Application Note

Channel:CaV3.2Cells:
HEK293Tools:
SyncroPatch® 384PE

High Throughput Pharmacology of CaV3.2
Channels on Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Millipore.

SummaryResults

The CaV3.2 channel is one of the three low voltage

For the evaluation of the performance of HEK cells

currents differ from the high voltage activated (HVA)

determined from one experiment and are shown in Fig. 1calcium currents in their activation and inactivation

at the start and end of the experiment.

activated (LVA) T-type calcium channels. The LVA

expressing CaV3.2 cells, Seal Resistance values werekinetics. LVA currents are activated at lower voltages
1006040

muscle contraction and hormone secretion. Morerecently, CaV3.2 has been shown to play a role innociception and pain3,4,5.

Here we present high quality data with reliable
pharmacology on CaV3.2 expressing HEK cells at ahigh throughput collected on the SyncroPatch® 384PE.

Current-voltage

plotsandconcentration

response

curves for the compounds nitrendipine, nifedipine,
mibefradil and amiloride are shown. The IC50 values forthese compounds are within the expected range2,6,7 andsuccess rates of up to 79% for completed experiments
were recorded.

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Ω

ΩM
00
>50
10

physiological processes including neuronal firing, smooth

diorgans. They have been implicated in a variety ofM

0tissue, heart, kidney, smooth muscle, and many endocrine

00organs throughout the human body, including nervous-520sablT-type channels are expressed in a wide variety ofΩCACNA1H gene on the human chromosome 16p13.3 .
2Mchannel contains the α1H subunit, encoded by theCells (%)

ions as compared with the HVA currents1. The CaV3.2

start of experimentend of experiment80

0slower deactivation and a smaller conductance of Ba2+

<10at around -20 mV), they display faster inactivation,ed(typically activating above -50 mV and peaking

Seal Resistance
Figure 1: Statistics of CaV3.2 expressing HEK293cells recorded on one
NPC-384 chip on the SyncroPatch® 384PE. Success rate (seal resistance)of individual HEK cells on the SyncroPatch® 384. Shown is a bar graph ofseal resistances at the start (light blue) and end of the experiment (darkblue).

Application NoteCurrents

mediatedbyCaV3.2

could

bereliably

recorded on the SyncroPatch 384PE with a high success®

rate. Figure 2 shows a screenshot of the data acquisition
and analysis software of the SyncroPatch® 384PE during
an experiment recording the current-voltage relationshipof CaV3.2 expressed in HEK cells. In this experiment, 83%of cells had a seal resistance > 500 MΩ with a further 11%
with a seal resistance > 100 MΩ.

Figure 3: IV and inhibition of CaV3.2 channels expressed in HEK cells on theSyncroPatch® 384PE. A CaV3.2-mediated current responses to a voltage step
protocol (see Fig. 2). B The corresponding current voltage relationship from(A) shows the typical IV curve of CaV3.2 channels1,2. C Current response of an
exemplar cell elicited using a voltage step to -10 mV for 75 ms from a holding
potential of -120 mV. The red trace shows inhibition by 50 µM nitrendipine,
the cursors indicate the maximum peak amplitude used for plotting theonline analysis timeplot. D Corresponding online analysis showing peak
amplitude plotted against time. The online analysis is color-coded, the grey
region indicates the application of control solution including vehicle (1%DMSO, 0.01% Pluronic) and the blue region indicates application of 50 µM
nitrendipine.

Figure 2: Typical recording from CaV3.2 expressed in HEK cells on theSyncroPatch® 384PE. The screenshot shows the data acquisition and analysis
software used on the SyncroPatch® 384PE. 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(green: Rmemb > 500 MΩ, blue: Rmemb = 100 - 500 MΩ, light blue or grey:Rmemb < 100 MΩ or disabled). One highlighted experiment is displayed
at the bottom, 16 selected experiments are displayed on the right. Theexperiment shows the current voltage relationship elicited by depolarizing
steps from -60 mV to 40 mV in 10 mV increments from a holding potential of-120 mV. The black trace highlights the maximum current response followed
by a step to -10 mV.

A summary of IC50 values and success rates for the 4compounds is shown in Table 1. The estimated IC50 values
agree well with those found in the literature2,6,7. The successrates for completed experiments were between 69 and79%.

Figure 3 shows the current-voltage relationship for an
exemplar CaV3.2-expressing HEK cell and block ofthe current by nitrendipine (50 µM). Figure 3B and3D show the online analysis for the current-voltage
relationshipand

thepharmacology

experiment,respectively. In Panel D, the vertical lines and colorcoded regions show addition and incubation ofcompound.

The

greyregionshows

incubationin vehicle (1% DMSO, 0.01% Pluronic) and the blue region
incubation in 50 µM nitrendipine. Each cell was exposed
to a single concentration of nitrendipine and theconcentration
thewhole

response
plate.

curve

The

calculated

averageacrossconcentration

response curves for four calcium channel blockers areshown in Figure 4. Only cells which

Figure 4: Average concentration response curves for 4 different calcium
channel blockers on the SyncroPatch® 384PE. The concentration response
curves 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.

satisfied certain

quality control criteria (Rmemb, Rseries, Cm and peakcurrent amplitude) were included in the analysis.

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 CaV3.2 expressing HEK cells as recorded on one NPC-384 patch clamp chip. A single compound (in this case nitrendipine) was applied to individual
wells at varying concentrations (including control wells) across the plate. 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 (Rmemb > 500 MΩ), blue (Rmemb = 100 – 500 MΩ), light blue or grey(Rmemb < 100 MΩ or cells disabled). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs
show current amplitudes of CaV3.2 channels from the test pulse to -10 mV during application of control solution including vehicle (1% DMSO, 0.01% Pluronic;
grey region) and inhibition by nitrendipine (blue region).

Exp end and chipunload
6 - 8 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 control wash + 1 vehicle wash +1 compound 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 CaV3.2-mediated currents took approximately 13-15 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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

Application Note
CompoundIC50 (µM)

Successrate (%)

Literaturerange (µM)

Nitrendipine 6.5 (304)79

96

Nifedipine

71.9 (306)79216

Mibefradil

0.85 (298)77

1.2 - 1.42,7Amiloride

72.5 (265)691677colors. In this example, grey shows incubation in control(vehicle; 1% DMSO, 0.01% Pluronic) solution and blue isnitrendipine.
In conclusion, CaV3.2 expressed in HEK cells canbe recorded on the SyncroPatch® 384PE with highsuccess rates for completed experiments (typicallyTable 1: IC50 values for nitrendipine, nifedipine, mibefradil and amiloride
on CaV3.2-mediated 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. All IC50
values recorded on the SyncroPatch® 384PE agree well with the literature
values2,6,7.

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.

Anindividual

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

backgroundminutes (start – end) and included wash with vehicle
followed by incubation in 1 concentration of blocker. The
current-voltage relationship of CaV3.2 recorded on theSyncroPatch® 384PE is in good agreement with theliterature1,2. The IC50’s calculated using the SyncroPatch®

384PE’s analysis software, DataControl® 384, of four

calcium channel blockers were in good agreement withthe literature2,6,7.
The SyncroPatch® 384PE is a high throughput and highly
reliable automated patch clamp device for recording
CaV3.2 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.
Cell culture

References
1. Perez-Reyes, E. 2003. Physiol. Rev. 83: 117-161
2. Cribbs, L.L., et al., 1998. Circ. Res. 83: 103-109
3. Bourinet, E., et al., 2005. EMBO. J. 24: 315-324
4. Choi, S., et al., 2007. Genes, Brain and Behavior. 6: 425431
5. Liao, Y-F., et al., 2011. Mol. Pain. 7: 43-52
6. Perez-Reyes, E., et al.,>70%). The timeline of each experiment was about 13-15

2009. JPET. 328: 621-627

7. Lacinova, L. 2005. Gen. Physiol. Biophys. 24 (Suppl. 1):
1-78

Cells were cultured and harvested according toNanion’s standard cell culture protocol.
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch® 384PE. A voltage step protocol from-120 mV (holding potential) to -10 mV for 75 ms wasapplied to the cells every 10 s for pharmacology
experiments. Peak amplitude at -10 mV was used for

Methods

analysis. The current-voltage relationship was elicited byCells

increments from a holding potential of -120 mV.

depolarizing steps from -60 mV to 40 mV in 10 mV

HEK293 cells expressing CaV3.2.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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