High throughput automated patch clamp of ionchannels important in cardiac safety and drug
discovery
Rodolfo Haedo1, Andrea Brüggemann2, Tom A. Goetze2, Markus Rapedius2, Michael George2, Niels Fertig2
1Nanion Technologies Inc., Livingston, New Jersey 07039, USA, info@naniontech.com; 2Nanion Technologies GmbH, Munich, Germany

Simultaneous recording of 6 cardiac ion channels[hERG, NaV1.5, CaV1.2, KV-LQT1 (KV7.1), KV4.3 and Kir2.1]

Abstract
One important part of the drug discovery process is the early assessment of a drug’s safety profile. The new paradigm in cardiac drug
safety screening, the Comprehensive In-vitro Proarrhythmia Assay (CiPA) initiative, is being introduced to provide a more complete
assessment of proarrythmic risk by evaluating and implementing currently available high throughput methods. An important part of thisis an extension of the electrophysiological evaluation of ion channels beyond hERG to include other cardiac channels such as NaV1.5,
CaV1.2, KV-LQT1 (KV7.1) and Kir2.1.
The ion channel CaV1.2, however, is not only found in cardiac tissue but also in the CNS and smooth muscle cells, amongst others. It isthought to play a role in CNS function, cardiac and smooth muscle contraction, neuroendocrine regulation and a multitude of otherprocesses (Hofmann, Flockerzi, Kahl, & Wegener, 2014). Blockers of CaV1.2 are used for the treatment of hypertension and angina andpharmacology of two such compounds (verapamil and nifedipine) are shown here.
In this study, HEK or CHO cells expressing the ion channels hERG, NaV1.5, CaV1.2, KV-LQT1 (KV7.1), KV4.3 and Kir2.1 were recorded
simultaneously on an automated patch clamp instrument recording from 384 cells in parallel. Additionally, CHO cells expressing CaV1.2
were recorded using multi-hole chips and pharmacology of verapamil and nifedipine will be shown.

hCav1.2 – cell line

hCav1.2 – Pharmacology

Raw traces

The following data were recorded froma stably transfected CHO cell line
(ChanTestCT6004),
expressing
hCav1.2/β2 /α2δ1 after induction for 2448h (1µg/ml tetracycline)
Figure adapted from [2]Cardiacaction
potentialandunderlying currents, figure modifiedfrom [5]Voltage protocol applied
during compound application

Voltage protocol used to activate 6 distinct
cardiac ion channels. The top panel shows thevoltage protocol used on all wells tosimultaneously activate the 6 distinct cardiac ionchannel currents shown in the bottom panels.

Vhold: -80mV
Chip: NPC-384 (4 holes/well)hCav1.2 – IV curve

Six different cardiac channels recorded simultaneously on the SyncroPatch
384PE. Shown is a screenshot of the data acquisition and analysis software
showing an experiment recording six different cardiac channels at once. A
representative image of the current recording for each ion channel is shown at
the top.

PatchControl software
overview of 384 wells
color coded on the basis of statistics

close-up
selected traces

Temperature control

Online analysis
On the basis ofconfigurable quality
criteria(Rseal,capacity,Rseries,protocol results (e.g.
peak current)) during
the course of theexperiment, wells areautomaticallyselected for analysis.

control
0.3% DMSO

nifedipine

verapamil

Current clamp
V/I Curve

For patch clamp experiments at physiological temperatures the SyncroPatch 384PE and thecomplete robotic liquid handler is sitting in a housing to keep temperature constant +/-0.5°C.

vehicle only
0.3% DMSO

peak voltage
vs.
current injection

Control

Stable peak currents over time,
no run-down within >25minafter cell catch

1st, 2nd & 3rd addition(arbitrary units)
1st

1stexperimental
single well highlightedstatistics
toggle - raw trace/online analysis

IV curve (averaged across the chip)
Results of fitting parameter
V0.5: -5.1mV
Reversal potential: +59mV
n: 300error bars: SEM

2ndNifedipine

2nd 3rd addition

Stability over time

3rd addition

Online Analysis[representative well]

Stable action potentials
overlay of 30 sweeps

Dose Response Curve[averaged]

success rate: 74% (94/128)
IC50: 12 nM(literature: 10-100 nM[3])Verapamil
success rate: 72% (92/128)
IC50: 7.6 µM(literature: 0.3-5 µM [4])References:

Erythromycin

Stable membrane potential

success rate: 84% (214/256)
IC50: 43 μM(literature: 39-59 μM [6-7], 257 μM [8]), at physiologicaltemperature

Cor4U cardiomyocytes
kindly provided by Axiogenesis[1] Hofmann F., Flockerzi V., Kahl S., & Wegener J.W. (2014). Physiological Reviews, 94(1), 303–326.
[2] Catterall W. A. (2011). Cold Spring Harb Perspect Biol; 3:a003947[3] Liao P., Yu D., Li G., Yong T.F., Soon J.L., Chua Y.L., Soong T.W. (2007). J. Biol. Chem., 282 (48): 35133-42[4] Johnson B.D., Hockerman G.H., Scheuer T., Catterall W. A. (1996). Mol. Pharmacol., 50 (5): 1388-400[5] Sager, P.T., et al., (2014). Am. Heart J. 167(3): 292-300[6] Stanat S.J.C., Carlton C.G., CrumbJr W.J., Agrawal K.C., Clarkson C.W. (2003), Mol. a. Cel. Biochemistry, 254 (1),1-7[8] Duncan et al. (2006). Biochem. and Biophys. Res. Comm. 341(2), 500–506[8] Polonuck (2012). Frontiers in pharmacology, vol. 3 (3), 54-60

Unless stated otherwise
all cell lines kindly provided by