Application Note

Channel:Cells:
Tools:

K2P3.1 (TASK-1)
HEK293
SyncroPatch 384PE

Activation and Inhibition of TASK-1 on Nanion’s SyncroPatch 384PE
The

electrophysiology

teamatNanion

Technologies

GmbH,Munich.

Cells kindly provided by SB Drug Discovery, Glasgow.

SummaryResults

The resting membrane potential of excitable cells is

For the evaluation of the performance of TASK-1 express-

determineding HEK293 cells Seal Resistance (Rseal) at the start andbyleakconductances

predominantly
potas-

end of one experiment using single hole chips were de-sium channels (K2P)1. K2P channels are character-termined (Fig. 1). Cells were captured with a high successized by the presence of two pore forming regions

rate (94% of cells had a seal >500 MΩ at the start of theand four trans-membrane spanning (4TMS) regions

experiment)

currentsofthat

backgroundact

toessentialforleak

type

potassium

resting

membraneregulatethe10080

potential and levels of cellular excitability1. The TWIK-

implicated in atrial fibrillation (AF) pathophysiology andwas suggested as an atrial-selective antiarrhythmic
drug target9-10. TASK-1 is activated by extracellularacidosis and inhibited by anandamide and by local
anesthetics including bupivicaine. Volatile general anestethics such as halothan and xenon stimulate TASK-111.
Here,

we

demonstrate

manTASK-1

expressed

recordings
inHEKofhu-cellson

the SyncroPatch 384PE with good success rates. hTASK-1
was activated by extracellular alkalization and blockedby tetrahexylammonium chloride (THA).

Download more Application Notes from www.nanion.de00kidney, intestine and prostate6-8. TASK-1 has been

20

10such as in the heart, adrenal gland, lung, pancreas,M

expressed throughout the CNS3-5 but also in other tissues

4000

encoded by the KCNK3 gene2. TASK-1 is ubiquitously<11) is a member of the K2P channel family and is60
Cells (%)

related acid-sensitive K+ channel 1 (K2P3.1 or TASK-start of experimentend of experimentΩproduction

areMchannels

00

These>5in each channel subunit and form functional dimers.

Ωtwo-pore-domain

Mand00KCNK-5byΩ

mediated

Seal Resistance
Figure 1: Statistics of TASK-1 expressing HEK293 cells recorded on one
NPC-384 chip on the SyncroPatch 384PE. Success rate (seal resistance) ofindividual HEK cells on the SyncroPatch 384. Shown is a bar graph of sealresistances at the start (light blue) and end of the experiment (dark blue).

Application NoteTASK-1 is sensitive to extracellular pH in the narrow physio-Under physiological conditions, activation of TASK-1 elic-logical range, activated by extracellular alkalization andits a typical outward rectifying leak conductance, which

inhibited by acidic pH . Relatively large changes in extra-

is not entirely voltage and time independent. Figure 2

cellular pH can take place, for example, during hypoxia,demonstrates the activation of TASK-1 channels byischemia or during epileptic seizures, pathophysiologicalextracellular alkalization and their current-voltage behav-conditions in which TASK-1 is proposed to be involved .
ApH 8.9
25 mV
pH 8.7
100 mspH 8.2
THA- 80 mV
pH 7.0

ior under physiological potassium gradient conditions.

1213,14

In a study with TREK-1, it has been shown that quaternery ammonuim ions, e.g. tetrahexylammonium (THA)
bind to the pore of K2P channels with high affinity andblock the channel15. Figure 2 shows subsequent block ofpH-activated hTASK-1 by 5 μM THA (red). As expected,
THA at this concentration blocked the hTASK-1-mediatedcurrent. Figure 2B shows the online analysis values for the0.2 nA

500 msoutward current of an example cell at +60 mV and thecorresponding amplitudes depicted as a bar graph foran average of 93 cells (Figure 2C). The outward current0activated by external alkalization with pH 8.9 was 578 ± 40pA compared with 256 ± 69 pA in control conditions and333 ± 73 pA after block with THA. THA blocks all K2P channels and is not K2P subtype specific. To isolate hTASK-1
0.8

from other K2P channel subtypes in native cells, the novel

0.6

TASK-1 specific blocker A293 appears to be a useful com-0.20CFigure 3 shows a screenshot of the SyncroPatch 384 soft0100200300 400Time (s)500600ware during an experiment. The user can choose whether
to visualize raw traces or online analysis. On the left, theraw trace view is chosen. On the right, the online analysis

0.6view is shown representing current amplitude plottedagainst time for 16 selected wells. An individual well can0.5

be highlighted to monitor the progression of the experi-

0.4

ment. In the online analysis view, the time points at which

0.3

solution additions have been made are indicated byTH
A

98.
pH

78.

con

pH

8.

2

vertical lines, as well as different background colors. In thistr
7. ol (0) pH

Amplitude (nA)pound16.

0.4

pHOA-Result (nA)B

Figure 2: Activation and inhibition of hTASK-1. A. Raw traces of an exemplar
cell in control conditions (grey), in the presence of alkaline pH (differentshades of blue) and in the presence of the inhibitor, THA (5 μM; red). Multiple
traces in the presence increased pH are included to show developmentof activation over time. The inset shows the ramp pulse protocol which
was used to monitor the voltage dependence of current during current
activation with different pH solutions. B. Online analysis current amplitude at60 mV is plotted versus time. Light grey background shows current responseover time during application of increased pH. Application of THA is shownin dark grey. C. Bar graph of the amplitude values for an average of 93 cells
is shown in control (light grey), pH 8.2 (light blue), pH 8.7 (blue), pH 8.9 (darkblue) and THA (red). Shown are average ± S.E.M.

Download more Application Notes from www.nanion.decase, white shows baseline amplitudes in control solution,light grey indicates activation with different pH (8.2, 8.7and 8.9) and dark grey shows inhibition by THA (5 μM). A
single concentration of THA was used in this experiment
but different concentrations of activator or inhibitor could
be used to investigate concentration response relationships.

Application Note

Figure 3: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot of depiction of online analysis
data of hTASK-1 expressing HEK293 cells as recorded on one NPC-384 (single-hole) patch clamp chip. Three hundred and eighty-four small color-coded
pictures as seen in the upper left part display 384 recordings. One highlighted experiment is displayed at the bottom, 16 selected experiments are displayedon the right. Graphs show raw traces and online analysis plots of current amplitudes versus time of hTASK-1 after activation with different pH and following
block with 5 μM THA (red trace). Two and a half minutes of baseline current was recorded before current activation with increased pH.

Exp end and chip
unload5 min

10 - 12 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 3 control applications (pH 7.0,8.2, 8.7, 8.9) plus 1 full block addition.

Analysis

Figure 4: Timeline of an experiment on the SyncroPatch 384PE. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for
three additions of activator plus full block of TASK-1-mediated currents took approximately 10-12 min.

Download more Application Notes from www.nanion.deApplication Note
In conclusion, hTASK-1 expressed in HEK cells can be

The SyncroPatch 384PE is a high throughput and highly

recorded on the SyncroPatch 384PE with high successreliable automated patch clamp device for recording

rates for RSeal > 500 MΩ at the start of the experimenthTASK-1 currents. User-friendly software, excellent success(typically >90%). hTASK-1 was activated by alkaline pHrates, single additions or multiple additions of compoundand inhibited by THA. The timeline of each experimentto each cell, and easy analysis result in reliable high qual-

was about 18-20 minutes (start – end) and included wash

ity data at an increased throughput with an economicalwith control solution to establish a stable baseline, acti-cost per data point.

vation by external application of pH and full block using
THA (Figure 4).
The typical outward rectification of hTASK1 mediatedcurrents in asymmetric potassium concentration, the activation by extracellular alkalization and the inhibition by
THA recorded on the SyncroPatch 384PE are as expected1,3,12,15.

References

Methods

1. Goldstein SA, et al. 2001. Nat Rev Neurosci 2:175-184

Cells

2. Manjunath NA. et al. 1999. Cytogenet. Cell Genet., 86

HEK293 cells expressing hK2P3.1 (TASK-1)(3-4): 242-3
3. Duprat F, et al. 1997. EMBO J. 16:5464–5471

Cell culture

4. Leonoudakis D, et al. 1998. J. Neurosci., 18 (3): 868-77

Cells were cultured and harvested according to

5. Reyes R, et al. 1998. J Biol Chem. 273:30863–30869

Nanion’s gentle cell culture protocol.

6. Bandulik, et al. 2015. Pflugers Arch 467(5), 1027–1042
7. Kim Y, et al. 1999. Am. J. Physiol., 277 (5): H1669-78

Electrophysiology

8. Czirják G, et al. 2000. Mol. Endocrinol., 14 (6): 863-74

Whole cell patch clamp recordings were conducted

9. Chai S, et al. 2017. Am J Physiol Heart Circ Physiol 312:

according to Nanion’s standard procedure for theH1144-H1153

SyncroPatch 384PE. Cells were held at -80 mV. A voltage

10. Schmidt C, et al. 2015. Circulation 132: 82-92

ramp protocol from -120 mV to +60 mV in 450 ms was

11. Patel AJ , et al. 1999. Nat Neurosci 2: 422–426

applied to the cells every 10 s. Current amplitude at 60

12. Enyedi P. & Czirjak, G. 2010. Physiol Rev 90, 559-605

mV was used for analysis. To calculate the pH response

13. Demaurex N, et al. 2010. J Physiol 588:4659–4665

curve, cells were activated with increasing pH and data

14. Heurteaux C, et al. 2004. EMBO J 23: 2684–2695

were normalized to the maximum response at pH 8.9.

15. Piechotta, P.L., et al. 2011. EMBO J. 30: 3607-3619

Cumulative pH response curve was calculated and fitted

16. Kiper AK, et al. 2015. Pflugers Arch. 467:1081-1090

with a Hill equation.

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