Application Note

Channel:
TREK
Cells:
HEK293Tools:
SyncroPatch® 384PE

Activation and inhibition of TREK-1 on
Nanion’s SyncroPatch® 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly provided by SB Drug Discovery.

Summary

Results

The TWIK-related Kchannel (TREK-1) is a two pore

For the evaluation of the performance of hTREK-1

1domain (K2P) K channel is encoded by the KCNK2 gene .

(HEK293)cells, Seal Resistance (Rseries) at the start andThe protein is a homodimer, each subunit comprised ofend of one experiment using single hole chips wastransmembrane (TM) helical regions (M1-M4), two pore

determined (Fig. 1). HEK293 cells expressing hTREK-1 could

domains (P1 & P2), an extracellular region with 2 helices,be captured to the patch clamp aperture with a highand intracellular N and C termini1,2. The pore helices andsuccess rate (>93% of cells had RSeal > 500 MΩ at thepore loops form the K selectivity filter . First discovered

start of the experiment).

+++1in 19962, it plays an important physiological role in back100

regulating resting membrane potential. TREK-1 widely is80

expressed throughout the CNS and spinal cord, particularly

by volatile anaesthetics9,10 and is likely to be an important
target for these agents10. TREK-1 has been proposed
to play a pivotal role in cerebral ischemia , epilepsy ,1010

depression11, pain perception12 and temperature sensing4
and is an interesting therapeutic target.
Human TREK-1 expressed in HEK cells were recorded on
the SyncroPatch 384PE with good success rates. hTREK-1
was activated by BL-1249 and blocked by THA.
Download more Application Notes from www.nanion.dech/
cato

phospoholipids4,6,8. It has also been shown to be openedN

cellular acidosis7, poly-unsaturated fatty acids6,8 andO
M

diincluding mechanical (stretch)4,6,7, temperature4, intra-

500sabby a variety of different physical and chemical stimulihm0>

membrane potential prior to labor. TREK-1 is modulatedhm

nancy5 and may play a role in maintaining a negative

20MO

human myometrium where it is up-regulated during preg-40

0regions such as lung, heart, kidney, skeletal muscle and2droot ganglion (DRG) neurons4. It is also expressed in other60

leexpressed in high levels in small and medium sized dorsal% Cells

in the cortex, hippocampus, thalamus, hypothalamus,
cerebellum and basal ganglia2,3. Additionally, TREK-1 isstart of experimentend of experiment<50ground K+ conductance2 and thus plays a major role inSeal Resistance

Figure 1: Statistics of hTREK-1 expressing HEK293 cells recorded on one
NPC-384 chip on the SyncroPatch® 384PE. Success rate (seal resistance)of individual HEK cells on the SyncroPatch® 384PE. Shown is a bar graph ofseal resistances at the start (light blue) and end of the experiment (darkblue).

Application NoteBL-1249 is a K+ channel opener with bladder relaxant

are potential therapeutic targets for treating theproperties13. It is proposed to exert it’s effects via activa-

overactive bladder and because of it’s preferential

tion of TREK-113. Expression of TREK-1 is relatively high inexpression in bladder smooth muscle cells, TREK-1 is an

bladder smooth muscle cells versus human aortic cells

attractiveand BL-1249 has been shown to act as a potassium

condition, without significant effects on blood pressure13.

channel activator exhibiting bladder versus vascular

Figure 2 shows current responses of an exemplar cell to aselectivity both in vitro and in vivo. K+ channel openers

voltage ramp protocol from -120 mV to 60 mV over450A

mstargetin

controlforthe(black)treatment

conditions

ofand

thisafter

activation with 10 µM BL-1249 (blue). As expected,
activation of TREK-1 results in an outwardly rectifying

ControlBL-1249
THAcurrent2.

Severaltracesinthepresenceof BL-1249 are included in the figure from the start toend of incubation to show development of current
activation. Quaternery ammonuim ions, e.g. tetrahexylammonium (THA) have been shown to bind to
the pore of TREK-1 with high affinity and block the2 nA
100 ms0channel14. Figure 2 shows subsequent block of BL-1249activated hTREK-1 by 10 µM THA (red). As expected, THA
at this concentration completely blocked the hTREK-1mediated current. Figure 2B shows the online analysis
values for the positive current of an example cell and thecorresponding amplitudes for an average of 368 cells. TheAmplitude (nA)B

outward current activated by BL-1249 was 5.9 ± 0.1 nA

6compared with 352 ± 15 pA in control conditions and4540 ± 20 pA after block with THA.

2

Figure 3 shows a screenshot of the SyncroPatch® 3840software during an experiment. The user can choose

ATH

9l

tro

24-1

onBLC

Figure 2: Activation and inhibition of hTREK-1. A Raw traces of an exemplar
cell in control conditions (black), in the presence of the activator, BL-1249(10 µM; blue) and in the presence of the inhibitor, THA (10 µM; red). Multiple
traces in the presence of the activator are included to show developmentof activation over time B Online analysis (left; current amplitude at 60 mV)
is plotted versus time and application of BL-1249 is shown in blue and THA
shown in grey. A bar graph of the amplitude values for an average of 368
cells is shown on the right in control (black), BL-1249 (blue) and THA (red).
Shown are average ± S.E.M.(10 µM; blue) and in the presence of the inhibitor,
THA (10 µM; red).

whether to visualize raw traces or online analysis. In
this case,online analysis is chosen and the graphs

represent current amplitude plotted against time. An
individual well can be highlighted to monitor theprogression of the experiment. In the Online Analysis view,the time points at which solution additions have been
made are indicated by vertical lines, as well as different
background colors. In this case, white shows baseline
amplitudes in control solution, blue indicates BL-1249(10 µM) and grey shows inhibition by THA (10 µM). A
single concentration of BL-1249 and THA were used in thisexperiment but different concentrations of activator orinhibitor could be used to investigate concentration
response relationships.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

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 hTREK-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 current amplitudes of hTREK-1 in control solution (white region), after activation with 10 µM BL-1249 (blue region) and following
block with 10 µM THA. Almost 5 minutes of baseline current was recorded prior to application of BL-1249.

Exp end and chip
unload15 - 18 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 2 control washes, activation using
external application of compound, full blockAnalysis

Figure 4: The completion of 1 experiment on the SyncroPatch® 384 patch clamp chip (384 wells) for a single addition of activator plus full block of hTREK-1
took approximately 22-25 min.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application NoteIn conclusion, hTREK-1 expressed in HEK293 cells can

The SyncroPatch® 384PE is a high throughput and highly

be recorded on the SyncroPatchreliable

automated

excellent success rate for RSeal > 500 MΩ at the start

recordinghTREK-1of the experiment (>93%). hTREK-1 was activated byexcellent success rates, single additions or multiple

BL-1249 and blocked by THA. The timeline of each

additionsexperimentand®

(Figure4)

wasabout

384PE with an

22-25

minuteseasyofpatch
currents.

compoundanalysis

resultclamp

deviceUser-friendlyforsoftware,toeachcell,

inreliablehigh(start – end) and included wash with control solution toquality data at an increased throughput with anestablish a stable baseline, activation by external

economical cost per data point.

application of BL-1249, time to reach a stable current
and full block using THA, all performed on each individual
well, drastically reducing the consumable cost per data
point to < $0.60.
The

outwardrectification

propertiesofhTREK-1

recorded on the SyncroPatch® 384PE, activation andblock of the channel are in excellent agreement with theliterature2,12,13.

References

Methods

1. Vivier, D., et al. 2016. J. Med. Chem. 59(11): 5149-5157

Cells

2. Fink, M., et al. 1996. EMBO J. 15(24): 6854-6862

hTREK-1 expressing HEK293 cells were supplied by SB Drug

3. Hervieu, G.J., et al. 2001. Neurosci. 103(4): 899-919

Discovery, Catalog Ref: SB-HEK-TREK1.

4. Maingret, F., et al. 2000. EMBO J. 19(11): 2483-2491
5. Buxton, I.L.O., et al. 2010. PLoS ONE 5(8): e12372.

Cell culture

doi:10.1371/journal.pone.0012372

Cells were cultured according to the manufacturer’s

6. Patel, A.J., et al. 1998. EMBO J. 17(15): p.4283-4290

recommendations and harvested according to Nanion’s

7. Maingret, F., et al. 1999. JBC. 274(38): 26691-26696

standard protocol.

8. Maingret, F., et al. 1999. JBC. 275(14): 10128-10133
9. Patel, A.J., et al. 1999. Nat. Neurosci. 2: 422-426

Electrophysiology

10. Heurteaux, C., et al. 2004. EMBO. J. 23(13): 2684-2695

Whole cell patch clamp recordings were conducted

11. Borsotto, M., et al. 2015. Br.J. Pharmacol. 172: 771-784

according to Nanion’s standard procedure for the12. Alloui, A., et al. 2006. EMBO J. 25: 2368-2376

SyncroPatch® 384PE using single-hole chips. A voltage

13. Tertyshnikova, S., et al. 2005. JPET. 313: 250-259

ramp protocol from -120 mV to +60 mV over 450 ms, with

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

70 ms at -120 mV and +60 mV at the start and end of theramp, respectively, was applied to the cells every 10 s.
Intersweep holding potential was -80 mV. Current
amplitude at +60 mV was used for analysis. Compounds
were applied via the external solution.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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