Application Note

Channel:Cells:
Tools:hERG
HEK293Port-a-Patch

Temperature controlled hERG recordings onNanion’s Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by EMD Millipore.

SummaryThehERG

Results

gene

encodeschannelCurrent responses of an individual cell expressing theresponsible for the repolarization of the IKr current in

hERG channel to the voltage protocol (described in

cardiac cells1. This channel is important in the re-Methods) at room temperature (RT) and physiologicalpolarization of the cardiac action potential. Ab-temperature are shown superimposed in Figure 1. The

normalities

inshortsyndrome,ormost striking effect of raising the temperature to 35°C wasthat the peak amplitude was increased by almost 2-foldcardiac arrhythmia. Given the importance of this(63 ± 9 % increase, n = 13). Additionally, the rise time waschannelanddecreased from 15.7 ± 0.7 ms at RT to 4.8 ± 0.3 ms at 35°C.

disturbances of channel activity by certain compounds

The decay phase could be fit using a single exponential

such as anti-arrhythmias and anti-psychotics, it has

equation and the time constant was decreased from 251

become an important target in compound safety± 18 ms at RT to 126 ± 9 ms at 35°C.

inchannel

potassium

fatal

QT

thisa

leadingmaintaining

cantocardiac

cause

long

potentiallyfunction,

screening. It is a desirable option to study this channel
at physiological temperature since compounds can25°C
35°C

display different actions or potencies at physiologicaltemperature2,3.
Here we present data collected on the Port-a-Patchusing the External Perfusion System coupled with Nanion’s
Temperature Control. Cells were captured and sealedat room temperature and control recordings made before raising the temperature to 35°C so that parameterssuch as current peak amplitude could be compared at
the two different temperatures. Furthermore, the hERG
active compound, quinidine, was used at physiologicaltemperature and a full dose response curve wasachieved.

This

demonstrates

thestabilityof

therecordings on the Port-a-Patch at this temperature. The
concentration response curve generated an IC50 similar

to that obtained at room temperature, and similar to thatpublished in the literature2,4-7.

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500pA
200ms
Figure 1: Example traces for hERG-mediated currents at 25 ± 2°C and 35 ±2°C. Peak amplitude was increased at 35°C, the rise time and decay time
constants were faster at physiological temperature compared to RT.

Application Note
100Figure 2 shows representative traces from an individual cellin the absence and presence of increasing concentrationsof quinidine at 35 ± 2°C. Quinidine was applied using theperfusion system and the temperature maintained at 35 ±2°C during perfusion of the drug. The recording was stable
and a full concentration response of quinidine ranging
from 0.1 µM to 10 µM was achieved. The effect of quinidine
was reversible and approximately 60-70% of the current
could be recovered upon washout (data not shown).
Control
0.1 µM Quinidine
1 µM Quinidine
3 µM Quinidine
10 µM Quinidine% Inhibition

80604025 ± 2°C
35 ± 2°C

2000.01

0.1
110Quinidine Concentration (µM)

100Figure 3: : Concentration response curves at 25°C and 35°C for quinidine.
The IC50 for quinidine at physiological temperature was estimated to be1.3 ± 0.2 μM (n = 5), similar to that obtained at RT (1.00 ± 0.03 μM, n = 3).

Concentration response curves for quinidine at RT and35°C are shown superimposed in Figure 3. The twoconcentration response curves are almost identical. TheIC50 for quinidine at 35°C was estimated to be 1.3 ± 0.2 µM(n = 5) and at RT was estimated to be 1.00 ± 0.03 µM (n =3). The estimation of the IC50 is in good agreement withthose reported in the literature2,4-7.
200pA

In conclusion, we have demonstrated stable recordings of

200ms
Figure 2: A full concentration response curve to quinidine acting on thehERG channel was achieved at physiological temperature. Example traces, in the absence and presence of increasing concentrations of quinidine,from one cell are shown here.

the hERG channel at physiological temperature on aplanar patch clamp system. The effect of temperature
had a dramatic effect on peak amplitude but did notaffect the IC50 of quinidine, as expected2.
Electrophysiology

ReferencesWhole cell patch clamp recordings were conducted

1. Sanguinetti, M. C., et al., 1995. Cell. 81: 299-307

according to Nanion’s standard procedure for the Port-

2. Kirsch, G.E., et al., 2004. JPTM 50: 93-101

a-Patch. Currents were elicited every 20 s stepping from

3. Guo, J., et al., 2005. Heart Rhythm. 2(8): 860-866
4. Paul, A.A., et al., 2002. Br. J. Pharmacol. 136: 717-729
5. Limberis, J. T., et al. 2006. Clin. Exp. Pharmacol. Physiol. 33: 1059-1065
6. Wolpert, C., et al., 2005. J. Cardiovasc. Electophys. 16(1): 54-58
7. Yang, B.F., et al., 2004. Acta Pharmacol. Sin. 25(5): 554-560

a holding potential of -80 mV to +40 mV for 200 ms
followed by a step to -40 mV for 200 ms and back to -80
mV. Currents were recorded at RT until they were stable.
The temperature control device from Nanion was thenswitched on. A stable temperature of 35 ± 2°C wasMethods

reached within 2 mins and maintained. In the case ofCells

quinidine stock was diluted in external solution andquinidine concentration response curves, a 1 mM

HEK293 cells stably expressing hERG were supplied by
EMD Millipore.

applied via the perfusion system. Quinidine was applied
for at least 2 mins at each concentration.

Cell culture
Cells were cultured and harvested according toNanion’s standard cell culture protocol.

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