Application Note

Channel:Cells:
Tools:nAChα7R
HEK293Patchliner

Nicotinic AChα7R activation and pharmacology recorded on Nanion’s  Patchliner
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Galantos Pharma, GmbH, Germany.

Summary

Results

The nicotinic acetylcholine receptor (nAChR) is a member

Nicotinic AChα7R were activated by increasing concen-of the ligand-gated ion channel superfamily which

trations of ACh or nicotine (Figure 1). A concentration

includes GABAA, 5HT3, NMDA and glycine receptors. It

response curve for ACh (Fig. 1B) revealed an EC50 of 304

is a cation-permeable ion channel activated by the± 42 µM (n = 11) in good agreement with the range foundneurotransmitter acetylcholine and the natural alkaloid,in the literature8-10. Raw traces from an example cell are

nicotine. Neuronal nAChR are pentameric and functional

shown in Fig. 1A. The concentration response curve forchannels are formed from a repertoire of nine α (α2 tonicotine is shown in Fig. 1C which reveals an EC50 = 70α10) and three β subunits (β2 to β4). Most nAChR exist± 14 µM (n = 3), in good agreement with the literature .

as heteromers with the stoichiometry 2α to 3β, howeverAsome α subunits function as homomers, these being α77-9AChor α9 (for reviews see Refs. 1 & 2). nAChR have been

1 µM ACh
30 µM
100 µM
300 µM
1 mM
3 mM

proposed to play a role in many neurological disorders
such as Alzheimer’s Disease, Parkinson’s, schizophrenia
and depression1-4. nAChα7R are widely distributed in
the mammalian brain including in the cerebral cortex,
andcould be a potential therapeutic target in cognitive
disorders such as Alzheimer’s Disease or schizophrenia3,5,6.
Here we present data collected on a ­Patchliner

B
1.0
0.8
0.6

0.2

record fast desensitizing nAChα7R currents activated by

0.0

thepotential­

acetylcholine (ACh) or

useofthePatchliner

nicotine. ACh and nicotine

activated nAChα7R in a concentration-dependent
manner with an EC50 similar to those reported in theliterature7-10. nAChα7R could be repetitively activatedby nicotine and were enhanced by the Type I positiveallosteric modulator (PAM), NS1738, with an EC50 in good
agreement with the literature11.

Download more Application Notes from www.nanion.deC

0.4

toshowing

50 ms
Acetylcholine
EC50 = 304 ± 42 µM (n = 11)

I/Imaxevidence that nAChα7R play a role in cognition

5,60.2 nA

I/Imaxhippocampus, basal ganglia and cerebellum . There is
4

1.0 Nicotine
EC50 = 70 ± 14 µM (n = 3)
0.8
0.6
0.4
0.2

1 µM

10 µM 100 µM 1 mM
[ACh]0.01 µM

10 µM
100 µM 1 mM
[Nicotine]

Figure 1: Activation of nAChα7R by increasing concentrations of AChor nicotine. A Raw traces from an example cell showing activation ofnAChα7R by increasing concentrations of ACh. B Concentration response
curve for ACh activation for an average of 11 cells, EC50 = 304 ± 42 µM (n= 11). C Average concentration response curve for nicotine activation,
EC50 = 70 ± 14 µM (n = 3).

Application NoteFigure 2 shows the repetitive activation of nAChα7R.

B 1.0

NS1738300 µM AChCurrents were activated with a similar peak amplitude

0 µM NS17380.1 µM NS1738
1 µM NS1738
3 µM NS1738
10 µM NS1738

when challenged 8 times with 100 µM nicotine.

ANS1738 (n = 4)
EC50 = 2.6 ± 1.1 µM

0.80.2 nA
100 ms

I/ImaxA0.6
0.4

0.2
0.0

10 µM
100 nM[NS1738]
Figure 3: Enhancement of nAChα7R by NS1738. NS1738 at increasing
concentrations (0.1 µM - 10 µM) was pre-incubated and then co-appliedwith 300 µM ACh. NS1738 enhanced the peak of the ACh response ata concentration of 1 µM or above. B Concentration response curve forNS1738 enhancement, EC50 = 2.6 ± 1.1 µM (n = 4).
1 nM

Imax (nA)B

200 pA5s

0.0-0.2-0.4-0.6-0.8-1.0200 pA

incubated and then co-applied with acetylcholine

5 ms(Fig. 3). A full concentration response curve to NS1738
1237

4
56
Sweep number

8Figure 2: A Nicotinic AChα7R could be repetitively activated by 100µM nicotine. Inset shows nAChα7R activation of the first application
expanded B Timecourse of the experiment. Nicotinic AChα7R currents ofapprox. 1 nA were activated 8 times reproducibly in the same cell.

NS1738 is a Type I PAM of nAChα7R which increases
amplitude

without

desensitization11.

Itanyhasprofound

been

shown

effects
toon

producecognitive enhancement in rats. We show that NS1738
enhanced nAChα7R-mediated responses when pre-References

was performed revealing an EC50 of 2.6 ± 1.1 µM(n = 4), in excellent agreement with the literature11.

In summary, nAChα7R stably expressed in HEK293
cells can be reliably activated by nicotine or AChand enhanced by the Type I PAM, NS1738, with EC50
values in good agreement with published literature

7-11.

Therefore, the Patchliner provides a viable, higher
throughput alternative to conventional patch clamp
for the discovery of agonists of nAChα7R as potential
therapeutics to enhance cognitive function.
Cell culture

1. Colquhoun et al., in Burger’s Medicinal Chemistry, Vol II
Drug Discovery and Drug Development, Chapter 11, pp357-405. Ed. Abraham, D., J. Wiley, New York.
2. Albuquerque et al., 2009. Physiol Rev. 89: 73–120

Cells were cultured and harvested according to Nanion‘s standard cell culture protocol.
Electrophysiology

3. Araki et al., 2002. Jpn. J. Pharmacol. 88: 133 – 138

Whole cell patch clamp recordings were conducted

4. Hurst et al., 2013. Pharmacol. & Therapeut. 137: 22–54

according to Nanion’s standard procedure for the

5. Leiser et al., 2009. Pharmacol. & Therapeut. 122: 302–Patchliner. Cells were held at a holding potential of311-80 mV. To achieve short exposure times, solutions were

6. Pohanka, M. 2012. Int. J. Mol. Sci. 13: 2219-2238

stacked in the robotic pipettor. First, wash solution (280 µl)

7. Fu et al., 2008. Am. J. Resp. Cell & Mol. Biol. 41: 94-99

was aspirated followed by aspiration of the agonist-

8. Corradi & Bouzat. 2016. Mol. Pharmacol. 90:288–299

containing solution (30 µl) and then rapidly applied to the

9. Virginio et al., 2002. Eur. J. Pharmacol. 445: 153–161

cell at a speed of 171 µl/s. Wash solutions contained the10. Andersen et al., 2013. PNAS. 110 (51): 20819–20824

PAM in the NS1738 experiments. The cells were pre-11. Timmermann et al., 2007. JPET 323: 294–307

incubated in NS1738 before co-application with 300 µM
ACh.

Methods
Cells
An inducible HEK293 cells stably expressing humannAChα7R were used.
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