Activation of CFTR channels in absence of internal fluoride using a highly parallel automated patch clamp system
Andrea Brueggemanna, Søren Friisa, Tim Strassmaierb, Markus Rapediusa, Tom Goetzea, Claudia Haarmanna, Ilka Rinkea, Atsushi Ohtsukic, Takayuki Okac, Marius Vogela, Timo Stengela, Johannes
Stiehlera, Michael Georgea and Niels Fertigaa Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germanyb Nanion Technologies USA, 1 Naylon Ave # 1, Livingston, NJ 07039, New Jersey, USAc Nanion Technologies Japan, MIL5, TWIns 3F, 2-2, Shinjyuku-ku, Tokyo, Japan

Activation of CFTR with internal exchange
from 0 to 60 mM F-

Activation of CFTR with forskolin

Abstract

0100
30-5100

10End/max ratio

0,0100 mV

Voltage protocols
CFTR currents were monitored using a 2 second ramp protocol from -100 to 100
mV every 15 seconds. A second longer ramp protocol was also used later in theexperiment series (see below) where the voltage was ramped down again to theholding potential of -80 mV. This protocol allowed to distinguish the proportion ofunblocked current as opposed to leak in the full block period.

120 F-0 F-

120 F-0 F-

60 mM FCells were patched in F--freeconditions.
Internalsolution
exchange to 60 mM F- was started
after 300 seconds (red region in ITplot above).
CFTR was blocked with 200 uM
glibenclamide (grey region). It was
found that CFTR activated withkinetics that was similar to forskolin.
The plate view above illustates theuniformity of the activation and thesuccess rate of this experiment. In
this case it was 89%.
To the left are typical sweeps andIT-plots shown.

Blocker dose response of CFTR activated with
Internal exchange Application of cAMP

Activation of CFTR with forskolin vs. internal F0 F-

60 mM F-n = 356
IC50(-80 mV):
63 µM

Time (sec)Inthefollowing
experiments 10 or 20 µM
forskolin was used.

0,2-100 mV

Themaxcurrent
response measured at 100 and +100 mV to thetested concentrations isshown to the left with anapparent EC50 of app.
0.5
µM
in
good
agreementwithprevoius reports 1,2.
Below is the fraction ofrundown measured fromthe max peak responseto the end of the 10 minperiod.

11010,4

101000,61000,8100 µM

Current (A)10 1551000-100

1Current (nA)

Mean max current response to 4 different
concentrations (µM) of Forskolin

1010 µM

1,0Cells
The experiments presented here were done using CHO cells expressing wild type
CFTR kindly provided by Charles River. The cells were cultured according to themanufacturers recommendations and harvested according to Nanion’s standard
protocol using trypsin.

300

300

1 µM

Current stability

Methods

Glibenclamide conc. (µM)
30103100DC0,1Cystic fibrosis is caused by malfunction of the chloride channel
Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressedin the apical membrane of epithelial cells where it is involved in theregulation of fluid transport across the epithelium. A large numberof mutations of the protein are known to cause CFTR to become
dysfunctional and only very few pharmaceutical compounds havebeen developed to treat the disease by restoring the chloride
conductance of the channel. CFTR is activated by cAMPdependent phosphorylation and is gated by ATP. Activation istypically achieved using forskolin that activates adenylate cyclase
which leads to phosphorylation of the channel via protein kinase A(PKA). An alternative mode of activation is via application of
intracellular F-. However, intracellular F- has also been widely usedon most automated patch clamp (APC) platforms due to it’s
stabilizing effects during the giga-seal formation. On the otherhand, this also makes it difficult to control the degree of activationof the channel and represents a general limitation in the use of
APC platforms in drug discovery for CFTR.
We now here present data from Nanion’s SyncroPatch 384PE
where we activate the channel with forskolin under F--freeconditions at a high troughput. Further, CFTR could also beactivated by first patching the cells in F--free conditions and thenexposing the cell to intracellular F- using the internal solution
exchange function. Our results show that the activation of CFTR byeither forskolin or intracellular F- is sensitive to Glibenclamide in adose – and voltage dependent manner. Taken together, theseexperiments firstly show a fluoride-free approach to study thepharmacology of CFTR at high throughput that might empowers
new ways in the drug discovery on CFTR.

Cells were stimulated with 0.1, 1, 10, 100 µM
forskolin and the response was monitored for
10 minutes. The experiment was ended withan addition of 100 µM Glibenclamide.
A part of the plate view is shown to the left
and below are 16 individual IT-plots from 3
concentrations.

Inhibitor dose response experiments

0 mM Inh-172

120 F-50 mM Inh-172

IC50(+80 mV):
205 µMn = 135

IC50(-80mV):
2.7 µM

IC50(+80 mV):
4.1 µM

Glibenclamide and Inh-172 weretested using forskolin (10 or 20µM) to activate CFTR. The
compounds were tested with
one concentration pr. site acrosseither a full or a half plate as
shown above.
Atest
concentrationofglibenclamide or Inh-172 wasadded after 5 min followed by afull block concetration of either
compound(300µMglibenclamide or 10 µM Inh-172
respectively). The derived Hillplots are shown to the left.
Glibenclamide was found to bevoltage senstive and Inh-172 was
not with IC50’s that was in good
agreement with litterature3,4,5.

Potentiator dose response of CFTR activated withinternal addition of cAMP0 nM Ivocaftor 200 nM Ivocaftor
n = 122

Electrophysiology
Whole cell patch clamp recordings were conducted according to Nanion’s
standard procedure for the SyncroPatch® 384PE using multi-hole chips. All
experiments followed a typical sequence: filling and patching and whole cell
formation followed by a baseline recordings for 60-120 seconds, an activation
period with either internal exchange and/or external addition of forskolin, anaddition of test compound for 3-5 minutes which was followed by full block ofeither glibenclamide or Inh-172. Total experiment lenght including filling andpatching was app. 20 minutes.

EC50(-80):
41 nM

100 µM cAMPFB

EC50(+80):
26 nM

Inh-172 (mM)
00 F-20 µM
Forskolin

On the right are two examples of a leaky (left) and a non-leaky (right) well. Note the difference in tailcurrent revealing leak vs. residual current. CFTR was measured at ±100 or 80 mV respectively.

optimal access and seal resistances for different cell lines

 Upgradable Current Clamp Option
 Upgradable Temperature Control Option
 Internal Solution Exchange

SyncroPatch ® 384PE

150 µMGlib.

150 µMGlib.

900
800
700
600
5000F

400120 F

3002001000prepost
forskolin forskolin

165000.5

1.651650

The comercially available potentiatior VX-770
or Ivocaftor was tested using 100 µM cAMP toactivate CFTR (second white period). A clear
potentiation of the curent was seen but withmore variation compared to the blockers as
displayed on the representative IT-plots to theleft and the derived EC50 above6.

block

A fourth method of activating
CFTR was used in this set ofexperiments. 100 uM cAMP wasapplied using internal exchange
to active the PKA pathway. Thiswasfollowed
byatestconcentration of Inh-172 and afull block in the end – as shown
above left.

The effect of internal F- on CFTR
activation was tested in anexperiment where a plate wasfilled with either 0 (col. 1-12) or120 F- (col. 13-24) as shown onplate view above left. 20 µM
Forskolin were added after 250
seconds (blue region), and 150µM Glibenclamide was addedin the end of the experiment.
Typical sweeps and IT-plots ofcurrents picked up at -100 mV
showing the effect of forskolin 0(left) and 120 F- (right) are also
show in the plate view.

1000Avg. single cell eq. resistance(MΩ)

 384 Amplifiers, 384 Pipetting Head High Gigaseal rate on HEK, CHO, LTK, RBL, iPS and RBC
 Fast solution exchange for ligand gated channels
 Partial Usage of the Chips Possible (32 wells per run) Temperature controlled cell hotel (4 - 70 OC)
 Customized Chips for different hole sizes, allowing

20 µM
Forskolin

5150 µM
Glib.

20 µM
Forskolin

120 F-Used voltage prototocols and typical current responses. In red is preactivation, in dark activated current.

0.5 1.6

All cells responded to 150 µM
Glibenclamide. Activation of
CFTR with forskolin and Factivation was found not to beadditive.
Avg. Seal ± SEM resistance pr.
cell before forskolin, withforskolin and after full block isshown to the left.

The derived Hill-fits from two halfplate experiments are shown
below. The potency of thecompound were found to be
idential between the two runs
and also comparable to theresults
foundusingforskolin
activationandingood
agreement with previous reported
values3,4. The plate view to theright shows the success ratemeasured as cells > 500 Mohm
before stimulation was 91%.

IC50(-80):
1.6 µM

IC50(-80):
2.2 µM
IC50(+80):
2.6 µM

IC50(+80):
1.9 µM

SummaryIn conclusion, CFTR expressed in CHO cells can be recorded on theSyncroPatch® 384PE with good success rates, typically >80% for RSeal > 500
MOhm at the start of the experiment. The internal perfusion feature of theSyncroPatch® 384PE could be used to activate CFTR using internal F- or cAMPand the increase in the current could be monitored online. CFTR was also
activated by external application of forskolin when using a F--free internalsolution. We could show that CFTR is activated by internal F-7, internal cAMP1,2 orexternal forskolin as expected, inhibited by glibenclamide and inh-172 in a
voltage dependent and independent manner and potentiated by VX-7703,4,5,6.
This demonstrates that the SyncroPatch® 384PE is a high throughput and highly
reliable automated patch clamp device for recording CFTR currents.

References
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4: Ma, T., et al. 2002. J. Clin. Invest. 110: 1651-1658
5: Van Goor, F., et al. 2009. PNAS. 106(44): 33487-334969
6: Sheppard et al, 1997, Journal of Phys, 503, 2 333-346
7: Berger, H.A., et al., 1998. Lung Cell. Mol. Physiol. 18: L305-L312