Application Note

Channel:Cells:
Tools:CNT1
CHO
2
SURFE R N1

Electrophysiological recordings of CNT1(SLC28A1) activity on Nanion’s SURFE2R N1The electrophysiology team at Nanion Technologies GmbH, Munich.

Results

Summary
The concentrative nucleoside transporter 1 (CNT1) isTo activate CNT1 on the SURFE2R N1, a sensor with at-a sodium-dependent uptake transporter encoded bytached CNT1-containing membrane fragments was in-

the SLC28A1 gene1. CNT1 functions as a co-transporter,

serted into the device and perfused with a buffer con-coupling the uphill nucleoside transport into the cells

taining NaCl and uridine. When the nucleoside is present,

to the electrochemical gradient of sodium . The

sodium movement across the membrane can be ob-

stoichiometry of transport is proposed to be 1:13, but

served until an electrochemical equilibrium is reached. To

a stoichiometry of 2 Na : 1 nucleoside has also been

generate sodium gradients, necessary as a driving force,suggested4. CNT1 is an electrogenic transporter, generat-

the sensor was flushed with KCl before and after activa-

ing a net charge flow. It plays a major role in the uptake oftion of CNT1 (Figure 1). Uridine elicits a larger response ofpyrimidines, including uridine and cytidine, from the extra-

CNT1 compared with cytidine but CNT1 has a higher ap-

cellular milieu into the cytoplasm1 in nucleoside salvage

parent affinity for cytidine vs uridine (Figure 2).

2+pathways which is the first step of nucleoside biosynthesis .
2

The transporter is expressed in epithelial tissues including
liver, kidney and small intestine where it is localized to theapical membrane2. CNTs are important targets for many
antiviral and anticancer agents5,6, and CNT1 has been
proposed to play a role in tumor biology via a mechanism
beyond nucleoside transport7. In fact, tumors expressing
high levels of CNT1 can indicate a higher risk of relapse forbreast cancer patients8, suggesting that nucleoside salvage may interfere with chemosensitivity8. On the other
hand, high expression of the CNT1 protein could promotedruginduced cytotoxicity if patients were treated withsuitable hCNT substrates1. In any case, hCNT1 is an
important mediator in the transport of anticancer andantiviral nucleoside drugs

1,3,5,6

by mechanisms that require

further study.
Here we present CNT1 activity measurements on the SURFE2R N1 instrument using purified plasma membrane of CH
cells expressing CNT1.

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Figure 1: Typical CNT1 current response on the SURFE2R N1. Uridine wasused as a substrate for CNT1. After establishing a sodium gradient (firstpeak), uridine was applied to the sensor (second peak). Uridine or cytidine
can be used as the substrate, uridine eliciting a larger response thancytidine (inset).

Application Note

The effect of different ion gradients on CNT1 activity
was also investigated. Figure 3 shows charge values for
CNT1 under different ionic conditions: Na+, K+, Li+ andcholine were used as the co-transported ion in symmetrical conditions. Significant ionic transfer by CNT1 only oc-The apparent affinity of CNT1 to the substrates uridine

curs in the presence of Na+, demonstrating strict coupling

and cytidine was investigated. Apparent Km values ofof nucleotide and sodium transport and a high Na+ speci-78.67 ± 21.16 µM (n = 6) for uridine, and 8.24 ± 1.38 µM

ficity of the transporter. Application of a sodium gradient(n = 5) for cytidine were determined (Figure 2) in good

(NaClKCl) increases the current amplitude, illustrating theagreement with range found in the literature

resulting increase of driving force.

3,4,9,10.

Figure 2: Increasing concentrations of 2 different substrates were added
cumulatively on the same population of membrane fragments and theaverage concentration response curves for cytidine (n = 5) and uridine (n =6) were constructed. When fitted with a Hill equation, apparent Km valuesof 8.24 ± 1.38 µM (n = 5) and 78.67 ± 21.16 µM (n = 6) for cytidine and uridine,
respectively, were obtained.

Figure 3: CNT1 co-transports Na+, Li+ acts as a weak substrate, K+ andcholine cannot be co-transported by CNT1. A Na+ gradient (NaClKCl)
increases the signal amplitude.

In conclusion, the SURFE²R N1 can be used to reliably
measure CNT1 activity. This has important implications
for drug discovery targeting CNT1 because nucleosideanalogues are used as anticancer and antiviral therapies.

References

CNT1 experiments were performed by the exchange of a

1. Pastor-Anglada, M., et al., 2008. Xenobiotica. 38(78):972-994
2. Gray, J.H., et al., 2004. Pflugers Arch. 447:728-734
3. Smith, K.M., et al., 2004. J. Physiol. 558.3:807–823
4. Larráyoz, I.M., et al., 2004. JBC. 279(10): 8999–9007
5. Pastor-Anglada, M., et al., 1998. TiPS. 19:424-430
6. Pastor-Anglada, M., et al., 2005. Vir. Res. 107:151-164
7. Pérez-Torras, S., et al., 2013. Cell Death and Disease 4,e648; doi:10.1038/cddis.2013.173
8. Gloeckner-Hofmann, K., 2006. Oncology. 70:238-244
9. Graham, K.A., et al., 2000. Nucleosides Nucleotides
Nucleic Acids. 19(1-2):415-34
10. Griffith, D.A., & Jarvis, S.M. 1996. Biochimica et Biophysica Acta 1286:153-181

sodium containing (“control”) buffer and afterwards a
nucleoside and sodium containing (“activating”) buffer.
Resting buffer contained: 140 mM KCl, 5 mM MgCl2,30mM HEPES, pH 7.4 with NMG. Activating buffer contained: 140 mM NaCl, 5 mM MgCl2, 30 mM HEPES, pH 7.4
with NMG, x mM uridine/cytidine.
SURFE2R sensor preparation
According to the Nanion standard procedure “SURFE2RSensor Preparation”. Sensors are prepared in resting
buffer, membrane is diluted 1:10 with resting buffer.
SURFE2R N1 measurement workflow
CNT1 can be activated by providing uridine and cytidine

Methods

as a nucleoside. A sodium gradient must be establishedin advance of nucleoside addition. Therefore, anyPlasma membrane preparation
Accordingsodium and nucleoside free (“resting”) buffer for atotheNanion’s

standard

procedure3-buffer Nanion standard protocol is suitable.

(“Quickguide Membrane Preparation from CHO cells”).
Total protein concentration was between 5 - 10 µg/µl.
Buffers
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