Application NoteChannel:
Cells:
Tools:nAChR α3β4HEK293
Patchliner

Nicotinic α3β4 receptors recorded onNanion´s Patchliner
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

The nicotinic acetylcholine receptor (nAChR) is a member

Current responses of an individual cell expressingof the ligand-gated ion channel superfamily which

α3β4 receptors to increasing concentrations of nico-includes GABAA, 5HT3, NMDA and glycine receptors. Ittine are shown in Figure 1. A concentration response

is a cation-permeable ion channel activated by thecurve revealed an EC50 for nicotine activation ofneurotransmitter acetylcholine and the natural alkaloid,

31 ± 2 µM (n = 11), in excellent agreement with the litera-nicotine. Neuronal nAChR are pentameric and functional

ture .

5,6channels are formed from a repertoire of nine α (α2 toα10) and three β subunits (β2 to β4). Most nAChR exist
as heteromers with the stoichiometry 2α to 3β, howeveror α9 (for reviews see Refs. 1 & 2). nAChR have been
proposed to play a role in many neurological disorderssuch as Alzheimer’s Disease, Parkinson’s, Tourette’s
Syndrome and depression. mRNA for the α3 and β4
subunits is found in the mammalian CNS and PNS, inparticular automomic ganglion cells and chromaffincells. nAChR containing the α3 subunit are essential for
mediating fast synaptic transmission in the autonomic
nervous system and is essential for survival3. Block of
nicotinic α3β4 receptors by methadone has also beensuggested to play a potential role in analgesia4.
Here we present data collected on a 4- or 8-channel
Patchliner showing the p­­ otential use of the Patchliner
to record nAChR α3β4 currents activated by nicotine.
Nicotine activated α3β4 receptors in a concentrationdependent manner with an EC50 similar to thosereported in the literature5,6. Nicotinic α3β4 receptors
could be repetitively activated by nicotine and blocked
by mecamylamine, a ganglionic blocker with clinically relevant hypotensive actions, with an IC50 in good
agreement with the literature4,5,7.

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1.0Nicotine
0 µM
1 µM
3 µM
10 µM
30 µM50 µM
100 µM
300 µM

1 nA
500 ms

0.8I/Imax

some α subunits function as homomers, these being α7

0.6
0.4
0.2
0.00.1

10[Nicotine (µM)]

Figure 1:
A Activation of nicotinic α3β4 receptors by increasing concentrationsof nicotine. B Concentration response curve for nicotine activation,
EC50 = 31 ± 2 µM (n = 11).

Application Note
Figure 2 shows the repetitive activation of nicotinic α3β4

Mecamylamine
50 µM Nicotine

receptors. Currents were activated with a similar peak

0 µM
0.1 µM
1 µM
10 µM
100 µMamplitude when challenged 10 times with 50 µM
nicotine.

A0.5 nA
500 ms

Ipeak (nA)B

0.5 nA0-1-2-3-45s

B

1.00.81-(I/Imax)

A

0.6
0.4
0.2
0.0
10.01
100
[Mecamylamine (µM)]

Figure 3:
Block of nicotinic α3β4 receptors by mecamylamine. Mecamylamine atincreasing concentrations (0.1 µM - 100 µM) was pre-incubated and thenco-applied with 50 µM nicotine. Some recovery from block was achieved
upon washout (approx. 20%; data not shown). B Concentration response
curve for mecamylamine block, IC50 = 2.1 ± 0.7 µM (n = 4), in good agreement with the literature4,5,7.

In summary, nicotinic α3β4 receptors stably expressed in246Sweep Count

810Figure 2:
A Nicotinic α3β4 receptors could be repetitively activated by 50 µMnicotine. B Timecourse of the experiment. Nicotinic α3β4 currents ofapprox. 4 nA were activated 10 times reproducibly in the same cell.

A full concentration response curve to mecamylamine
was performed (Fig. 3). Mecamylamine blocked thenicotine activated current with an IC50 of 2.1 ± 0.7 µM(n = 4), in good agreement with the literature4,5,7.

References

HEK293 cells can be reliably activated by nicotine
and blocked by the non-specific nAChR blocker,
mecamylamine. The data shown here agrees well
1-7

with published literature for nicotinic α3β4 receptors .
Therefore, the Patchliner provides a viable, higher
throughput alternative to conventional patch clamp forthe discovery of agonists and antagonists of nicotinic
α3β4 receptors as potential therapeutics for analgesia
and Tourette’s Syndrome, amongst others .

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., S
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. Xu et al., 1999. PNAS. 96: 5746-5751

Whole cell patch clamp recordings were conducted

4. Xiao et al., 2001. JPET. 299: 366-371

according to Nanion’s standard procedure for the

5. Xiao et al., 1998. Mol. Pharmacol. 54: 322-333

Patchliner. Cells were held at a holding potential of

6. Colquhoun & Patrick., 1997. J. Neurochem. 69: 2355--70 mV. To achieve short exposure times, solutions were2362

stacked in the robotic pipettor. First, wash solution (185 µl)

7. Papke et al., 2001. JPET. 297: 646-656

was aspirated followed by aspiration of the agonistcontaining solution (40 µl) and then rapidly applied to thecell at a speed of 171 µl/s. Wash solutions contained theMethods

antagonist in the mecamylamine experiments. The cells
were pre-incubated in mecamylamine before co-Cells

application with 50 µM nicotine.

HEK293 cells stably expressing nicotinic α3β4 were used.

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