Application Note

Channel:Cells:
Tools:PepT1
CHO
2
SURFE R N1& N96

Electrophysiological recordings of PepT1(SLC15A1) activity on Nanion’s SURFE2R
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

The human peptide transporter PepT1 is anGlycine, which does not act as a substrate for PepT1,

uptake transporter responsible for initial absorption

was first used as a control in order to separate possible

and renal reabsorption of dietary oligopeptides1. It ismechanical disturbances (perfusion) from the signal. The

primarily located in the plasma membranes of enterocytes

substrate, glycyl-glycine, was then added causing protonof the small intestine as well as the renal proximal tubular

movement across the membrane which was observed

cells1. PepT1 functions as a co-transporter, coupling the up-until electrochemical equilibrium was reached (Fig. 1A).

hill peptide transport into the cells to the electrochemical
proton gradient. Due to the movement of protons, PepT1 isan electrogenic transporter. PepT1 shows a very high capacity but a low affinity and substrate specificity1. Its ability to transport a large range of compounds has enabled
the rational design of drugs and pro-drugs (e.g. penicillins,ACE inhibitors) which have good oral bio-availability using
delivery via PepT11. Designing pro-drugs with higher affinity
for PepT1 is a successful strategy to increase the bio-availability of poorly absorbed drugs.
Here we present electric real-time PepT1 activity measurements on the SURFE2R instruments using purified plasma
membranes of CHO cells overexpressing PepT1. Peptide
transport was activated on the SURFE2R N1 using a sensorwith attached PepT1-containing membrane fragments
which was inserted into the device. This was perfused witha buffer containing the dipeptide glycyl-glycine as the substrate. The data presented here show activation of PepT1
by glyclyglycine and inhibition by Lys[Z(No2)]-Val on theSURFE2R N1 and scale-up of the assay on the SURFE2R N96.

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A PepT1 current responses on the SURFE2R N1 were activated by glycylglycine
but not glycine. B Inhibition of PepT1 proton current by Lys[Z(No2)]-Val.
Increasing concentrations of the inhibitor were tested cumulatively on thesame population of membrane fragments C Concentration response
curve for Lys[Z(No2)]-Val giving an IC50 of 580 ± 34 µM (n = 5).

Application Note
The rationally designed oligopeptide Lys[Z(No2)]-Val
was tested as an inhibitor of PepT1. By adding different
concentrations of the peptide via the buffer solution anIC50 of 580 ± 34 µM (n = 5) was determined (Fig. 1B & C).
To increase throughput, PepT1 measurements werealso performed on the SURFE2R N96 in 96-well-plate format (Fig. 2).

Figure 3: Screenshot of the analysis software, DataControl 96
showing inhibition of PepT1 by Lys[Z(No2)]-Val. 96 individual
concentration-response curves were generated.

PepT1 activity was inhibited by Lys[Z(No2)]-Valon the SURFE2R N96. An individual IC50 determination was performed in each well. The data
were automatically analyzed with Nanion’s
DataControl 96 (Fig. 3).
In conclusion, the SURFE2R N1 and N96 platforms
can be used to measure activation and inhibition of transporters such as PepT1. The ability toreliably record peptide transporters is critical forthe rational design of compounds to improve

Figure 2: Screenshot of the data acquisition software of the SURFE2R N96
showing PepT1 activation by glycyl-glycine.

SURFE2R sensor preparation

References
1. Daniel, H & Kottra, G. 2004. Pflugers Arch. 447(5):610-8

Methods

According to the Nanion standard procedure “SURFE2R
Sensor Preparation”. Sensors are prepared in resting
buffer, membrane is diluted 1:50 with control buffer.

Plasma membrane preparation
Accordingoral bio-availability.

totheNanion’s

SURFE2R N1 measurement workflow
standardprocedure(“Quickguide Membrane Preparation from CHO cells”).
Total protein concentration was between 5 - 10 µg/µl.

PepT1 can be activated by providing glycyl-glycine as asubstrate, no further driving force is necessary. Therefore
any standard two step protocol is suitable

Buffers
PepT1 experiments were performed by the exchange ofa substrate-free (“control”) buffer for a substratecontaining(“activating”) buffer. Control buffer contained: 140 mM
KCl, 25 mM HEPES, 25 mM MES, 4 mM MgCl2, 20mM
glycine, pH 6.7 with KOH. Activating buffer contained:
140 mM KCl, 25 mM HEPES, 25 mM MES, 4 mM MgCl2,20mM Glycyl-glycine, pH 6.7 with KOH. Both buffers wereprepared from a 2 x stock solution.
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