Application Note

Channel:CaV1.2Cells:
CHO®
Tools:
SyncroPatch 384PE

Stability and Pharmacology of CaV1.2
Channels on Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Charles River.

SummaryThe

CaV1.2

Results
channelisa

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

For the evaluation of the performance of CHO-CaV1.2
cells, seal resistance values were determined fromtissues and is essential for multiple processes including CNS

one experiment (Fig. 1) at the start and end of thefunction, cardiac and smooth muscle contraction andexperiment. Experiments were performed in perforated

neuroendocrine regulation . VGCCs are composed of 4patch mode using multi-hole (4 holes per well) chips andsubunits, which include an alpha 1 subunit which forms theseal resistance values were calculated and displayed

Ca -selective pore, contains the voltage sensor and many

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 . The
1

by the CACNA1C gene on the human chromosome

12p133. Mutations in the L-type Ca2+ channels have been
associated with inherited arrhythmic disorders such as80Cells (%)

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

60
4020In addition, in the light of the CiPA initiative, the L-type0

compounds

thenifedipineand verapamil are shown. The IC50 values for thesecompounds are within the expected range7-11 andsuccess rates of >70% for completed experiments wererecorded. Importantly, CaV1.2 recorded on the Syncro-Patch 384PE exhibited stable peak amplitudes during thecourse of the experiment and displayed little or no rundown.

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responsecurves

concentration

hmandOplots

M

Current-voltage

00high throughput collected on the SyncroPatch 384PE.
®

M-5

pharmacology on CaV1.2 expressing CHO cells at a000hm

O

M

Here we present high quality data with reliable

>5

100

cardiac safety testing5,6.

0<1Timothy, Brugada and early repolarization syndromes4.
channel is likely to become an important target forstart of experimentend of experiment

Seal Resistance

Figure 1: Statistics of CaV1.2 expressing CHO cells recorded on one NPC384 chip on the SyncroPatch® 384PE. Success rate (seal resistance) ofindividual CHO cells on the SyncroPatch® 384. Shown is a bar graph ofseal resistances at the start (light blue) and end of the experiment (darkblue). Multi-hole (4X) chips were used and the seal resistance values perhole are displayed.

Application Note
Currents

mediatedbyCaV1.2

could

bereliably

recorded on the SyncroPatch 384PE with a high success®

rate (>70% for completed experiments). Figure 2 showsa screenshot of the data acquisition and analysis software of the SyncroPatch® 384PE during an experimentrecording the current-voltage relationship of CaV1.2
expressed in CHO cells. The current-voltage plot for an

average of 272 cells is shown in Panel C. A Boltzmann
equation reveals a V0.5 of activation of -4.8 mV with areversal potential of 57 mV in good agreement with theliterature for L-type Ca2+ channels recorded from humanventricular myocytes12-14.

Figure 3: Recordings of CaV1.2 channels expressed in CHO cells on theSyncroPatch® 384PE are stable over time. Shown are online analysis values
peak amplitude versus time for 16 example cells. The recordings are stablefor >25 min. The inset shows an enlarged graph.

Rundown of current is a phenomenon common to VGCC
channels15 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 >25 min (Figure 3).
CaV1.2 currents were

blocked by nifedipine andverapamil in a concentration-dependent manner. The
average concentration response curves are shown inFigure 4 and the IC50 values summarized in Table 1. The

estimated IC50 values agree well with those found in theliterature7-11.
AFigure 2: Typical recording of CaV1.2 expressed in CHO 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 fromA1 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 an
example cell elicited by depolarizing steps from -60 mV to 40 mV in 10 mV
increments from a holding potential of -80 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 272 cells. ABoltzmann equation fit revealed a V0.5 of activation of -4.8 mV.

BFigure 4: Average concentration response curves for 2 different calcium
channel blockers on the SyncroPatch® 384PE. Cumulative concentration
response curves were performed and the concentration response curves
were constructed and averaged for 8 columns of the NPC-384 plate as
shown in Figure 5. The SyncroPatch® 384PE analysis software (DataControl®
384) was used to calculate the average concentration response curves. The
points were normalized to control and fitted with a standard Hill-equation
with minimum and maximum fixed to 0 and 1, respectively. A summary of theIC50 values and success rates is shown in Table 1.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 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 CHO cells as recorded on one NPC-384 patch clamp chip (multi-hole, 4X). Control (0.3% DMSO), nifedipine or verapamil inincreasing concentrations was applied to eight columns for each compound. Three hundred and eighty-four small color-coded pictures as seen in theupper left part display 384 recordings. Depending on the seal resistance, pictures are green (Rmemb > 50 MΩ), blue (Rmemb = 10 – 50 MΩ), light blueor grey (Rmemb < 10 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 at the test pulse to 10 mV during application of reference solution (white region), and 3 applicationsof control (0.3% DMSO) or 3 different concentrations of nifedipine or verapamil. CaV1.2-mediated currents were stable over the entire experiment whichlasted over 20 min.

Exp end and chip
unload20 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 2 control washes followed by 3compound additions

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 cumulative (3 concentrations per well) concentration response curve on CaV1.2-mediated currents took approximately 27 min. Each concentration ofcompound was incubated for approximately 4 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application Note
CompoundIC50

Success
rate (%)

Literature range

solution and blue is vehicle (0.3% DMSO) applied 3 times.

Nifedipine

12 nM (94)74

9 - 220 nM7-10In conclusion, CaV1.2 expressed in CHO cells canVerapamil

7.6 µM (92)72

0.17 - 110 µM9-11

Table 1: IC50 values for nifedipine and verapamil on CaV1.2mediated currents recorded on the SyncroPatch® 384PE. Shown
are IC50 values (number of cells shown in brackets), success rate forcompleted experiments and the expected literature IC50 values. IC50values recorded on the SyncroPatch® 384PE agree well with the literature
values7-11. Note that 1/3 of the plate received each compound, i.e. 128
wells.

Figure 5 shows a screenshot of the SyncroPatch® 384
software during an experiment. A color-coded overview (based on seal resistance in this case) of all 384
wells gives the user a good impression of the successrate of the experiment. The user can choose whether tovisualize raw traces or online analysis. Here, online analysis
is chosen and the graphs represent current amplitude
versustime.

An

individual

wellcanbehighlighted to monitor progression of the experiment.
In the Online Analysis view, the time points at which
solution additions have been made are indicated byverticallines,

aswellasdifferent

backgroundcolors. In this example, white shows incubation in control

References

be recorded on the SyncroPatch® 384PE with highsuccess rates for completed experiments (typically>70%). The timeline of each experiment was about 27
minutes (start – end) and included wash with vehicle
followed by incubation in 3 concentrations of blocker (orcontrol solution). Importantly, CaV1.2 displayed little or no

rundown and current-voltage relationship recorded on
the SyncroPatch® 384PE is in good agreement with theliterature12-14. The IC50’s calculated using the Syncro-Patch® 384PE’s analysis software, DataControl® 384, of two
calcium channel blockers were in good agreement withthe literature7-11.
The SyncroPatch® 384PE is a high throughput and highly
reliable automated patch clamp device for recording
CaV1.2 currents. User-friendly software, excellent successrates, single additions or multiple additions of compoundto each cell and easy analysis result in reliable highquality data at an increased throughput with an
economical cost per data point.

Methods

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

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

CHO cells expressing CaV1.2/β2/α2δ1 provided by CharlesRiver, Catalog #CT6004 .

3. Schultz, D., et al., 1993. PNAS. 90: 6228-6232
4. Napolitano, C. & Antzelevitch, C. 2011. Circ. Res. 108: 607-618
5. Sager, P., et al., 2014. Am. Heart J. 167: 292-300

Cell culture

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

Cells were cultured and harvested according to

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

Nanion’s standard cell culture protocol.

8. Liao, P,. et al., 2007. JBC. 282: 35133-35142
9. Kang, J., et al., 2012. JPET. 341(2): 510-517

Electrophysiology

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

Perforated patch clamp recordings were conducted

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

according to Nanion’s standard procedure for the

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

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

13. Bénitah, J.P. et al, 1992. Pflügers Arch. 421: 176-187

current-voltage relationship was elicited by depolarizing

14. Belzmann, B. et al, 1998. Cardiovasc. Res. 38: 424-432

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

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

from a holding potential of -80 mV. A voltage stepprotocol from -80 mV (holding potential) to 10 mV for
200 ms was applied to the cells every 20 s forpharmacology experiments.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9

fax

80636 Munich, Germany

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