An emerging technique for the characterization oftransport proteins: SSM-based electrophysiology
Andre Bazzone, Maria Barthmes, Andrea Brüggemann, Niels Fertig
Nanion Technologies GmbH, Ganghoferstr 70A, 80339 München, Germany

About the technique:SSM-based electrophysiology

MembranepotentialGƹ increasingGƹ PD[

decreasing

On-signal

Current A

Off-signal

OCT2
Vmax and EC50 fordifferent organic cations
APEPT1
Inhibition assays for protoncoupled peptide transportBAC1nA
0.5 s

Start of perfusion

Noramlized Peak Current [%]B

Glycyl-glycine

100CA EC50 values and relative Vmax for three different
substrates using the SURFE2R N1. TEA is the highest affinity
susbtrate with lowest Vmax, while Metformin shows thelowest affinity and the highest Vmax.
B Example of transient currents recorded on two sensor
columns using the SURFE2R 96SE. Left: Incubated withOCT2 sample; Right: Control membrane without OCT2.
C Using the SURFE2R 96SE we performed measurementswith different speeds of solution flow. The faster thesolution flow, the higher the time resolution of solution
exchange. The time resolution affects the signal shape offast transient currents. The possibility to switch between
different time resolutions allows to resolve fast binding
kinetics or to reduce the impact of fast artifacts on thesignal shape.

SGLT1

Symporter (sugar/H+)

80
60
4020

IC50 = 580 mM

010

2.0control
100µM
500µM
1mM

1.5
1.0

Na+/K+ ATPase
Ion pump (3Na+/2K+)

ATP, Na+
ATP, Na+, K+

NhaA

Exchanger (Na+/2H+)

0.5 nA
250 ms100 µM Carbamoylcholine+ 1 µM MB327(enhancer)

A2B

internal: 20 mM X2+
external: 10 mM Na+
300 ms10C

0.5

1.1

1.2

1.3
1.4
Time [s]1.5

Verified targets
ATPases:NaK-ATPase,
HK-ATPase,
SERCA, v-ATPase, fof1-ATPase, KdpATPase, CopA, ATP7, PPase. Redox
driven pumps: Complex I, I/III, II/III, COX.
Light driven pumps: BR, KR2, HR, ChR,Acerhodopsin. Ion channels: P2X2,nAChR, AM2, UCP1, TRPC. Inorganic ion
transporters: NhaA, NhaP, NCX, Clc,NirC, Amt, SulP, NIS, NaPi. Amino acid /peptide transporters: PEPT1, YdgR, YhiP,PutP, GltP, EAAC1, PAT1, ArcD, Slc6a15,
CAT2B,GlyT.
Sugartransproters:
SGLT1/2, MelB, LacY, FucP, XylE, GlcP.
Organic ion transporters: OCT2, CNT1,ANT, GAT1, BetP, CHT.

Verified samples
Proteoliposomes, membrane vesicles,
plasma membrane fragments, synaptic
vesicles, lysosomes, microsomes, ER/SR
vesicles, mitochondrial membranes,nanodiscs, whole cells.

300 ms

C a2+ M g2+ S r2+ B a2+2300 ms

1

0.5
02
1.5

300 ms
C a2+ M g2+ S r2+ B a2+XylE
pH dependence of sugartransport and binding
ABCDinternal: 140 mM X+
external: 0.1 mM Ca2+

10.50Dinternal: 140 mM Na+
external: 0.1 mM X2+

1.5

A Whole trace of a standard solution exchange protocolon the SURFE2R N1 exchangig from 30 mM glycine
containing buffer to a 30 mM Gly-Gly containing buffer.
Gly-Gly is the substrate and activates the transporter.
B The same solution exchange was repeated withdifferent background concentrations of the inhibitor LyZ
to determine IC50 value.
C The concentration response experiment was repeatedusing the SURFE2R 96SE to measure 96 sensorssimultaneously. The screenshots show the measurement
software SurfControl (top) and the data analysis software
DataControl (bottom).

500 ms

Ion channel (cations)

-3

1010
LyZ Concentration [M]50 pAnAChR-5

NCX
Ion selectivity and modes of
Na+/Ca2+ exchange

0.5

Glycine

0.0-7

The SURFE2R 96SE was released this year to address therequirements of high throughput for industrial drug
screening purposes. It allows the simultaneous
measurment of 96 sensors in a 96-well plate format. The
electrophysiologicalhardware
fromNanion
Technologies is integrated into the robotic liquidhandling platform CyBio Felix.

1.5

Stop of perfusion

Current Response [nA]

Glycine

SURFE2R 96SE:
high throughput instrumentation

21.5

N a+

K+

R b+

Li+

internal: 20 mM Ca2+
external: 10 mM X+

10.5
0N a+

K+

R b+

L i+

The SURFE2R N1 allows preloading of the vesicle withCa2+ or Na+ using the double solution exchange
protocol. This allows the observation of different transport
directions with positive or negative current amplitudes.
Different ions were used to test the ion selectivity of thetransporter. Black bars represent measurements using
negative control membranes without NCX.
A: Selectivity for internal divalent cations against an
inward directed Na+ flow.
B: Selectivity for internal monovalent cations against an
inward directed Ca2+ gradient.
C: Selectivity for external divalent cations against an
outward directed Na+ flow.
D: Selectivity for external monovalent cations against an
outward directed Ca2+ gradient.

A Sugar induced transient currents at different pH for wild
type XylE (H+/xylose symport) using the SURFE2R N1.
B Kinetic model of H+/sugar symport in XylE. There are
two electrogenic reactions: one sugar binding induced
conformational transition and one conformationaltransition following proton release reflecting thesteady-state electrogenicity.
C The E206A mutation inside the sugar binding pocket
decreases Vmax of sugar transport. At acidic pH proton
release is blocked and a fast sugar binding current
becomes visible, which is also observed in wild type XylE.
D The mutation also yields a pK shift in transport activity
compared to wild type XylE. E206 therefore plays acrucial role in coupling between proton and sugartransport.

The future of transporter researchClassification of human transport proteinsV-type F-type
P-type ATPases ATPases
ATPases
ABC transporters
otherion channels

3%
6%

3% 2%

62 % transporters + ATPases
38% ion channels

12%
48%

ligand-gatedion channels

Number of publications

Events @electrodeDuring the experiment a fast solution exchange provides
the substrate and activates the electrogenic transport
reaction. This is accompanied by the charging of themembrane which is detected as a transient current. The
high sensor stability allows for different solution exchange
experiments using the same sensor. Both transport andbinding can be resolved and kinetic parameters like rate
constants, KD, KM or IC50 can be determined in a fastand easy workflow. One single measurement takes onlyseconds.

Normalized Current PeakNon-activating

Normalized Current Peak

Activating

500 pA

Non-activating

500 pA

Perfusion

0HDVXUHPHQW
HOHFWURGH

500 pA*ROGVXUIDFHZLWK660/LSLGOD\HU660 7KLROOD\HUGoldAThe SURFE2R N1 device contains electrophysiologicalhardware, liquid handling components and thecomputer running the data recording and analysis
software SurfControl. It’s an easy-to-learn, all-in-one
robotic workstation which can measure 150 data points
a day in a fully automated manner. The solution
exchange is controlled by the Ionjet which loads
solutions from storage containers and injects them to thesensor well enclosed by a Faraday cage. An
autosampler on top of the device allows placing up to 53
solutions for automated sequential measurements.

500 pA

Reference
HOHFWURGHIn SSM-based electrophysiology any membranes
containing the protein of interest are adsorbed onto asolid supported membrane (SSM). The SSM is assembled
on a gold coated sensor chip and consists of a thiol layer
with a lipid monolayer on top. The large sensor of 3 mm indiameter allows the simultaneous analysis of up to 109transporters. The possibility to use purified samples further
increases the signal to noise ratio which allows themeasurement of targets with lower turnovers comparedto ion channels, such as uniporters, symproters,exchangers and ion pumps.

Normalized Current Peak

/LSRVRPHVSURFE2R N1:
compact solution for research purposes

Normalized Current PeakThe sensor

About the instrumentation:
22SURFE R N1 and SURFE R 96SE

SLC transporters

9%

17%

voltage-gatedion channels

Hediger et. al. The ABCs of membrane transporters
in health and disease (SLC series): Introduction

Analysis of the human transport proteins shows that 62%
are potential targets for SSM-based electrophysiology
and only 38% are ion channels which are analyzed
mostly using conventional electrophysiology. It is
known that SLC transporters play a crucial role inhuman health and disease. But the tools for functional
characterization of transporters are rare. SSM-based
electrophysiology may be the missing tool to pushtransporter research to the next level.

SSM-based electrophysiology
on the fast track
50454035302520151050year
SSM-based electrophysiology has already been
proven as an efficient tool for the characterization ofelectrogenic membrane proteins. Until now more than
100 different proteins have been tested; almost 100peer reviewed papers have been published. The
number of publications using this technique steadily
increased over time proving the acceptance of thistechnique in the scientific community.

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