Application Note

Channel:INa, IK, GABA
Cells: iPS cell-derived neurons
Tools:Patchliner®

Stem cell derived neurons recorded onNanion´s Patchliner®
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults
iCell®

Cellular Dynamics International is developing

Voltage-gated Na+ currents of an individual cell are

Neurons, human iPS cell-derived neurons. These neurons

shown in Figure 1. Not every cell showed expression of ahave been used on Nanion’s Patchliner®, an automatedNa+ current and in this example the current is quite large.

patch clamp device for recording from up to 8 cells

Typically, when observed, the maximum peak amplitude

simultaneously. Cells were thawed and cultured as per thewas between -100 and -500 pA. A full concentration re-manufacturer’s instructions. After plating, cells developed

sponse curve to TTX was performed on this cell revealing

neuronal outgrowth within 24 hours and could be kept

an IC50 for TTX of 5 nM (Figure 2). The voltage-gated Na+

in culture for 2 weeks. An investigation into voltage- andligand-gated ion channels expressed in these cells wascurrent is TTX sensitive although the exact Na+ channel
subtype present was not yet investigated further.

®. The first experiments areundertaken using the PatchlinerABcellular model for neurons and an increased throughput
patch clamp device.

Control
1 nM TTX
10 nM TTX
100 nM TTX
1µM TTX

B

I/ImaxA

1.5

0.5 nA

1.05 ms

0.5Voltage (mV)
1 nA-80-40-0.5

405 msFigure 1:
A Voltage-gated Na+ current recorded in iCell® Neurons. Current
responses to a voltage step protocol. A large inward Na+ current can beseen in this cell with a K+ outward current present at positive voltages. B
Normalised IV plot from an average of 4 cells.

Download more Application Notes from www.nanion.de

1.0

Normalised blockshown here and offer promising results for combining a0.8

TTX (n=1)
Max=1, IC50 = 4.9 nM

0.6
0.4
0.2
0.0

10 pM

1 nM
[TTX]

100 nM

Figure 2:
A Block of Na+ current by increasing concentrations of TTX. Current
was blocked by low nM concentrations of TTX indicating a TTX
sensitive Na+ channel type expressed in this cell. B Concentration response
curve for TTX inhibition, IC50 = 4.9 nM (n =1).

Application Note
An outward K+ current was also seen in some cells. An
example of the K+ current recorded is shown in Figure 3.

Bicuculline
GABA

The current could be blocked by approximately 50% by
30 µM GABA
1 µM Bicuculline(+ 30 µM GABA)

B

1.0
0.8
0.6

I max (nA)

1 mM TEA (data not shown).
A

0.4 nA
500 ms0.4
0.2

0.4 nA-4050 ms

040
Voltage (mV)

80Figure 3:
A Voltage-gated K+ current recorded in iCell® neurons. Current responses
to a voltage step protocol. An outward K+ current can be seen in this cell.
B Corresponding IV plot from an average of 7 cells.

To investigate the expression of ligand-gated ion channels
in iCell® Neurons, a single concentration of GABA (30 µM)
was applied to the cells for ~500 ms (Figure 4). The current
could be blocked by ~50% by 1 µM bicuculline.

Methods

Figure 4:
Activation of GABA currents by 30 µM GABA and partial block of theGABA response by 1 µM bicuculline. Bicuculline was pre-applied for atleast 30 s before co-application with GABA (30 µM). Approximately 50% of
the current was blocked by 1 µM bicuculline.

In summary, iCell® Neurons from Cellular Dynamics
International can be successfully used on the Patchliner®
to combine a cellular neuronal model with higher
throughput automated electrophysiology. Such a cell
model provides an alternative to primary neuronal cell
cultures for studying neuronal toxicity, disease researchand drug discovery.

increasing in 10 mV increments with each sweep up to60 mV. For TTX application, cells were stepped fromCells-100 mV to -20 mV for 10 ms repeated every 5 s. For K+

Human iPS cell-derived neurons (iCell®

Neurons) fromcurrents, cell were stepped from a holding potential of

Cellular Dynamics International were used.

-80 mV to -60 mV for 200ms and then increasing in 20 mV

Cell culture

increments with each sweep up to 60 mV. For GABA

Cells were received as frozen aliquots and were plated
and cultured according to the manufacturer’s instructions. Cells were harvested using a combination of trypsinand TrypLE.

experiments, cells were held at a constant holding
potential of -70 mV and GABA was applied for ~500 ms
using a stacked solutions protocol. Bicuculline was preincubated for at least 30 s before co-application with
30 µM GABA.

Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for thePatchliner®. For Na+ currents, cells were stepped from aholding potential of -100 mV to -80 mV for 20 ms and thenNanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax80636 Munich, Germany

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