Application Note

Transporter:
Sample:Origin:Tools:

Ec-ClC
Proteoliposomes
E. coli
SURFE2R N1Electrophysiological recordings of the H+/
Cl- exchanger Ec-ClC on the SURFE2R N1The electrophysiology team at Nanion Technologies GmbH, Munich.
Samples kindly provided by Dr. Merritt Maduke, Stanford University, USA

Summary

Results

ClCs are a family of chloride ion channels and transport-

To activate Ec-ClC on the SURFE2R N1, a sensor withers with important physiological roles including regulation

attached Ec-ClC proteoliposomes pre-incubated inof the membrane potential, transepithelial salt transportchloride-free buffer solution was inserted into the de-and ion homeostasis1,2. To date, 9 members of the ClC

vice and perfused with a buffer containing chloride.

family have been identified in mammals , the first 4 (ClC-Application of chloride leads to transient currents with

1, ClC-2, ClC-Ka, and ClC-Kb) are located on the plas-

negative amplitude, reflecting the net transport of one

ma membrane where they act as chloride ion channels

negative charge into the proteoliposomes. The sensor

whereas the remaining 5 are located in intracellular or-was flushed with chloride-free buffer solution before andganelles (ClC-3-7) and are chloride-proton exchangers1-3.

after Ec-ClC activation (Figure 1A). The half saturation of

These transporters are important for endosome, lyso-

Ec-ClC mediated chloride translocation was investigatedsome and synaptic vesicle acidification1,2, and mutations

by applying different chloride concentrations sequential-in, e.g. ClC-5 underlie the rare chronic kidney disorder,

ly on the same sensor. An EC50 value of 12.3 ± 0.6 mM (n =Dent’s Disease, and mutations in ClC-7 underlie osteopetrosis2,4, a rare inherited bone hardening disorder. Given
their ubiquitous expression and importance in physiological processes, they are important potential drug targets.
ClC from Escherichia coli (Ec-ClC or ClC-ec1) is closely
related to its mammalian counterparts and is a Cl-/H+ exchanger3,5. It transports 2 Cl- into the cell, coupled to theefflux of 1 H+3,5. Therefore Ec-ClC is an electrogenic transporter, generating a net charge flow. In E. coli, the Ec-ClC
mediates acid resistance of enteric bacteria by promoting H+ extrusion1,5,6.
Here we present Ec-ClC activity measurements on theSURFE2R N1 instrument using proteoliposomes reconstituted with purified Ec-ClC at different lipid-to-protein ratios.

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4 sensors) was determined (Figure 1B).
AB
NaCl
300 mM
75 mM
19 mM
4 mM
5 nA

Current (nA)

1,250
4030
20
10025 ms

Imax = 40.9 ± 0.5 nA
EC50 = 12.3 ± 0.6 mM
0 50 100 150 200 250 300[Chloride (mM)]

Figure 1: Typical Ec-ClC current response on the SURFE2R N1. A When
applying chloride to the sensor a transient negative current occurs. B
Concentration response curves for chloride was tested cumulatively on
the same population of proteoliposomes. Data are average ± S.E.M of 4sensors.

Application Note
Since the direction of transport is dictated by theIn addition to inhibition of the current, we also investigatedelectrochemical gradients, we were able to detect

the impact of lipid-to-protein ratio on the Ec-ClC

the opposite exchange activity by pre-loading theresponse. The signal-to-noise was clearly dependent onproteoliposomes with chloride and activating Ec-ClC by

the Ec-ClC density where increasing the lipid-to-protein

removing chloride from the external buffer solution. In thisratio resulted in smaller current responses. Liposomes notcase we measured transient positive currents, reflecting

containing Ec-ClC on the other hand do not generate

the net charge translocation of one negative charge out

any current response in the SURFE2R assay (Figure 3B).

of the proteoliposomes (Figure 2).

BANaCl
19 mM
4 mM
1 mM

Control
100 µM OADS (5 min)Washout after OADS
1 nA

5 nA
25 ms

Figure 2: Typical Ec-ClC current response on the SURFE2R N1. When preloading proteoliposomes with chloride and removing chloride in the external buffer solution a positive current occurs.

To confirm that the currents were indeed mediated byEc-ClC, we applied the specific Ec-ClC inhibitor, OADS7,
at 100 µM in all buffer solutions. After just 1 s after adding
OADS, we observed 50% inhibition, and after 5 minutes incubation we obtained up to 80% inhibition. After rinsingwith OADS-free buffer solution we could partially recover
the signal (approx 50%; Figure 3A).

References

Lipid-to-protein ratio(w/w)
20:1
100:1
500:1
2000:1
Empty
5 nA

25 ms

25 ms

Figure 3: A Transient currents mediated by Ec-ClC were blocked by OADS.
Current traces are shown after activation by chloride before (dark blue),after 5 min incubation in 100 µM OADS (light blue), and after washout (red).
B Current response on the SURFE2R N1 for proteoliposomes with different
protein densities attached to different sensors. Lipid-to-protein ratio (w/w)
is shown and the lower the protein density (i.e. higher ratio) the smaller thecurrent response. Empty liposomes generate no current response (red).

In conclusion, the SURFE²R N1 can be used to reliably
measure Ec-ClC activity. This assay can easily be adopted
for other transporters of the ClC family and provides a useful tool in future drug discovery.

activating solution, followed by activation using the1. Zifarelli , G. & Pusch, M. A. 2007. In: Amara S. (eds) Rev. Physiol., Biochem. Pharmacol. Vol 158. Springer, Berlin, Heidelberg: 23-76
2. Jentsch, T.J., et al. 2002. Physiol. Rev. 82: 503–568
3. Garcia-Celma, J., et al. 2013. J. Gen. Physiol. 141 (4): 479–491
4. Schulz, P., et al. 2010. PLoS ONE 5(9): e12585
5. Accardi, A. & Miller, C. 2004. Nature. 427: 803- 807
6. Iyer, R., et al. 2002. Nature. 419: 715-718

control solution. Control buffer contained: 10 mM MES, 5mM MgSO4, (100 + x) mM K2SO4, pH 5.2. Activating buffer
contained: 10 mM MES, 5 mM MgSO4, 100 mM K2SO4, 2*x

mM KCl, pH 5.2.

SURFE2R N1 sensor preparation

7. Howery, A.E., et al. 2012. Chem. Biol. 19(11): 1460–1470

According to the Nanion standard procedure “SURFE2R

Methods

N1 Protocols: Sensor Preparation Guide”. Sensors areProtein purification and reconstitution

prepared in control buffer. For the reverse transport

Protein was purified according to ref. 3. For reconstitution,mode, sensors are prepared in activating buffer.

E.coli polar lipid extract (Avanti) was used. Samples wereProteoliposomes were diluted 1:10 before sonication in akindly provided by Dr. Merritt Maduke, Stanford University,water bath sonicator.

USA.
Buffers

SURFE2R N1 measurement workflow

Ec-ClC experiments were performed by the exchange ofClC can be activated by providing chloride. There is noa chloride free (“control”) buffer for a chloride containing

additional driving force required. Therefore, any 2-buffer(“activating”) buffer. When the reverse transport mode

Nanion standard protocol is suitable. ClC activity is higher

was investigated, the sensor was first flushed within the acidic pH range.

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