Application Note

Channel:Cells:
Tools:

hClC-1
CHO
SyncroPatch 384

Characterization of hClC-1 on the SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by Charles River.

SummaryThe

Results

voltage-gatedchloridechannelgene(ClC)

We first studied the properties of ClC-1 mediated currents

family is highly conserved and their members are present

in CHO cells. The current was isolated from KV by using

in both pro- and eukaryotes. In mammals, nine different

Cs+ in the internal solution. Currents were obtained by

ClC subtypes have been identified, which differ accord-

application of a voltage step protocol (Fig. 1A)

ing to tissue distribution and subcellular location. ClC-1 isandexclusively expressed in skeletal muscles1.

deactivation at hyperpolarization and saturation at

ClCs function as homodimers2-4, allowing chloride andpositive potentials (Fig. 1B). To study the gatingother anions to be conducted through each single proto-bahaviour of ClC-1 deactivation curves were further ana-

pore. Gating of the single monomer is fast while the com-lyzed. Half maximal activation (Vhalf) of the tail current (Itail)cellular5 and extracellular chloride6,7, as well as pH8.
ClC proteins mediate chloride flux across cellular
membranes in most cell types and participate in maintenanceofrestingmembranepotential.

Plasmamembrane chloride channels play an important rolecharacteristic

B

-50
-100

0100

200 300 400 500 600
Time (ms)Cmembrane repolarization and stabilizes the membrane-100

voltage in skeletal muscle. Experimental block of themuscle hyperexcitability, manifested as myotonia .
9properties

andpharmacology

biophysicalof

hClC-1expressed in CHO cells. We applied voltage protocols
includingvarioustest

potentials

tostudy

thevoltage dependence of compounds. In order toinvestigate compound binding properties we used
the internal perfusion system of the SyncroPatch 384
for direct application of anthracene-9-carboxylic acid
(9-AC) to the intracellular environment. Moreover, we
also investigated the effect of 9-AC and niflumic acid(NFA) when they are applied from the extracellular side.
Download more Application Notes from www.nanion.de5
V (mV)
50-50100-10

Here we present data conducted on the Syncrocharacteristic

IsstItail-5

chloride conductance mediated by ClC-1 facilitatedshowingand500in reducing muscle excitability . ClC-1 contributes to384

activation

was estimated to be -2.6 ± 1.6 mV (n = 384).
A1Patchfast

5 nA
0.1 s

-15
-20

I (nA)

is slow. The channel gating can be modulated by intra-

Amplitude (mV)

mon gate to open and close the pores simultaneously

showedFigure 1: Voltage-dependent activation of hClC-1. Currents were elicitedby 300 ms voltage steps from 80 mV decreasing in 20 mV increments
to -120 mV following a pre-pulse from a holding potential of -100 mV to
60 mV for 100 s, as illustrated in A. B. Example traces of currents obtained.
The inward current typically deactivates with persisting hyperpolarization.
Mean currents obtained at the end of the various voltage step (Isst) andminimum current 1 ms after stepping the voltage back to -100 mV (Itail)were further analyzed. C. Average current plotted against voltage of384 cells. Blue indicates the average steady-state current of various test
potential, black the average tail current. Data were fitted by a Boltzmann
equation. Errors represent SD in all graphs.

Application Note
We used a low intracelular chloride concentration whichAAmplitude (mV)explains the discrepancy of Vhalf compared to literature
values as ClC gating has been shown to be strongly de10

pendent on ionic strength.
Niflumic acid (NFA) and anthracene-9-carboxylic acid(9-AC) are common known pharmacological tools thatare used to gain detailed insights into ClC-1 channel
models11,12andtoinduce

myotonia. Both compounds havein500-50-100
0100200

B

300400Time (ms)500600100 µM 9-ACex

9-ACinanimalbeen shown to2 nA

biophysics

100affect ClC-1 mediated currents by acting from either-2

the intracellular or extracellular side. For 9-AC, a hydrophobic binding pocket inside the ClC-1 channel has
inflammatory drug, the binding site within the channel isstill unknown.

-4I-125mV (nA)been proposed13, whereas for NFA, a non-steroidal anti-50 mscontrol
10 µM 9-ACin
30 µM 9-ACin
60 µM 9-ACin100 µM 9-ACex-6-8

In the experiment in Figure 2, different concentrations(10 µM, 30 µM, 60 µM) of 9-AC were perfused on theintracellular side of the cells, while currents were-10

continuously recorded over time. The voltage protocol toactivate ClC-1 mediated currents is given in Figure 2A.
9-ACin blocks ClC-1 mediated currents at -125 mV in a0C
1.0

concentration-dependent manner yielding an IC50 ofIn comparison, Figure 3 shows concentration response
curves for 9-AC and NFA after extracellular application.
The current at -125 mV was blocked by 75 % with 300 µM
NFA and completely abolished after 60 µM 9-AC
confirming the hypothesis that both compounds caneasily permeate the plasma membrane to enter thenormalized current

10.2 ± 2.7 µM (n = 320).

200

400time (s)

600

9-ACi
IC50 = 10.2 ± 2.7 µM (n = 320)0.5

inner side of the cell and reach their binding pockets15.
NFA yielded an IC50 of 99.6 ± 6.7 µM (n = 184), 9-AC an IC50of 2.4 ± 0.4 µM (n = 174). These values are consistent withthose found in literature1,14.

010-7

10-6

9-ACi (M)

10-5

10-4

Figure 2. Intracellular perfusion of 9-AC blocks ClC-1 in a concentrationdependent manner. A. A triple-voltage step protocol was used to activate
hClC-1. From a holding potential of -100 mV, the first step was made to+60 mV for 10 ms, followed by a step to +100 mV for 300 ms and a third
potential to -125 mV for 100 ms. B. Average traces from 335 cells expressing
hClC-1 activated by the voltage protocol shown in A. Time plot for an
average of 82 (black), 82 (dark blue), 77 (blue) and 66 (light blue) cells
receiving the indicated compound concentration from the intracellularside. Grey indicates full block by 100 µM extracellular 9-AC. Shown aremean of peak current recorded at -125 mV ± S.D. C. Concentration
response curve was calculated across the plate. Intracellular applicationof 9-AC yielded an IC50 of 10.2 ± 2.7 µM (n = 320).

Download more Application Notes from www.nanion.deApplication NoteASteady-state current recorded at -125 mV was normal1.0

ized to the maximum current amplitude of the same po-9-AC
IC50 = 2.39 ± 0.42 µM (n = 174)

tential and plotted against the voltage step of the prepulse. Increasing concentrations of 9-AC shifted the Vhalf

been made for NFA. Vhalf values for respective 9-AC andNFA concentrations are summarized in Table 1.
1.0

0.5

I/Imax

normalized currentof deactivation to the right. Similar observations have

010-8

B

1.0

10-7

10-6
9-AC (M)

10-5

9-AC

0.5

10-40 µM
1 µM

NFA
IC50 = 99.6 ± 6.74 µM (n = 184)

3 µM
10 µM
30 µMnormalized current

60 µM

0.5-100-50V (mV)50100

Figure 4: 9-AC shifts the Vhalf to depolarized potentials in a concentration
dependent manner. Steady-state current at -125 mV normalized to themaximum current amplitude recorded at -125 mV was plotted against thevarious pre-pulses. For concentrations indicated, Vhalf was calculated byfitting the data points with a Boltzmann equation and summarized in Table
1.
010-8

10-7

10-6

10-5

10-4

NFA (M)
Figure 3: Extracellular application of NFA and 9-AC block ClC-1 mediatedcurrents. The concentration response curves were calculated across theplate using a full block concentration (100 µM 9-AC) at the end of theexperiment for normalization. A. The concentration response curve for 9-AC
yielded an IC50 of 2.39 ± 0.42 µM (n=174). B. The concentration response
curve for NFA revealed an IC50 of 99.6 ± 6.74 µM (n=184). Each compound
was applied on one half of a single-hole NPC-384.

On the other hand, currents at +100 mV were lessefficiently blocked by NFA: IC50 = 389 µM and9-AC: IC50 = 7.1 µM, confirming the voltage-dependent
nature of the current block by these compounds . Figure
164 shows the effect of 9-AC on the ClC-1 gating.

Download more Application Notes from www.nanion.deCompoundConcentration(µM)

Vhalf ± SD (mV)n9-AC0-7.7 ± 1.5

311-0.3 ± 1.732NFA311.8 ± 1.9

3210

17.6 ± 2.43230

70.6 ± 6.93260

174.3 ± 124

320-6.5 ± 1.5323-4.8 ± 1.43210-1.5 ± 1.73130

13.5 ± 2.93210020.7 ± 3.232300

32.6 ± 2.731

Table 1: Summary of Vhalf of deactivation for different concentrations of9-AC and niflumic acid (NFA) recorded in one experiment. Vhalf values arepresented as mean ± SD, n is the number of cells included for analysis. Theexperiment was done on one single-hole NPC384 chip.

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of online analysis dataof hClC-1 expressing CHO cells as recorded on one NPC-384 (single-hole) patch clamp chip. Three hundred and eighty-four small color-coded pictures
as seen in the upper left part display 384 recordings. One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on
the right. Graphs show current amplitudes after vehicle addition (white region), followed by block by different concentrations of NFA or 9-AC (blue region;concentrations indicated) and finally full block with 100 µM 9-AC (grey region).

Exp end and chipunload
5 min

15 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash, one compound
addition followed by fullblock addition.

Analysis

Figure 6: The completion of one experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a single point concentration response curve including
full block at the end of the experiment took approximately 23 minutes.

Download more Application Notes from www.nanion.deApplication Note
Figure 5 shows a screenshot of the SyncroPatch 384

effect from increase in leak or loss of seal andsoftware during an experiment. The user can choose

canwhether to visualize raw traces or online analysis. In

Voltage step protocols before and after intracellular

this case,bea

benefit

for

assay

development.

online analysis is chosen and the graphs

application of 9-AC were applied to investigate therepresent current amplitude at hyperpolarization plottedvoltage dependence of the compound. The timeline

against time. An individual well can be highlighted toof each experiment was about 23 minutes (start – end,

monitor the progression of the experiment. In the Online

Fig. 6) and included wash, block with internal perfusion

Analysis view, the time points at which solution additionsof 9-AC, single compound concentration application

have been made are indicated by vertical lines, as well asand full block, all performed on each individual well,different background colors. In this case, white shows

drastically reducing the consumable cost per data point

activated hClC-1 recorded in vehicle solution, blue shows

to < $0.60.

inhibition by different concentrations of NFA or 9-AC andgrey shows full block with 100 µM 9-AC.

The SyncroPatch 384 is a high throughput and highly
reliable automated patch clamp device for recording

In conclusion, hClC-1 expressed in CHO cells can berecorded on the SyncroPatch 384 . The internal
perfusionfeatureofusedblock

hClC-1totheSyncroPatchusing

different

384

was

concen-trations of 9-AC and the typical slow decrease in thecurrent could be stably monitored online for more

hClC-1 mediated chloride currents. User-friendly software,
excellent success rates, single additions or multiple
additions of compound to each cell, perfusion of theinternal solution and easy analysis result in reliable highquality data at an increased throughput with an
economical cost per data point.

than 20 minutes. This continuous monitoring of current
amplitude is important for distinguishing compound

Cell culture

References

Cells were cultured according to the manufacturer’s

1. Steinmeyer, K., et al. 1991a. Nature 354:301-304.
2. Lorenz, C., et al. 1996. Proc Natl Acad Sci USA 93:13362–13366.
3. Weinreich and Jentsch. 2001. J Biol Chem 276:2347-2353.
4. Scholl, U., et al. 2006. Proc Natl Acad Sci U S A.;103(30):11411-11416.
5. Lobet, S. & Dutzler, R. 2006. EMBO J. 25(1):24-33.

recommendations and harvested according to Nanion’sstandard protocol using TrypLE.
Electrophysiology
Whole cell and perforated patch clamp recordings wereperformed according to Nanion’s standard procedure

6. Pusch, M., et al. 1999. J Physiol. 515 (Pt 2):341-353.

for the SyncroPatch 384 using single hole chips. Currents

7. Niemeyer, MI., et al. 2003. J Physiol. 553:873-879.

were elicited by voltage steps of 300 ms from 60 mV to8. Yusef, YR., et al. 2006. J Physiol. 572:173-181

values between 80 mV and -120 mV in steps of -20 mV

9. Steinmeyer, K., et al. 1991b. Nature 354:304-308.
10. Accardi, A. & Pusch, M. 2000. J Gen Physiol.;116(3):433-444.
11. Bryant, SH. & Morales-Aguilera, A. 1971. J Physiol 219: 367–383.
12. Desaphy, JF, et al. 2014. Exp Neurol 255: 96–102.

following a prepulse from a holding potential of -100 mV
to 60 mV for 100 s. The intersweep interval was 20 s.
Pharmacological experiments were performed every
20 s by continuous recording of the current elicited by a

13. Estévez, R., et al. 2003. Neuron 38, 47–59.
14. Astill, DS., et al. 1996. Biochim Biophys Acta. 1280(2):178-86.
15. Altamura, C., et al. 2018. Br J Pharmacol. 175(10):1770-1780.
16. Pusch, M., et al. 2002. Mol Membr Biol, 19:4, 285-292.

voltage step protocol starting from a Vhold of -100 mV,
followed by a pre-pulse to +60 for 10 ms and a step to 120
mV for 300 ms, followed by a tail potential of -125 mV for
100 ms before going back to Vhold.

Methods
Cells

hClC-1 expressing CHO were provided by Charles River.

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