Application Note

Channel:Cells:
Tools:nAChR α1β1γδ
TE671
SyncroPatch 384

Reproducible activation and pharmacologyof α1β1γδ nAChR on the SyncroPatch 384
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were obtained from CLS.

SummaryResults

Nicotinic acetylcholine receptors (nAChR) are acetyl-

Nicotinic AChα1βγδ endogenously expressed in TE671

choline- (ACh) and nicotine-gated cation permeable

cells was activated by acetylcholine (ACh) in a concen-ion channels, which mediate fast synaptic transmission

tration-dependent manner (Fig. 1). Fitting the normalized

at central synapses and neuromuscular junctions. Neu-data by applying a Hill equation revealed an EC50 of 3.6 ±romuscular nAChR form heteromeric proteins composed

0.5 µM (n = 361), which is in good agreement with valuesof four subunits: α, β, γ (or ε) and δ. Depending on thereported in manual patch clamp studies8. Success rate

developmental stage, the AChR subunit stoichiometry

for completed experiments was 94%.

changes from α1β1γδ (embryonic) to α1β1εδ (adult)1.
Several inherited and acquired diseases are associated1.0with nAChR dysfunction, most of which lead to impaired
neuromuscular transmission and muscle weakness. The
acquired autoimmune disease myasthenia gravis (MG)

Acetylcholine
EC50 = 3.6 ± 0.5 µM (n = 361)

is caused by autoantibodies targeting muscle nAChRs2
muscle weakness and fatigue3,4. Inherited diseases called
congenital myasthenic syndromes (CMS) are associated

I/Imaxthat disrupts nerve-muscle communication resulting in

0.1 µM
1 µM
3 µM
10 µM
30 µM

0.5

with several abnormalities affecting ACh-release, acetylcholinesterase activity, nAChR function and/or nAChR

1 nA

number5,6,7. Treatment has been limited to nonselective,
chronic immunosuppressive therapies which have longterm toxicities. More selective and targeted therapies are
now under development6.
Here we present data collected on the SyncroPatch
384 showing activation and block of nAChRα1β1γδ
expressed in human TE671 cells with rapid application of ligand or co-application with blockers. We found that ACh
activates nAChRα1β1γδ receptors with an EC50 value

similar to those reported in the literature8. We recorded
highly reproducible currents in response to ACh andobtained IC50 values for mecamylamine and α-conotoxin
GI that were in good agreement with the literature9,10,12,13,.

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1s

0.0
10-7

10-6

10-5

Acetylcholine (µM)
Figure 1: Cumulative activation of nAChR α1β1γδ by acetylcholine
recorded on the SyncroPatch 384. The concentration response curve
for acetylcholine for an average of 361 wells is shown with the average
traces for each concentration shown in the inset. Multihole chips (4 holesper well) were used, and nAChR α1β1γδ was recorded using a constantholding potential of -80 mV. Five concentrations of ACh (0.1 - 30 µM)
were cumulatively added to each well and the average concentration
response curve calculated from the plate. Currents from each well werenormalized to the maximal response within the well, averaged and fittedwith a Hill equation revealing an EC50 of 3.6 ± 0.5 µM (n = 361). Data
represent mean ± SEM.

Application Note
In order to minimize desensitization of the channel

1.0and to allow for repetitive activation, nAChα1β1γδ
receptors were activated by brief exposure (0.5 secs)
asmallapproachamountcombined

applications,ofligandwith

nAChα1β1γδ

(2µl).

Using

twowashes

betweencouldberepetitivelyactivated by repeated stimulation with 3 µM ACh andlower concentrations (Fig. 2) allowing inhibition to bestudied.

Acontrol

3 µM

3 µM

3 µM

3 µM

0.8

thisACh Max

Normalized BlocktoMecamylamine hydrochloride
IC50 = 2.2 ± 1.1µM(n = 181 wells)
0 µM
1 µM
3 µM
10 µM
30 µM100 µM

0.60.40.5 nA

0.21s

0.03-1.0-1.5

3010-2.02s

100-2.5200400

600 800time (s)1000 1200

Figure 2: Repetitive activation of nAChR α1β1γδ by acetylcholine.
Nicotinic AChα1β1γδ could be repetitively activated by 6 µM andlower concentrations of ACh at least 4 times in the same well with apeak amplitude consistent with prior applications (<10% change in peakamplitude). A, shows raw current traces in response to 3 µM ACh from anexemplar cell. B, the average timecourse of the experiment for all AChconcentrations used is shown. Each ACh concentration was applied 4times to the same well. Different concentrations varied across the plate.

To record concentration-response curves, each wellreceived 3 applications of 3 µM ACh followed by co-application of ACh and a single concentration of either mecamylamine hydrochloride or α-conotoxin GI. Mecamylamine is a noncompetitive antagonist targeting all

10-4

Figure 3: Average concentration response curve for mecamylamine. A single
concentration of mecamylamine hydrochloride was co-applied with 3 µM
acetylcholine and the concentration response curve for an average of 181
wells is shown. Average traces in different concentrations of mecamylamine
are also shown in the inset. The IC50 for mecamylamine was 2.2 ± 1.1 µM (n =181) giving a success rate of 94% for completed experiments.

Alpha (α)-conotoxin GI is a competitive antagonist withan IC50 in the nM range which does not block neuronalnicotinic acetylcholine receptors11. In our experiments,
nAChα1β1γδ activated by 3 µM ACh was blocked byα-conotoxin GI with an IC50 = 6.8 ± 2.9 nM (n = 171 wells; Fig.
4), which agrees withthe range reported for other

α-subunits12,13.
1.00.8
Normalized Block-0.50.5 nA

ACh (µM)
0.1
1B

Ipeak (nA)

10-8
10-7
10-610-5[Mecamylamine hydrochloride (M)]α-Conotoxin (GI)IC50 = 6.8 ± 2.9 nM(n = 171 wells)
0 nM
1 nM
3 nM
10 nM
30 nM
100 nM

0.6
0.4
0.5 nA

0.21snAChR subtypes . Figure 3 shows the concentration
10response curve for mecamylamine for an average of181 wells. The IC50 for mecamylamine was calculated tobe 2.2 ± 1.1 µM. For comparison, the effect of mecamyl-amine isomers were examined on neuronal and muscularnAChR subtypes blocking α3β4 containing nAChR withan IC50 of ~0.6 µM, α7 containing nAChR with an IC50 of~7 µM and adult muscular α1 containing nAChR with anIC50 of 2.2 µM9.

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0.0
10-10

10-910-8[α-Conotoxin GI (M)]

10-7

Figure 4: Average concentration response curve for α-conotoxin GI. A single
concentration of α-conotoxin GI was co-applied with 3 µM ACh and theconcentration response curve for an average of 171 wells is shown. Average
traces in different concentrations of α-conotoxin GI are also shown in theinset. The IC50 for α-conotoxin GI was 6.8 ± 2.9 nM (n = 171).

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384. Screenshot of depiction of raw current tracesof nAChα1β1γδ endogenousy expressed in TE671 cells as recorded on one NPC-384 patch clamp chip (4x holes per well). Three hundred and eightyfour small color-coded pictures as seen in the upper left part display 384 recordings. Wells used for analysis are shown in green. Grey wells indicate failed
wells based on the quality control criteria. One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right.
Graphs show current traces of nAChα1β1γδ after activation with 3 µM acetylcholine (red trace) and co-application with mecamylamine hydrochloride orα-conotoxin GI at the given concentrations (black trace).

Exp end and chip
unload5 min~25 min

2 min
1 min

Chip load andpriming

Patch clamp experiment: 1 control (no current activation), 3
applications of 3 µM ACh made 4.5 mins apart, 1 compoundco-application and 1 full activation. Between applications cells are
washed twice with buffer.

Analysis

Figure 6: The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a single point concentration response curve plus
repeated activation of nAChα1β1γδ with acetylcholine took approximately 33 min.

Download more Application Notes from www.nanion.deApplication NoteFigure 5 shows a screenshot of the SyncroPatch 384

single compound concentration to each well and a fullsoftware during an experiment. A color-coded over-activation with 100 µM ACh.

view (based on seal resistance in this case) of all 384
wells gives the user a good impression of the suc-

Using the LigandPuff application procedure of thecess rate of the experiment. The user can choose

SyncroPatch 384, nAChα1β1γδ could be repetitively

whether to visualize raw traces or online analysis. Inactivated.

Asexpected,nAChα1β1γδ

this case, raw current traces are shown, red traces

activatedbyAChandinhibited

indicate control (3 µM ACh) and black traces indi-aminehydrochlorideandα-conotoxin

cate the presence of either mecamylamine hydro-concentration-dependent manner with IC50 values similar

chloride or α-conotoxin GI. An individual well can bebycouldbemecamylGI

inato those reported in the literature9,10,12,13.

highlighted to monitor the progression of the experiment.
The SyncroPatch 384 is a high throughput and highly
In conclusion, nAChα1β1γδ endogenously expressed

reliable automated patch clamp device for recording

in TE671 cells wasrecorded on the SyncroPatch 384

nAChα1β1γδ currents. User-friendly software, excellent

with a good success rate for completed experiments

success rates, single additions or multiple additions of com-(>89%). The timeline of each experiment was about 33

pound to each cell and easy analysis result in reliable, highminutes (start–end) and included activation with AChquality data at an increased throughput with an(repeated three times), two washes between ligand

economical cost per data point.

application for complete ligand washout, application of aReferences

Methods

1. Mishina M., et al. 1986. Nature. 321(6068), 406–411.

Cells

2. Patrick J. & Lindstrom J. 1973. Science. 180(4088), 871–nAChRα1β1γδ endogenously expressing TE671 cells were872.

obtained from CLS.

3. Fambrough D. M., et al. 1973. Science. 182(4109), 293–295

Cell culture

4. Gilhus N. E., et al. 2016. Nat. Rev. Neurol. 12(5), 259–268

Cells were cultured and harvested according to5. Ohno K. et al. 1995. Proc. Natl Acad. Sci. USA. 92. 758–

Nanion’s standard cell culture protocol for gentle cell762

handling .

6. Lindstrom J. M. 2000. Muscle & Nerve. 23(4), 453–477
7. Engel A. G., et al. 2003. Nat. Rev. Neurosci. 4(5), 339–

Electrophysiology

352.

Whole-cell patch clamp recordings were conducted

8. Shao Z., et al. 1998. J. Pharmacol. Exp. Ther. 286(3):1269-according to Nanion’s standard procedure for the76.

SyncroPatch 384. A constant holding potential of -80 mV

9. Papke R.L., et al. 2001. J. Pharmacol. Exp. Ther.

was used and a small volume of ligand (2 µl) was applied297(2):646-56

to activate nAChRα1βγδ. Multihole chips were used with

10. Nickell J. R., et al. 2013. Pharmacol. Biochem. Behav.

4 holes per well. For pharmacology experiments the108, 28–43.

inhibitor was co-applied with activator after 3 control

11. Ning J., et al. 2018. Mar. Drugs. 16(12):507.

applications of ligand and data normalized to peak

12. Azam L. & McIntosh J. 2009. Acta Pharmacol. Sin. 30,

amplitude in the absence of the blocker. A single

771–783

concentration of blocker was applied to each well and

13. Lebbe E. et al. 2014. Mar. Drugs. 12(5), 2970–3004.

the concentration response curve calculated acrossmultiple wells.

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