Application Note

Channel:Cells:
Tools:

CaV1.2
CHO
SyncroPatch 384

Stable Recordings and High Throughput
Pharmacology of Assay Ready CaV1.2 Cells
on the SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by NMI TT Pharmaservices, Steinbeis-Innovationszentrum
Zellkulturtechnik and acCELLerate.
SummaryThe

CaV1.2

Results
channelisa

voltage-gatedcalciumchannel (VGCC) expressed in a variety of mammalian

CHO cells stably expressing CaV1.2 were provided asfrozen stocks (Assay Ready Cells) that were thawed outtissues and is essential for multiple processes including CNS

and used without prior cultivation. Multi-hole chips werefunction, cardiac and smooth muscle contraction andused on the SyncroPatch 384 (4 holes per well) and cells

neuroendocrine regulation . VGCC are composed of 4

were captured with good success rates (94% of wells

subunits, which include an alpha 1 subunit which forms the

had RSeal >100MΩ). Rundown of current is a phenomenon

1

Ca -selective pore, contains the voltage sensor and many
2+common to VGCC channels12 and can be a problem for2of the binding sites for regulatory modulators and drugs

electrophysiological measurements. Using the perforated

and accessory subunits α2δ, β and γ which are involved inpatch mode and multi-hole chips we could reliably re-anchorage, trafficking and regulatory functions . The

cord CaV1.2-mediated currents for >10 mins with <10%1

CaV1.2 channel contains the alpha-1C subunit, encod-

ed by the CACNA1C gene on the human chromosome

rundown (Figure 1).
A

12p133. Mutations in the L-type Ca2+ channels have been
associated with inherited arrhythmic disorders such as
Timothy, Brugada and early repolarization syndromes4. In

0.2 nA
Control at 120 s
50 ms
Control at 200 s
Control at 500 s
Full block (200 µM verapamil)

addition, the L-type Ca2+ channel is an important target
for cardiac safety testing, especially in the light of theCiPA initiative5,6.
Here we present high quality data with reliable pharma-Band concentration response curves for the compounds
nifedipine and verapamil are shown. The IC50 values forthese compounds are within the expected range7-11 andsuccess rates of >75% for completed experiments wererecorded. Importantly, CaV1.2 recorded on the SyncroPatch 384 exhibited stable peak amplitude during thecourse of the experiment and displayed low rundown.

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Normalized Ipeak

cology on Assay Ready CaV1.2 Cells at a high throughput
collected on the SyncroPatch 384. Current-voltage plots

Full block

Control

1.0
0.8
0.6
0.4
0.2
0.0
100

200

300
400
Time (s)500600700

Figure 1: CaV1.2 expressed in CHO cells were reliably recorded on theSyncroPatch 384. A Traces from an example cell at 120 s, 200 s and500 s overlay almost exactly. The current was blocked by 200 µM verapamiland the remaining current subtracted. B Timecourse of the experiment foran average of 26 wells. Rundown was low, <10% over 500 s.

Application Note
1.0Figure 2 shows a screenshot of the data acquisition andanalysis software of the SyncroPatch 384 during an experiexpressed in CHO cells. The current-voltage plot for anaverage of 327 wells is shown in Panel C. A Boltzmann
equation reveals a V0.5 of activation of -11.9 ± 0.4 mV witha reversal potential of 56.0 ± 0.5 mV in good agreementwith the literature for L-type Ca2+ channels recorded

0.8

Normalized Blockment recording the current-voltage relationship of CaV1.2

Nifedipine
IC50 = 6.6 nM (n = 162 wells)

0.6
Control
10 nM nifedipine

0.4

0.2 nA
100 ms0.2

from human atrium cells13, rat neonatal ventricular cardiomyocytes7, human ventricular cardiomyocytes14 and

0.0

alpha-1C expressed in HEK cells15.

10-11

10-10

10-9
10-8
[Nifedipine (M)]

A10-7

10-6

Figure 3: Average concentration response curve for nifedipine block of CaV1.2
recorded on the SyncroPatch 384. The cells received a single concentrationof nifedipine and the concentration response curve constructed acrossmultiple wells. Example traces from a single well are shown in the inset withblock by 10 nM nifedipine. The IC50 was calculated using a Hill Equation andfound to be 6.6 nM.

CaV1.2currents

wereblockedbynifedipine

andverapamil in a concentration-dependent manner. Eachwell received one concentration of either nifedipine (0300 nM) or verapamil (0-100 µM) and the concentration
response curves calculated across multiple wells. The
average concentration response curve for nifedipine isshown in Figure 3. The IC50 was 6.6 nM in good agreementwith the range found in the literature7-10. The average con-centration response curve for verapamil is shown in Figure-60

4. The IC50 was 1.61 µM in good agreement with the range

Voltage (mV)
-40-20-200-400

204060-600

0.5 nA
20 ms

found in the literature9-11. The IC50 values and success rates
are summarized in Table 1.

-800
Vhalf = -11.9 ± 0.4 mV (n = 327 wells)

Figure 2: Typical recording of CaV1.2 expressed in CHO cells on theSyncroPatch 384. A The screenshot shows the data acquisition and analysis
software used on the SyncroPatch 384i. Three hundred and eighty-four
small color-coded pictures shown in the upper left part from A1 to P24
display 384 recordings. Wells are color-coded based on seal resistance.
One highlighted experiment is displayed at the bottom, 16 selected
experiments are displayed on the right. The online analysis values are shown
for a current-voltage experiment. B The raw traces from an example cell
elicited by depolarizing steps from -60 mV to 40 mV in 10 mV incrementsfrom a holding potential of -90 mV are shown. C The normalized currentvoltage plot for an average of 327 wells. A Boltzmann equation fit revealed
a V0.5 of activation of -11.9 ± 0.4 mV.

Download more Application Notes from www.nanion.deNormalized Block

CIPeak (pA)B

1.0 Verapamil
IC50 = 1.61 µM (n = 175 wells)
0.80.60.40.20.0Control
1 µM verapamil
0.2 nA
100 ms10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
[Verapamil (M)]

Figure 4: Average concentration response curve for verapamil block of CaV1.2
recorded on the SyncroPatch 384. The cells received a single concentrationof verapamil and the concentration response curve constructed acrossmultiple wells. Example traces from a single well are shown in the inset withblock by 1 µM verapamil. The IC50 was calculated using a Hill Equation andfound to be 1.61 µM.

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of online analysis dataof CaV1.2 expressing CHO cells as recorded on one NPC-384 patch clamp chip (multi-hole, 4X). A single concentration of compound (in this case either
verapamil or nifedipine) was applied to individual wells. Three hundred and eighty-four small color-coded pictures as seen in the upper left part display
384 recordings. Depending on the seal resistance, pictures are green (RSeal > 100 MΩ), blue (RSeal = 25 – 100 MΩ), light blue or grey (RSeal < 25 MΩ or wells
disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show current amplitudes of
CaV1.2 channels from the test pulse to 0 mV during application of reference solution (white region), inhibition by different concentrations of nifedipine orverapamil (blue region; concentrations indicated) and full block by 200 µM verapamil (dark grey).

Exp end and chip
unload5 min~12 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash, 1 compoundaddition, full block addition

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch 384. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for asingle point concentration response curve on CaV1.2-mediated currents took approximately 19 min.

Download more Application Notes from www.nanion.deApplication Note
CompoundIC50

Successrate (%)

Literature range

In the Online Analysis view, the time points at which
solution additions have been made are indicated byNifedipine

6.6 nM (162) 84

9 - 220 nM7-10

Verapamil1.7 µM (175) 91

0.17 - 110 µM9-11

verticallines,

aswellasdifferent

backgroundcolors. In this example, white shows incubation in controlsolution, blue is verapamil or nifedipine and grey is full

Table 1: IC50 values for nifedipine and verapamil on CaV1.2mediated currents recorded on the SyncroPatch 384i. Shown are
IC50 values (number of wells shown in brackets), success rate forcompleted experiments and the expected literature IC50 values. IC50
values recorded on the SyncroPatch 384 agree well with the literature
values7-11.

block (200 µM verapamil).

Figure 5 shows a screenshot of the SyncroPatch 384

line of each experiment was about 19 minutes (start –software during the experiment where half of theend) and included wash with control solution followed

plate (192 wells) received nifedipine and the otherby incubation in a single concentration of blocker andhalf received verapamil. A color-coded overview

full-block at the end of the experiment. Importantly,(based on seal resistance in this case) of all 384 wells

CaV1.2 displayed little rundown (<10% rundown overgives the user a good impression of the success rate

In conclusion, CaV1.2 Assay Ready Cells can berecorded on the SyncroPatch 384 with high success rates

for completed experiments (typically >84%). The time-10 mins) and current-voltage relationship recordedof the experiment. The user can choose whether toon the SyncroPatch 384 is in good agreement with thevisualize raw traces or online analysis. Here, online analysis

literature7,14,15. The IC50s calculated using the Syncro-ischosenandthegraphs

represent

current

Patch 384’s analysis software, DataControl 384, of twoamplitude versus time. An individual well can becalcium channel blockers were in good agreement withhighlighted to monitor progression of the experiment.

the literature7-11.

References

which prevent gene silencing of neighboring transgenes

1. Hofmann, F., et al. 2014. Physiol. Rev. 94: 303–326

and

2. Welling, A., et al., 1993. J.Physiol. 471: 749-765

combination of the IGAMI technology with an inducible

3. Schultz, D., et al., 1993. PNAS. 90: 6228-6232

gene expression of CaV1.2 protein subunits led to the4. Napolitano, C. & Antzelevitch, C. 2011. Circ. Res. 108: 607-618

therefore

allow

astableco-expression.

Thegeneration of Assay Ready CaV1.2 Cells which can be

5. Sager, P., et al., 2014. Am. Heart J. 167: 292-300

used instantly after thawing without prior cultivation. The

6. Colatsky, T., et al., 2016. J. Pharmacol. Toxicol. Meth. 81: 15-20

standardized production and freezing process of Assay

7. Pignier, C. & Potreau, D. 2000. Am. J. Physiol. Heart Circ. Physiol. 279(5): H2259-2268

Ready Cells significantly increases the reproducibility of

8. Liao, P,. et al., 2007. JBC. 282: 35133-35142

experiments since all variations occurring from cell culture

9. Kang, J., et al., 2012. JPET. 341(2): 510-517

can be excluded.

10. Kuryshev, YA., et al., 2014 Assay Drug Dev. Tech. 12(2): 110-119
11. Cai, D., et al. 1997. Mol. Pharm. 51: 872–881

Electrophysiology

12. Belles, B., et al. 1988. Pflügers Arch. 1988. 411: 353 - 360

Cells were thawed out and used directly for experiments.

13. Mewes, T. & Ravens, U. 1994. J. Mol. Cell. Cardiol. 26 (10): 1307-1320

Perforated patch clamp recordings were conducted

14. Pelzmann, B., et al. 1998. Cardiovasc. Res. 38: 424–432

according to Nanion’s standard procedure for the

15. Hu, H. & Marban, E. 1998. Mol. Pharmacol. 53: 902–907

SyncroPatch 384 and multi-hole chips (4X). The currentvoltage relationship was elicited by depolarizing steps

Methods

from -60 mV to 40 mV for 200 ms in 10 mV increments fromCells

a holding potential of -90 mV. A voltage step protocol

This recombinant CHO-CaV1.2 cell line was developed

from -90 mV (holding potential) to 0 mV for 200 ms wasby Steinbeis-Innationszentrum Zellkultur and NMI TT using

applied to the cells every 20 s for pharmacology

their proprietary IGAMI® expression system for theexperiments. The current remaining after full-blockdevelopment of stable cell lines. This technology uses

application was subtracted from current in control andspecial stabilizing and expression enhancing sequences,drug conditions.

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