Characterization of ion channel currents endogenously expressed in
Neuro2A cells using automated patch clamp
Alison Obergrussberger1, Maria Giustina Rotordam1, Francesc X. Sureda2, Nadine Becker1, András Horváth1, Niels
Fertig1
1Nanion Technologies GmbH, Munich, Germany; 2 Pharmacology Unit, Department of Basic Medical Sciences, Universitat Rovira i Virgili, Reus, Spain

Introduction

A

Neuro2A cells are a mouse neuroblastoma cell line used extensively
to investigate neuronal differentiation, axonal growth and cellsignalling pathways . They are also used as an expression system forstudying ion channels. Neuro2A cells have been shown toendogenously express NaV channels, predominantly NaV1.7 but also
NaV1.2, 1.3 and 1.4 (Lou et al., 2005), mechanosensitive channels
Piezo 1 (Coste et al., 2010; Rotordam et al., 2019), purinergic
receptors (Vetter & Lewis, 2010) and glutamate receptors (Van derValk & Vijverberg, 1990). In this study, we used the medium and highthroughput automated patch clamp devices, Patchliner andSyncroPatch 384, to record different ion channels endogenously
expressed in Neuro2A cells.

NaV recorded from Neuro2A is TTX sensitive
B

NaV pharmacology: Tetracaine and LidocaineCDPatchliner Dynamite8
4 to 8 recording channels

SyncroPatch 384
384 recording channels

Piezo1 activated by Yoda1 recorded on theSyncroPatch 384
AFigure 1: A voltage-gated sodium current was recorded from Neuro2A cells. A We calculated a Vhalf of activation of 19.4 ± 0.6 mV (n = 8 cells) using a Boltzmann equation. B The NaV current was completely blocked by nM
concentrations of TTX with an IC50 or 4.8 ± 0.5 nM (n = 9 cells). Multiple concentrations of TTX were added to each wellof the NPC-16 chip of the Patchliner and complete concentration response curves were constructed from 9 individualcells. C & D The NaV current was also blocked by Tetracaine (C) and Lidocaine (D) with an IC50 of 11.2 ± 2.0 µM(n = 12) and 654 ± 58 µM (n = 12), respectively. Holding potential was -100 mV.

Heat-activated response is mediated by TRPV3 and TRPV4

BFigure 3: Piezo1 channels endogenously expressed in Neuro2A cells wererecorded on the SyncroPatch 384. A Screenshot of PatchControl 384 software
during an experiment. The wells are colour-coordinated based on seal
resistance (green wells have RSeal >500 MΩ, blue wells have RSeal 5 – 500 MΩ).
Sixteen wells are shown enlarged on the right and one well is highlighted at thebottom of the screenshot. The black trace is control and red trace in thepresence of 10 µM Yoda1. B Statistical analysis of the currents at -100mV (left)
and at 80 mV (right). 140 out of 384 Neuro2A cells (37%) passed the quality
criteria and 85 cells (60% of the valid cells) were considered as Yoda1responders.
Data from Rotordam et al, 2019.

References

Figure 2: TRP channels are expressed in Neuro2A cells. External solution was heated in the pipette of the Patchliner andrapidly applied to the cells. A heat-activated response was seen in 8/8 cells at 42°C. Following this, different TRP
channel activators were applied. No activation was seen with capsaicin in 8/8 cells, whereas all cells (8/8) respondedto GSK1016790A and 4/8 cells responded to 2-APB. Shown are raw current traces from an example cell (left) and thetimecourse of the experiment (right). This indicates that TRPV4 and in some cells, TRPV3, is expressed in Neuro2A cells
but not TRPV1.

Conclusions• Neuro2A cells can be used on automated patch clamp devices with success rates of 60-80% for >1
GΩ seals

Coste
B, MathurJ, Schmidt
M, Earley
TJ, Ranade
S, PetrusS,MJ,
Dubin
AEDubin& Patapoutian
Coste
B, MathurJ, Schmidt
M, Earley
TJ, RanadePetrus
MJ,AE & A(2010).
Piezo1 andessential
distinct components
mechanically activatedPatapoutian
A Piezo2(2010).arePiezo1andcomponents
Piezo2 are ofessentialof distinct
cationchannels. Science(80- cation) 330, 55–60.
mechanicallyactivatedchannels. Science (80- ) 330, 55–60.

• Voltage-gated Na currents were recorded with a Vhalf of activation of -19.4 ± 0.6 mV and blocked
by nM concentrations of TTX

Lou
JY,JY,Laezza
F, Gerber
BR, Xiao
YamadaKA, Hartmann
H, Craig AM,NerbonneLou
LaezzaF, Gerber
BR, M,Xiao
M, YamadaKA, Hartmann
H, Craig
AM, JM &Ornitz DM (2005). Fibroblast growth factor 14 is an intracellular modulator of voltage-gatedNerbonne JM & Ornitz DM (2005). Fibroblast growth factor 14 is an intracellularsodium channels. J Physiol 569, 179–193.

• The NaV current was also blocked by lidocaine and tetracaine at µM concentrations

Rotordam MG, Fermo E, Becker N, Barcellini W, Brüggemann A, Fertig N, Egée S, Rapedius M,
Rotordam
Fermo
E, Becker
N, gain-of-function
Barcellini W, BrüggemannA, Fertig
N, Egée S,Bianchi P & MG,KaestnerL (2019).
A novelmutation of Piezo1
is functionally
Rapedius
M,bloodBianchi& high-throughput
Kaestner L (2019).
A novelgain-of-function
mutationaffirmedin redcellsPbypatchclamp.
Haematologica 104,e179.

• A heat activated response was measured after application of external solution at 42°C. This wasmediated by TRPV4 or TRPV3 but not TRPV1

Van der Valk JBF & Vijverberg HPM (1990). Glutamate-induced inward current in a clonal
clamp. Haematologica
e179. 185, 99–102.
neuroblastoma
cell line. Eur J104,Pharmacol• Piezo1 was recorded from Neuro2A cells on the SyncroPatch 384 following application of Yoda1

modulator of voltage-gated sodium channels. J Physiol 569, 179-193.

of Piezo1 is functionally affirmed in red blood cells by high-throughput patch

VanI derValkRJJBF& Vijverberg
HPM (1990).
Glutamate-induced
inwardcurrent in
Vetter& Lewis(2010).
Characterizationof endogenouscalcium responses
in neuronalcellclonal Pharmacol
neuroblastoma
cell line. Eur J Pharmacol 185, 99–102.
lines. aBiochem
79, 908–920.
Vetter I & Lewis RJ (2010). Characterization of endogenous calcium responses inneuronal cell lines. Biochem Pharmacol 79, 908–920.

• Neuro2A cells are a suitable cell type for investigating endogenous NaV currents, as well as Piezo1
and some TRP channels.