichiometry GABA:Na+=1:2.

1Bibliography:• Bicho et al., “Rapid Substrate-Induced Charge
Movements of the GABA transporter GAT1” DOI:
https://doi.org/10.1016/j.jbc.2021.101220• Gonzales et al., “Turnover rate of the gammaaminobutyric acid transporter GAT1” DOI:
10.1007/s00232-007-9073-5• Thomas et al., “A solid-supported membraneelectrophysiology
assayforefficientcharacterization of ion-coupled transport” DOI:
https://doi.org/10.1016/j.jbc.2021.101220