Application Note

Channel:Cells:
Tools:nAChR α4β2HEK
SyncroPatch 384PE

Pharmacology of human α4β2 nAChR
recorded on the SyncroPatch 384PE
The

electrophysiology

teamatNanion

Technologies

GmbH,Munich.

Cells kindly provided by SB Drug Discovery, Glasgow.

Summary

Results

Nicotinic Acetylcholine Receptors (nAChR) are cation-Currents mediated by nAChR were activated by add-

permeable ion channels, which mediate fast synaptic

ing ACh using the Ligand Puff function in PatchCon-transmission

endogenoustrol 384. A single concentration of ACh was applied toneurotransmitter acetylcholine (ACh) and the exogenous

each well, and cells were maximally activated with 200

naturalwhenalkaloid,activatednicotine.

bytheformµM ACh which was added to all wells. Currents were

pentameric channels which are composed of two α (α2NeuronalnAChR

normalized to the maximal response of each well andto α10) and three β subunits (β2 to β4) . Mutations ofconcentration response curve was calculated acrossnAChR are associated with some forms of epilepsy andthe whole plate. Data was fitted using a Hill equation

many other neurological disorders such as Alzheimer’srevealing an EC50 = 3.4 µM (342 wells), in excellent agree-

1,2Disease, Parkinson’s, Tourette’s Syndrome, Schizophrenia

ment with the literature3,7,8,9 (Figure 1). The success rate forcompleted experiments was 89%.

and depression .
3

The most abundantly expressed nAChR in the mammalian
receptors4. In contrast to the α7, a4β2 nAChR has aA

B
1.0

high affinity for nicotine . This property, the up-regulation
5

during chronic exposure to nicotine6, and the receptorbrain like the ventral tegmental area6, strongly indicatethat the a4β2 nAChR is a potential target for addiction
to nicotine3.
Here we present data collected on the SyncroPatch

384PE

showing

activation

andblockof

α4β2 nAChR currents expressed in HEK cells with
rapid

application

activates

α4β2ofligand

nAChR(‘LigandinaPuff’).

AChconcentration-

dependent manner with an EC50 value similar to thosereported in the literature3,7,8,9. Reproducible currents
were achieved when cells were preincubated withacetylcholinesterase (AChE). Finally, α4β2 nAChR were
blocked by dihydro-b-erythroidine hydrobromide (DHßE),a well known competitive antagonist of the α4 subunit3
with an IC50 in good agreement with the literature7,9.
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200 pA

expression location in addiction sensitive regions of the2s

0.3 μM
1 μM
3 μM
5 μM
10 μM
30 μM
200 μMnormalized current

brain are the α7 homomeric and α4β2 heteromeric

EC50= 3.4 µM ± 0.46 µM

0.8
0.6
0.4
0.2
10-6

10-5
10-4ACh (M)10-3

Figure 1: Activation of nAChR α4β2 expressed in HEK cells on the SyncroPatch
384PE by ACh. A. A single concentration of ACh was applied to eachwell and the average traces of all 342 cells are shown. The concentration
response curve was calculated across the plate and is shown in Panel B.
Currents from each well were normalized to the maximal response within
the well, averaged and fitted with a Hill equation revealing an EC50 of 3.4 µM(n = 342) in excellent agreement with the literature3,7,8,9.

Application Note
Repeated or extended exposure of ACh lead to desensiti- For pharmacological experiments, DHβE, a competitive
zation of the nAChR and resulted in receptor inactivation. nicotinic acetylcholine receptor antagonist, was coAChE, a natural enzyme located on the postsynaptic applied with ACh. Single point concentration response
membrane, hydrolyzes ACh, and the liberated choline is curves were calculated by combining the results of thethen recycled into the presynaptic membrane. Conse- application of nine different concentrations distributed
quently, the postsynaptic nAChRs quickly recover from de- across the plate (Figure 4). This gave an IC50 = 48.2 ± 14.8 nM

sensitization and ACh is resynthesized in the presynapse (n = 362), in excellent agreement with the literature7,9. The
which is able to reactivate nAChRs.

success rate for completed experiments was 94 %. For

Figure 3 shows a4β2 nAChR desensitization after repetitive normalization 1 µM ACh was applied first, followed by coactivation of the receptor with 2 µM ACh and recovery application of DHβE and 1 µM ACh.
from desensitization when ACh is co-applied with AChE.

AWhen ACh is co-applied with AChE peak amplitudes varied with each application by no more than 5%.

2 nA

A5s
1 μM ACh
1 μM ACh/600 nM DHβE

1s0.5 nAB

1.01s

normalized current

200 pA

B

C
2 µM ACh/
2 U AChE

Ipeak (nA)

02 µM ACh/
2 U AChE

2 µM ACh/
2 U AChE

-10.8

DHßE
IC50 = 48.2 ± 14.8 nM
n = 362

0.6
0.4
0.2
0.0
10-10-2-3-40200

400
600
Time (s)

800

1000

Figure 3: Desensitization and recovery of nAChR mediated currents
recorded on the SyncroPatch 384PE. A. Averaged raw current traces from64 wells showing current activation by repetitive application of 2 µM ACh.
B. Averaged raw current traces from 64 wells showing current activation byapplication of 2 µM ACh in the presence of 2 units AChE repeated 3 times inthe well. Peak amplitudes were very similar with each application of ACh/AChE, differing by no more than 5%. C. Online analysis plot showing peak
amplitude versus time for one cell.

10-910-8
DHβE (M)

10-7

10-6

Figure 4: Block of nAChR α4β2 by DHβE. A. Exemplary averaged traces after
addition of 1 µM ACh (black) and after co-application with 600 nM DHβE(blue). B. Currents in the presence of the antagonist were normalized to thecontrol current activated by ACh only. The concentration response curvefor DHßE was calculated across the plate. Data were fitted with standard Hillequation and IC50 calculated to be 48.2 ± 14.8 nM (362), in good agreementwith values7,9.

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 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot depicts raw data traces ofnAChR α4β2 expressing HEK cells as recorded on one NPC-384 patch clamp chip. A single point concentration response curve was performed for DHβE onindividual wells. One control application of 1 µM ACh was followed by one concentration of DHβE on each well and increasing concentrations across theplate. DHβE was co-applied with ACh. Multi-hole chips were used where 8 holes were present per well. The data of the 384 well plate representation in theupper left part are color-coded for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb > 70 MΩ), blue (Rmemb 10 - 70MΩ) or light blue (Rmemb > 10 MΩ). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs
show raw data traces of nAChR α4β2 channels following application DHβE co-applied with ACh.

Exp end and chipunload

15 - 20 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1 buffer application (baseline),
1 control application ACh (normalization), 1 compound,co-application, 2 washes between additions

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch 384PE. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for asingle point concentration response curve on α4β2 nAChR-mediated currents took approximately 20-25 min.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application Note
Figure 5 shows a screenshot of the SyncroPatch 384

recordingsoftware during an experiment. A color-coded over-physiologicalview (based on seal resistance in this case) of allof

384 wells gives the user a good impression of theconcentration

success rate of the experiment. The user can easily

maximum

concentration

toggle between raw traces and online analysis. In

typicallyapplied.

the example shown, raw traces are chosen and theresponse was inhibited by DHßE in a concentration-graphs show current responses to ACh followed bydependent manner as expected with an IC50 within theinhibition by

co-application of DHßE. The combina-tion of brief compound application andthe perfor-

mance of single point concentration response curves

AChafterapplication

solution,

followed

singlebyofofstandard

controlapplication

co-application

withsingle

antagonist.

Finally,

aofTheACh

α4β2(200µM)

wasnAChR-mediatedrange round in the literature7. The EC50 values for AChand the kinetics of desensitization were consistent with
α4β2 nAChR heteromers7.

provides an easy and quick way to discover agonists
and antagonists of fast desensitizing receptors such

The SyncroPatch 384PE is a high throughput and highly

as nAChR. The cells were stable for about 30 minutes

reliable automated patch clamp device for stud-and compound could be rapidly applied to the cells

ies

to minimize desensitization. An individual well can befriendly

highlighted to monitor progression of the experiment andadditions

is shown enlarged at the bottom of the screen.

each well and easy analysis result in reliable highon

α4β2
software,ornAChR-mediatedexcellent

multiplecurrents.

success

additionsofrates,

Usersingle

compoundtoquality data at an increased throughput with an
In conclusion, α4β2 nAChR expressed in HEK cells can be

economical cost per data point.

reliably and reproducible recorded on the SyncroPatch
384PE. The timeline of each experiment was approximately 20 minutes (start – end) and included baseline

References

Nanion’s standard cell culture protocol. Nicotinic ACh
α4β2 Receptor expression was induced by incubation in1. Matta et al.. 2017. Cell Reports 19, 688–6961 μg/ml tetracycline 18 - 24 hours prior to experiments.

2. Picciotto et al.. 2003. TiPS. 24, 493–499
3. Nelson et al.. 2003. Mol Pharmacol. 63(2):332-41.

Electrophysiology

4. Picciotto et al.. 1998. Nature. 391:173–7

Whole cell patch clamp recordings were conducted

5. Gopalakrishnan et al.. 1996. J. Pharm. Exp. Ther., 276

according to Nanion’s standard procedure for the(1): 289-97

SyncroPatch 384PE using multi-hole (8 holes per well)

6. Schwartz R. et al.. 1983. Science 220, 214–216

chips. Cells were held at -100 mV for the duration of the7. Buisson et al.. 1996. JNeurosci 16(24):7880–7891

experiment, if not state different. For ACh concentration

8. Wu et al., 2004. J Biol Chem. 279(36):37842-51.

response curves, AChE (1U/µM ACh) was used to prevent

9. Sidach et al.. 2009. J Neurosci Methods 182, 17-24

desensitization. For pharmacology experiments, cells
were pre-incubated in DHβE for 5 min and thenMethods

compound was co-applied with 2 µM ACh. Receptors
were activated using the ‘Ligand Puff’ command inCells
HEK293PatchControl 384: Agonist volume: 5 µM, wash volume: 7cells

expressingnAChR

withthesubunitµM, application speed 40 µl/s. Cells were then washed 3

configuration α4β2 are kindly provided by SB Drugx with external solution before re-application of agonist.

Discovery

Integral and the minimum peak current amplitudes wereanalyzed to determine the EC50 or IC50. Data were fitted

Cell culture

by Hill equation either to normalized values or raw

Cells were cultured and harvested according toamplitudes.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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