High throughput real-time measurement of electrogenic membranetransport driven by the SLC transporters PepT1 and OcT2.
Maria Barthmes1, Andre Bazzone1, Stephan Holzhauser1, Niels Fertig1, Michael George1, Andrea Brüggemann1
1Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany, contact: Info@nanion.deScreen shot of analysis software. For each well an individual
EC50 is calculated and for each substrate an averaged EC50.

resultexport

Screen shot of measuring software during substrate specificity comparison. Negative control(red), full activation (black), increasing substrate concentrations (blue).

Screen shot of measuring software during competitive inhibition experiment. Negative control (red),
positive control (blue), inhibitor addition (black).

Substrate specificity.
OCT2 was expressed in CHO cells and the membrane was purified. Threedifferent substrates are provided in increasing concentration cumulatively ineach well. The substrates show different kinetics and affinities. Parental
membrane was used as a negative control.

automated
analysisAnalysis of PepT1 peak currents over time.

Summed up IC50 calculation of 96 wells.

Evaluation of Target Scope

ChR2 H134R

ChR2 T159C

controlbacterial sugar transporter(XylE wt)

Signal Stability. RepeatedactivationofPepT1
demonstrates stable signal
amplitudes for more thanan hour. This enables
recordings of replicatesandcumulative
compound addition.

automated
analysisFirst it was examined whether

Membrane fragments from CHO cells overexpressing NCX (Charles River).
Cumulative addition of five increasing calcium concentrations after
application of a sodium gradient.

Membrane fragments form electroplaque tissue of T. californica. Activation
with 100 µM carbachol after application of a sodium gradient. Inhibition ofnative sodium channels with ambroxol. Negative control experiment isdisplayed in blue. Protocol after: Niessen et.al. Toxicology Letters 247 (2016).

Whole CHO cells expressing different mutations of channel rhodopsin 2.
Activation with a light pulse (470 nm, 200 ms). Sodium moves trough the open
channel into the cell, following the concentration gradient.

Inner mitochondrial membrane isolated from pig heart tissue. Complex I of therespiratory chain is activated by the application of NADH. Negative controls
are displayed in blue.

Mitochondrial F-Type ATPaseA
specializedrecordingchamberincorporating a 96 well sensor plate, a 96
channel low noise amplifier and respective
control electronics was included into aCybio Felix liquid handling robot. Optimized
assay workflows and software tools forrecording and analysis were developed,
enabling the recording of 96 data points inless than 5 minutes. Additionally thepreparation of the SSM sensor plates can be
performed by the platform itself.

Competitive Inhibition.
PepT1 was expressed inCHOcellsandthemembrane was purified.
IC50
determination:
Aninhibitor is applied in various
concentrations.
Inadvance, a negative (red)and a positive control(blue) were performed ineachwell.
The
fullexperiment takes 15 min.

Channel rhodopsin 2

Due to the growing interest in membranetransporters and pumps as drug targets
there is an increasing demand for reliablehigh throughput measuring systems. Here
we designed and validated an automatedfull parallel 96 well based platform,
employing SSM base elctrophysiology. The
method is not only unique in its target andassay flexibility, it’s also providing highquality data and it’s label- and radioactivity
free – Properties ideal for a high throughput
system.

Function: Renal secretion of organic cations
Mechanism: Polyspecific cation transport
Clinical significance: Renal clearance, drug interactions

Respiratory chain complex I(enzymatic proton pump)

Scale-up

OCT2/ SLC22A1

Function: Absorption of oligopeptides in the small intestines
Mechanism: Proton/Peptide symport into the cell
Clinical significance: Increasing the bioavailability of drugs

Sodium/Calcium
Exchanger (NCX1)“Solid Supported Membrane (SSM) based electrophysiology” is anestablished method, developed at the Max-Plank-Institut, which allows torecord low amplitude electrogenic events with a high amplification. The
method allows real-time activity measurement of targets for whichconventional electrophysiology is unsuitable, like many electrogenic
membrane transporters and pumps, or targets localized in intracellular orbacterial membranes.
This is achieved by theuse of a 1-3 mm gold
electrodewhichiscoated with an SSM andmembrane vesicles orliposomes containing thetarget protein. The proteinofinterest
isactivated by a fast solution
exchange.
Ineletrogenic transporters
this generates a membrane potential, which
can be measured at theelectrode.
Thepeak
current is proportional tothe turnover rate.

PepT1/ SLC15A1

Nicotinic acetylcholine
receptor

An electrophysiological method
suitable for transporters and pumps

XylE was purified from e. coli and reconstituted in a polar lipid extract with anlipid to protein ration of 5. The liposomes were added to the SSM. Activation oftransport with 30 mM D-Xylose. PH dependence of the transport wasdemonstrated: at acidic pH only sugar binding is observed, visible in acharacteristic fast signal.

Control

Inner mitochondrial membrane isolated from pig heart tissue. Activation of theFoF1 ATPase with 300 µM MgATP. A stable signal amplitude of the sensitive
pump for repetitive signal activation can be maintained. ANT, diemitochondrial nucleotide transporter was blocked by magnesium.