Application Note

Channel:
CFTR
Cells:
CHO®
Tools:
SyncroPatch 384PE

Different modes of activation of CFTR
recorded on Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by Charles River.

Summary

Results

Cystic Fibrosis (CF) is an autosomal recessive genetic

CFTR is activated by internal F-, in the presence of ATP6,

disorder which affects a number of organs, in particularand by external application of forskolin7,8. Figure 1 shows

the lungs, pancreas and sweat glands. It is caused bycurrent amplitude at -100 mV or +100 mV versus time

mutations in the gene encoding the cystic fibrosis trans-for F--free (columns 1 - 12) or F--containing (columns 13

1,2

membrane conductance regulator protein (CFTR) .

- 24) internal solution on a single plate. In F--free solution,

CFTR is a regulated epithelial chloride channel

andexternal application of forskolin increased current

mutations cause a reduction in activity of the channel

amplitude. In the presence of internal F-, forskolin did not

via a variety of mechanisms . This results in defective

further activate the current.

2,3

2electrolyte transport in airway epithelia and thereby,
chronic lung infection and premature mortality4. Therefore, compounds which increase activity of CFTR havetherapeutic potential for treating CF.
The CFTR protein is composed of 5 domains: there
are 2 transmembrane (TM) domains, 2 nucleotidebinding domains (NBDs) and 1 regulatory domain (R)5.
The TM domains form the pore of the channel, channel
activity is determined by phosphorylation of the R domain
and gating is controlled by hydrolysis of ATP at the NBD5.
CFTR is activated via a number of reagents includinginternal fluoride6, cAMP7,8 and external forskolin7,8.
Here we show activation of CFTR expressed in CHO
cells on the SyncroPatch 384PE by internal perfusion ofF- or external application of forskolin. The current wasblocked with the specific blocker, inh-172 with an IC50 ingood agreement with the literature9. In addition, CFTR
activatedbyinternalcAMPwaspotentiatedby

VX-770. Using F--free internal solution and activation bysubmaximal cAMP or forskolin, potentiators of CFTR canbe investigated as potential therapeutics to treat CF.

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Figure 1: Activation of CFTR using internal F- or external applicationof Forskolin. Screenshot of the data acquisition software during anexperiment. Half the plate (columns 1 - 12) received F--free internal solution
and half the plate (columns 13 - 24) had an internal solution containing
120 mM F-. The online analysis values of peak current at -100 mV (light bluepoints) and +100 mV (dark blue points) are shown. Forskolin was applied
via the external solution (blue region) which activated CFTR in cells which
had F--free internal solution but did not further activate CFTR in cells which
had F--containing internal solution. The current was blocked by external
application of 150 µM glibenclamide (grey region) regardless of activation
method.

Application Note
Currents

mediatedby

CFTR

wereactivatedbyapplication of 60 mM F in the internal solution. The-timecourse of activation of the current followinginternal perfusion of F- could be monitored online and isshown in Figure 2. Raw current traces to a voltage ramp
are shown in Panel A. The timecourse of the experimentand the increase in current amplitude at -100 mV and+100 mV upon perfusion of internal F- can be seen in
Panel B. The current was blocked by glibenclamide (greyregion).

Figure 3: Block of CFTR by inh-172. Shown are online analysis values at +80
mV (light blue) and -80 mV (dark blue) for 8 exemplar cells (top) and one
example cell (middle). In this experiment, cAMP perfused internally was used
to activate CFTR. The blue region indicates external application of inh-172
and grey is full block with 30 µM inh-172. A concentration response curve wasconstructed across the plate (half the plate was used for the experiment)
and is shown in the bottom panel. The IC50 for inh-172 at +80 mV was 1.6 µM
and at -80 mV was 1.9 µM. This experiment was performed in the whole cellconfiguration using multi-hole (8X) chips.

One potential therapeutic strategy for treating CF is toincrease CFTR function in patients with defective CFTR
channels due to mutations. VX-770, also known as
Ivacaftor, is a CFTR potentiator which increases the activityof wild type and mutant CFTR channels such as ∆F508 andG551D by increasing open probability11. Figure 4 shows activation of CFTR by internal perfusion of cAMP andFigure 2: Activation of CFTR by internal perfusion of F-. A Raw current traces
in response to a voltage ramp protocol of 16 example cells are shown. At
the beginning of the experiment the internal solution contains 0 mM F- (redtrace). CFTR is activated as the internal solution is perfused with F--containing
solution (blue and black traces). B Online analysis showing current amplitudeat +100 mV (dark blue) and -100 mV (light blue) versus time during perfusionof F- in the internal solution. Perfusion of F- is indicated by the red bar. Thecurrent was blocked by external application of 200 µM glibenclamide (greyregion). This experiment was performed in the whole cell configuration using
multi-hole (8X) chips.

subsequent potentiation of the response by VX-770. VX-770
potentiated the CFTR response by approximately 25 - 40%
at + 80 mV and - 80 mV, respectively.

CFTR was also activated using internal perfusion of cAMP(Figure 3). 100 µM cAMP was perfused via the internalsolution (F -free) and the CFTR-mediated currents were-

blocked by the specific inhibitor, inh-172. A single-point
concentration curve was performed and the average
concentration plot was constructed across the plate(Figure 3; half the plate was used for the experiment). TheIC50 calculated using a Hill equation was estimated to be

Figure 4: Potentiation of CFTR by VX-770. A CFTR was activated using internal
perfusion of cAMP. Online analysis values showing current amplitude at +80
mV (light blue) and -80 mV (dark blue) are shown versus time for an example
cell. Red bar indicates perfusion of cAMP in the internal solution. Blue region
indicates external application of VX-770 (in this example 200 nM) and thegrey region indicates external application of 40 µM inh-172 (full block). The
concentration response curve was constructed across the plate (half theplate was used for the experiment) and is shown in B. VX-770 potentiates thecAMP-induced response by approximately 40% at -80 mV (dark blue) and
25% at +80 mV with an EC50 of 34 nM and 17 nM, respectively.

1.6 µM at +80 mV and 1.9 µM at -80 mV in good agreement with the literature using a transport assay9 and wasvoltage independent as expected10.
Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch® 384PE. Screenshot of depiction of online analysis
data of CFTR expressing CHO cells as recorded on one NPC-384 (multi-hole, 8 holes per well) 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 of CFTR activated by external forskolin (10 µM; white region), potentiation by increasing
concentrations of genistein (1, 10, 100 µM; blue regions) and full block by 300 µM glibenclamide (grey region).

Exp end and chip
unload10 - 20 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash, activation using
internal perfusion of F- or cAMP or external application offorskolin, 1 - 3 compound additions, full blockAnalysis

Figure 6: The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) for a single or multiple point concentration response curve
plus activation of CFTR with internal F- or cAMP or external application of forskolin and full block at the end of the experiment took approximately 17-27 min.

Nanion Technologies GmbHphone +
 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note
Figure 5 shows a screenshot of the SyncroPatch® 384

leak or loss of seal and can be a benefit for assay

software during an experiment. The user can choosedevelopment.

whether to visualize raw traces or online analysis. In

external application of forskolin when using a F--free

this case,

CFTR

wasalsoactivatedbyonline analysis is chosen and the graphsinternal solution. The timeline of each experiment wasrepresent current amplitude plotted against time. An

about 17-27 minutes (start – end) and included wash,

individual well can be highlighted to monitor theactivation

progression of the experiment. In the Online Analysis

externalview, the time points at which solution additions havecurrent,

been made are indicated by vertical lines, as well as

concentration(s)applicationdifferent background colors. In this case, white shows

performed

each

activation of CFTR by external forskolin and thereducing the consumable cost per data point todifferent

increasing< $0.60. Importantly we could show that CFTR isconcentrations of the potentiator, genistein. Grey shows

activated by internal F-, internal cAMP or external

full block by 300 µM glibenclamide.

forskolin as expected6,7,8, inhibited by inh-172 in a voltage-

shadesofblue

representwithinternalF -,forskolin,

timetoreachsingleormultiple

on

cAMP
aor
stablecompoundandfullblock,individual

well,drasticallyall

independent manner10 and potentiated by VX-77011.
In conclusion, CFTR expressed in CHO cells can berecorded on the SyncroPatch® 384PE with goodsuccess rates (typically >80% for RSeal > 500 MOhmat the start of the experiment). The internal perfusionfeatureoftheSyncroPatch

384PE

wasusedtoactivate CFTR using internal F or cAMP and increase in-the current could be monitored online. This continuous
monitoringof

distinguishingcurrent

amplitude

compoundeffectisfromimportantfor

increase

inThe SyncroPatch® 384PE is a high throughput and highly
reliable

automatedrecording

CFTRpatchcurrents.

clamp

deviceUser-friendly

forsoftware,

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.
Cell culture

References

Cells were cultured according to the manufacturer’s

1. Riordan, J.R., et al. 1989. Science. 245: 1066-1073

recommendations and harvested according to Nanion’s

2. Welsh, M.J. & Smith, A.E. 1993. Cell. 73: 1251-1254
3. Gadsby, D.C., et al., 1995. Ann. Rev. Physiol. 57: 387-416
4. Al-Nakkash, L., et al. 2001. JPET. 296(2): 464-472
5. Sheppard, D.N. & Welsh, M.J. 1999. Physiol. Rev. 79, Suppl.: S23-S45
6. Berger, H.A., et al., 1998. Lung Cell. Mol. Physiol. 18: L305-L312
7. Haws, C.M., et al. 1996. Am. J. Physiol. 270: C1544-C1555
8. Al-Nakkash, L. & Hwang, T.C. 1999. Pflügers Arch. 437: 553-561
9. Caci, E., et al. 2008. Biochem. J. 413: 135-142

standard protocol using trypsin. Cells were allowed togrow to confluency and then induced for 24 hours prior
to harvesting for experiments.
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch® 384PE using multi-hole (8 holes per well)

10. Ma, T., et al. 2002. J. Clin. Invest. 110: 1651-1658
11. Van Goor, F., et al. 2009. PNAS. 106(44): 33487-334969

chips. A voltage ramp protocol from -100 mV to +100 mVover 100 ms was applied to the cells every 10 s. Intersweep

Methods

holding potential was -20 mV. Current amplitude atCells

were applied via the internal and external solutions.

+100 mV and -100 mV was used for analysis. Compounds

CFTR expressing CHO cells, catalog #: CT6113, weresupplied by Charles River.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax

80636 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960