Application Note

Channel:Cells:
Tools:hERG
HEK293Patchliner®

Effect of temperature on erythromycin
action on hERG currents recorded onNanion´s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by Millipore, UK.
SummaryResults

The hERG gene encodes a potassium channel responsi-

Current responses of two individual cells to 500 ms volt-

ble for the repolarization of the IKr current in cardiac cells

age pulses to +40 mV and then -40 mV in the presence(Sanguinetti et al, 1995). This channel is important in theand absence of 100 mM erythromycin at RT and 35°C are

repolarization of the cardiac action potential. Abnormali-shown in Figure 1. At RT (Panel A) 100 mM erythromycinties in this channel can lead to long or short QT syndrome,caused little reduction in current amplitude (at -40 mV;

leading to potentially fatal cardiac arrhythmia. Given

approx. 15%) compared with an almost 50 % reduction ofthe importance of this channel in maintaining cardiac

current amplitude at 35°C. This is in good agreement withfunction, and disturbances of channel activity by certain

the literature (Guo et al, 2005).

compounds such as anti-arrhythmias and anti-psychotics,
it has become an important target in compound safety
screening.
Compounds can display different properties or different

Control
100 µM
Erythromycin

potencies at physiological temperature (35°C) vs. room
temperature (RT) and therefore, it is a desirable option
to be able to study this channel electrophysiologically at
200 pAelevated temperature. One such compound which has
been shown to have an increase in potency at physiological temperature is erythromycin. Erythromycin is a200 ms

macrolide antibiotic which can cause QT prolongationand cardiac arrhythmia. Erythromycin has been shown
to block hERG channels at physiological temperature
with an IC50 of approximately 40 mM (Stanat et al, 2003;

Duncan et al, 2005). However, at RT erythromycin is muchless potent. At a concentration of 100 mM, erythromycincauses no significant block of hERG currents at RT but
significantly blocks currents at physiological temperature(Guo et al, 2005).
Here we present data collected on an 8-channel Patchliner® with temperature control at RT and at 35°C and theeffect this has on the potency of erythromycin.

Download more Application Notes from www.nanion.deA. Room Temperature

B. 35°C

Figure 1:
Effect of erythromycin on hERG-mediated currents at (A) room temperature and (B) 35°C. The graphs are shown on the same scale. 100 mM
erythromycin significantly blocked hERG currents at 35°C but had little
effect on hERG currents at RT.

Application Note1.0RT35ºC

0.8erythromycin at RT and at 35°C. At higher concentrations(300 mM), erythromycin did block hERG currents at RT by
approximately 40% and gave an IC50 of 427.5 mM calculated from the graph. At 300 mM, erythromycin blockedInhibition

Figure 2 shows the concentration response curves for0.60.4hERG currents at 35°C by 70% and the IC50 at this tem-perature was calculated to be 30.7 mM. This is in excellent
agreement with values reported in the literature (Stanat0.2

et al, 2003; Duncan et al, 2005).
0.0

Erythromycin at RT(n = 12), Max = 1
KD = 427.5 µM, Hill = 11.0

Erythromycin at 35ºC
(n = 18), Max = 0.7
KD = 30.7 µM, Hill = 0.97

1.0Inhibition

Inhibition

0.6

0.4

1000

0.4

Typically, single concentrations of erythromycin were

0.2

0.0

0.0
10 µM
Erythromycin

10100
Conc Erythromycin (µM)

0.60.2

10 nM

1Figure 3:
The IC50 of erythromycin is strongly dependent on temperature. When theconcentration response curves at room temperature and 35°C are overlaid, it is clear to see the shift in potency.

0.8

0.8

0.1

10 mM

applied to each cell. Average concentration responses were then plotted using 12 (RT) and 18 (35°C) cells.
The average concentration response curves are shown
100 nM

10 µM 1 mM
Erythromycin

above overlaid which clearly demonstrates that erythromycin is 10-fold more potent at 35°C.
In summary, it is possible to perform whole cell record-Figure 2:
Concentration response curves for erythromycin at room temperature andat 35°C. Erythromycin is approximately 10-fold more potent at 35°C compared with room temperature.

ings on the Patchliner® at physiological temperature. The
expected IC50 value for erythromycin at 35°C was calcu-lated. For safety screening, it may be critical to test compounds at 35°C and this makes the Patchliner® an ideal

tool for such experiments.

References

Cell culture

1. Sanguinetti, M. C., Jiang, C., Curran, M. E., Keating, M.
T., 1995. Cell. 81: 299-307
2. Stanat, S.J.C., Carlton, S.G., Crumb Jr, W.J., Agrawal,K.C., Clarkson, C.W., 2003. Mol. & Cell Biochem. 254 (1-2):
1-7
3. Duncan, R. S., Ridley, J.M., Milnes, J,T., Leishman, D.J.,
Hancox, J.C., Witchel, H.J., 2005. J. Physiol. 567P, PC8(poster abstract)
4. Guo, J., Zhan, S., Lees-Miller, J.P., Teng, G.Q., Duff, H.J.,
2005. Heart Rhythm. 2 (8): 860-866

Cells were cultured and harvested according to Nanion’s standard cell culture protocol.
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for thePatchliner®. Currents were elicited every 20 s by 500 ms
voltage steps to +40 mV followed by 500 ms step to-40 mV from the holding potential of -80 mV. Erythromycin
was made as a 100 mM stock in DMSO and diluted inexternal solution at the indicated concentrations (max.
DMSO concentration was 0.3%).

Methods
Cells
HEK293 cells stably expressing hERG were used.
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