Studying Electrogenic Transporters of the SLC Family in a Parallel
Way Utilizing Solid Supported Membrane Technology
Maria Barthmes, Melanie Giesler, Stephan Holzhauser, Andrea Brüggemann
Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany

Abstract

PepT1/ SLC15A1

The solute carrier (SCL) family of membrane transport proteins includes
over 300 members. Many of them are related to conditions and diseases,or are involved in the transport of pharmacological agents. Therefore,
there is a strong need for efficient and flexible assays to study properties
and interactions of those transporters.

Assay: Activation by application of
Glycyl-glycine as substrate. Gly-Gly
gradient is sufficient to generate a driving
force. Electric current is induced byproton cotransport.

We utilized solid supported membrane based electrophysiology to study
real-time SLC-transporter activity in a 96-well based format, introducing amethodalso suitable for drug screenings and safety tests. Hereapplications to study activation and inhibition properties of the SLC
transporters PepT1, SGLT1, OCT2 and ANT are presented.

Lipid coating

Immobilisation ofmembrane

SSM based biosensors
Membrane fragments or liposomes containing the target are immobilized
on a functionalized gold electrode of 3 mm diameter. One sensor
contains the protein of about 10000 cells.

Accumulated transport current
The protein on the sensor issynchronously activated by a fast
perfusion system. The transport ofcharged substrates over the membrane generates an electrical current.
It’s measured as a sum signal of all theprotein on one sensor, thereby
enabling a high amplification.

Robot arm withpipetting head
Wash
stationRecordingchamber

Buffers/
compounds

Platform
The experiments are performed by a96-well based robotic platform,integrating liquid handling, recording
and a matching software forexperiment setup and data analysis.

Function: Renal secretion of organiccations
Mechanism: Polyspecific cation transport
Clinical significance: Renal clearance,
drug interactions
Assay: Activation by fast application of acationic substrate, e.g. choline. The
substrate gradient also generates thedriving force.

Analysis

Experiment

MethodGold substrate

OCT2/ SLC22A1

Function: Absorption of dietaryoligopeptides in the small intestines
Mechanism: Proton/Peptide cotransport
Clinical significance: Bioavailability ofdrugs

Application: Inhibitor characterization
Glibenclamide was used as an inhibitor of PepT1 activity. In each well anindividual IC50 was determined by cumulative application of increasing
inhibitor concentrations.

Application: Substrate characterization
TEA, choline and amiloride were compared as cationic substrates.
Induced current amplitude and shape differ. The evoked amplitude at1mM was compared, and EC50 of TEA and Choline were determined.

SGLT1/ SLC5A1

ANT/ SLC25A4

Function: Sugar uptake in intestines andkidney
Mechanism: Sodium coupled cotransportClinical significance: Glucose-galactose
malabsorption, Oral rehydration therapy
Assay: Activation by fast application of αMDG. Optional a sodium gradient can begenerated in advance. Sodium and/or
sugar gradient generate a driving force.

Function: Mitochondrial ATP export/ADP
import, part of mPTP
Mechanism: Several modes of action
Clinical significance: Associated with several hereditary and autoimmune relateddiseases, e.g. dilated cardiomyopathy
Assay: ATP and/or ADP supply. Native
mitochondrial membranes as source
materialFirst it was examined
whether
Signal stability

Application: Inhibitor characterization
Phlorizidine is used as a competitive inhibitor of SGLT1 activity. After signal
stability is ensured, different compound concentrations are applied
sequentially.

Application: Transport modes
Different modes of action were compared. ANT is able to transport only
ATP or ADP in an bidirectional way over the membrane, but also toexchange ATP against ADP. The current characteristic and amplitudechange depending on the mode of action.