Application Note

Channel:CaV1.2Cells:
HEK293Tools:
SyncroPatch® 384PE

High Throughput Pharmacology of CaV1.2
Channels on Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by SB Drug Discovery.

SummaryThe

CaV1.2

Results
channelisa

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

For the evaluation of the performance of HEK CaV1.2 cells,

Seal Resistance values were determined from one experi-tissues and is essential for multiple processes including CNS

ment (Fig. 1) at the start, after compound addition and atfunction, cardiac and smooth muscle contraction andthe end of the experiment. Experiments were performed

neuroendocrine regulation . VGCC are composed of 4in perforated patch mode using multi-hole (4 holes persubunits, which include an alpha 1 subunit which forms thewell) chips and seal resistance values are calculated andCa -selective pore, contains the voltage sensor and many

displayed per hole.

12+of the binding sites for regulatory modulators and drugs2and accessory subunits α2δ, β and γ which are involved in100

anchorage, trafficking and regulatory functions . The80

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

ed by the CACNA1C gene on the human chromosome
12p13 . Mutations in the L-type Ca channels have been
32+

associated with inherited arrhythmic disorders such asTimothy, Brugada and early repolarization syndromes4.
In addition, in the light of the CiPA initiative, the L-type

60
40
200

nifedipine

and verapamil are shown. The IC50 values for thesecompounds are within the expected range7-11 andsuccess rates of >75% for completed experiments wererecorded. Importantly, CaV1.2 recorded on the SyncroPatch 384PE exhibited stable peak amplitude during thecourse of the experiment and displayed little or no rundown.

Download more Application Notes from www.nanion.dehm

compounds

theOforhm

response

curvesconcentration

OandM

plots00

Current-voltage

M-5high throughput collected on the SyncroPatch 384PE.
®

000

pharmacology on CaV1.2 expressing HEK cells at ahmOM

Here we present high quality data with reliable>5100

cardiac safety testing5,6.

start of experimentafter compoundend of experiment

0<1

channel is likely to become an important target for

Cells (%)

1

Seal Resistance

Figure 1: Statistics of CaV1.2 expressing HEK293 cells recorded on one
NPC-384 chip on the SyncroPatch® 384PE. Success rate (seal resistance) ofindividual HEK cells on the SyncroPatch® 384. Shown is a bar graph of sealresistances at the start (light blue), after compound addition (mid-blue)and end of the experiment (dark blue). Multi-hole (4X) chips were used
and the seal resistance values per hole are displayed.

Application NoteCurrents

mediatedbyCaV1.2

could

bereliably

recorded on the SyncroPatch 384PE with a high success®

rate (>75% for completed experiments). Figure 2 shows ascreenshot of the data acquisition and analysis softwareof the SyncroPatch® 384PE during an experiment recording the current-voltage relationship of CaV1.2 expressed

in HEK cells. The current-voltage plot for an averageof 279 cells is shown in Panel C. A Boltzmann equation
reveals a V0.5 of activation of -12 mV with a reversal

potential of 53 mV in good agreement with the literaturefor L-type Ca2+ channels recorded from human atrium
cells12, rat neonatal ventricular cardiomyocytes7 andalpha-1C expressed in HEK cells13.

Figure 3: Recordings of CaV1.2 channels expressed in HEK cells on theSyncroPatch® 384PE are stable over time. Shown are online analysis values
peak amplitude versus time for 16 example cells. A small amount of rundownis seen at the beginning of the recording but this stabilizes and recordings
are stable for >15 mins. The inset shows an enlarged graph. At the end of theexperiment, CdCl2 was used to fully block the current.

Rundown of current is a phenomenon common to VGCC
channels14 and can be a real problem for electrophysiological measurements. Using the perforated patch modeand multi-hole chips (4 holes per well) we could reliably
record CaV1.2-mediated currents for >20 mins (Figure 3).
CaV1.2 currents were blocked by nifedipine and verapamil in a concentration-dependent manner. The average concentration response curves are shown in Figure 4
and the IC50 values summarized in Table 1. The estimatedFigure 2: Typical recording of CaV1.2 expressed in HEK cells on theSyncroPatch® 384PE. A The screenshot shows the data acquisition andanalysis software used on the SyncroPatch® 384PE. Three hundred andeighty-four small color-coded pictures shown in the upper left part from
A1 to P24 display 384 recordings. Wells are color-coded based on sealresistance. 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 anexample cell elicited by depolarizing steps from -60 mV to 40 mV in 10
mV increments from a holding potential of -90 mV are shown. The black
trace highlights the maximum current response elicited by a step to +10
mV. C The normalized current-voltage plot for an average of 279 cells. ABoltzmann equation fit revealed a V0.5 of activation of -12 mV.

IC50 values agree well with those found in the literature7-11.
AB

Figure 4: Average concentration response curves for 2 different calcium
channel blockers on the SyncroPatch® 384PE. The concentration responsecurves were constructed across the whole plate as shown in Figure 5.
The SyncroPatch® 384PE analysis software (DataControl® 384) was used
to calculate the average concentration response curves, normalized tomaximum block and fitted with a standard Hill-equation. A summary of theIC50 values and success rates is shown in Table 1.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch® 384PE. Screenshot of depiction of online analysis
data of CaV1.2 expressing HEK cells as recorded on one NPC-384 patch clamp chip (multi-hole, 4X). A single compound (in this case nifedipine) wasapplied to individual wells at varying concentrations (including control wells, 0.3% DMSO) across the plate. Three hundred and eighty-four small colorcoded pictures as seen in the upper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 30 MΩ), blue (Rmemb= 1 – 30 MΩ), light blue or grey (Rmemb < 1 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 (whiteregion), inhibition by different concentrations of nifedipine (blue region; concentrations indicated) and full block by 200 µM CdCl2.

Exp end and chip
unload10 - 15 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1-2 control washes, 1-3 compound
additions, full block addition

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch® 384PE. The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) fora single point concentration response curve on CaV1.2-mediated currents took approximately 17 - 22 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax

80636 Munich, Germany

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

Application Note
CompoundIC50

Successrate (%)

Literature range

Nifedipine

17 nM (310)819 - 220 nM7-10

Verapamil

8.6 µM (287) 75

In conclusion, CaV1.2 expressed in HEK cells canbe recorded on the SyncroPatch® 384PE with highsuccess rates for completed experiments (typically0.17 - 110 µM9-11>75%). The timeline of each experiment was about 17 - 22
minutes (start – end) and included wash with vehicle

Table 1: IC50 values for nifedipine and verapamil on CaV1.2-mediated currents recorded on the SyncroPatch® 384PE. Shown are IC50values (number of cells shown in brackets), success rate for completed experiments and the expected literature IC50 values. IC50
values recorded on the SyncroPatch® 384PE agree well with the literature
values7-11.

followed by incubation in 1 - 3 concentrations of blocker

Figure 5 shows a screenshot of the SyncroPatch® 384

literature7,12,13. The IC50’s calculated using the Syncro-

software during an experiment. A color-coded over-(or control solution) and full-block at the end of theexperiment. Importantly, CaV1.2 displayed little or no rundown and current-voltage relationship recorded on theSyncroPatch® 384PE is in good agreement with thePatch® 384PE’s analysis software, DataControl® 384, of twoview (based on seal resistance in this case) of all 384

calcium channel blockers were in good agreement withwells gives the user a good impression of the successthe literature7-11.

rate of the experiment. The user can choose whether tovisualize raw traces or online analysis. Here, online analysis

The SyncroPatch® 384PE is a high throughput and highlyis chosen and the graphs represent current amplitude

reliable automated patch clamp device for recording

versus

CaV1.2 currents. User-friendly software, excellent successtime.

Anindividual

wellcanbehighlighted to monitor progression of the experiment.

rates, single additions or multiple additions of compoundIn the Online Analysis view, the time points at which

to each cell and easy analysis result in reliable highsolution additions have been made are indicated byquality data at an increased throughput with anverticaleconomical cost per data point.

lines,

as

well

as

different

backgroundcolors. In this example, white shows incubation in control(vehicle; 0.3% DMSO) solution and blue is nifedipine.

References

Cell culture

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

Cells were cultured and harvested according to

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

Nanion’s standard cell culture protocol.

3. Schultz, D., et al., 1993. PNAS. 90: 6228-6232
4. Napolitano, C. & Antzelevitch, C. 2011. Circ. Res. 108: 607-618Electrophysiology

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

Perforated patch clamp recordings were conducted

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

according to Nanion’s standard procedure for the

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

SyncroPatch® 384PE and multi-hole chips (4X). The

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

current-voltage relationship was elicited by depolarizing

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

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

10. Kuryshev, YA., et al., 2014 Assay Drug Dev. Tech. 12(2): 110-119

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

11. Cai, D., et al. 1997. Mol. Pharm. 51: 872–881

protocol from -90 mV (holding potential) to 0 mV for 200

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

ms was applied to the cells every 20 s for pharmacology

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

experiments.

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

MethodsCells

HEK293 cells expressing CaV1.2 provided by SB DrugDiscovery, Catalog Ref: SB-HEK-Cav1.2 .

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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