Application Note

Ion channel/Transporter: AMPA,GABAA, EAAT, GAT
Sample:Whole cells®
Cell type: iCell GlutaNeurons
Tools:SURFE2R N1, Patchliner,SyncroPatch 384PE

Electrophysiological recordings of LGICand AA transporters in iCell® GlutaNeurons
The electrophysiology team at Nanion Technologies GmbH, Munich.
iCell® GlutaNeurons kindly provided by FUJIFILM Cellular Dynamics, Inc.

Summary

Results

Human neurons derived from induced pluripotent stem

Tocells (hiPSCs) are becoming increasingly important forsensor

studying basic neuronal physiology and can provide

inserted into the device and perfused with a buffer

good models for studying neurological disorders. hiPSC-containing NaCl and glutamate. When the substrate isderived neurons provide a viable alternative to primarypresent, Na+ and K+ movement across the membranecells and animal models in the drug discovery industry

can be observed until an electrochemical equilibrium isfor finding novel therapeutics to treat seizure-related

reached. To generate Na+ and K+ gradients, necessaryand neurodegenerative disorders. iCell® GlutaNeurons

as a driving force, the sensor was flushed with KCl before

are glutamatergic-enriched cortical neurons derived

and after the glutamate transporter activation. The tracesfrom hiPSCs. Single cell gene transcription analysis hasand concentration response curve for glutamate for an

shown the presence of glutamate receptors: AMPA,

example sensor is shown in Figure 1, confirming the pres-kainate and NMDA, as well as glutamate and GABA

ence of a glutamate transporter in iCell® GlutaNeurons,

transporters1. Ionotropic glutamate receptors mediate thealthough the exact identity was not investigated further.

activate
withtransporton

theattachediCell®

SURFE2R

N1,GlutaNeurons

awasmajority of excitatory neurotransmission in the mammalian
CNS and removal of glutamate from the synaptic cleft by1.0

reuptake via glutamate transporters plays a role ininhibitory neurotransmitter in the brain and is importantin controlling excitability. After release, GABA is removed
from the extracellular space by GABA transporters(GATs), thus terminating inhibitory synaptic transmission.
Both GABA and glutamate transporters may provide
novel therapeutic targets for, e.g. Parkinson’s disease2,Alzheimer’s disease3, and epilepsy4.
Werecordedligand-gatedionchannel

currents

mediated by GABAA and AMPA receptors from iCell®
GlutaNeurons on the Patchliner and SyncroPatch 384PE.

Furthermore, we could measure GABA and glutamate
transporters in these neurons using the SURFE2R N1 device.

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Normalized Ipeak

regulating neuronal excitability. GABA is the major

Glutamate
EC50 = 0.392 mM

0.8
0.6
Control
0.1 mM Glutamate
0.5 mM Glutamate
1 mM Glutamate

0.4
0.2

0.1 nA0.00.01

0.1150 ms
10[Glutamate (mM)]
Figure 1: Glutamate transport was activated in iCell® GlutaNeurons
upon addition of glutamate. Peak amplitude increased with increasing
glutamate concentration with an EC50 of 0.391 mM. Traces recorded fromthe cells on the sensor of the SURFE2R N1 are also shown.

Application Note
GATs transport GABA in exchange for Na+ and Cl- withthe proposed stoichiometry 2 Na+: 1Cl-: 1 GABA2. Using
the SURFE2R N1, GABA transport could be observed in theABLY404187
100 µM glutamate

presence of Na+ and Cl- upon addition of GABA (Figure2), confirming the presence of a GABA transporter iniCell® GlutaNeurons although the exact identity of theGAT was not further investigated.

0 µM LY404187
100 µM LY404187

Control
10 mM GABA

Control (no ligand)
100 µM GABA

100 pA

100 pA1s

1s

100 pA
100 msFigure 2: A Addition of 10 mM GABA to sensors with attached iCell®
GlutaNeurons resulted in the activation of a current mediated by a GABA
transporter.

Figure 3: A In the presence of the AMPA-specific PAM, LY404187, a responseto glutamate in iCell® GlutaNeurons recorded on the Patchliner was seen.
B GABAA receptor mediated response was recorded on the SyncroPatch384PE

GABA induced an inward current when applied to iCell®
GlutaNeurons on the SyncroPatch 384PE mediated by
the GABAA receptor (Figure 3B).

iCell® GlutaNeurons were also used on the medium andIn conclusion, iCell® GlutaNeurons can be successfullyhigh throughput patch clamp instruments, the Patchliner

used on the SURFE2R N1 to measure transporter activity,

and SyncroPatch 384PE. Figure 3A shows the current

and on the Patchliner and SyncroPatch 384PE to measure

response of an iCell® GlutaNeuron to glutamate in thewhole cell currents using the patch clamp technique forabsence and presence of the AMPA specific positive

drug discovery and ion channel/transporter research.

allosteric modulator (PAM) LY404187 on the Patchliner.

References

For glutamate experiments, cells were held at -80 mV,
pre-incubated in LY404187 and this was then co-applied

1. Chen, Y. 2018. In: Neurotoxins. DOI: 10.5772/intechopen.77043
2. Jin, X-T. et al 2011. Frontiers in Systems Neuroscience. 5:Article
63
3. Fuhrer, T.E., et al. 2017. Neuroscience. 351: 108-118

with glutamate. For GABA experiments, cells were held at-80 mV and GABA was applied for ~5 s.
SURFE2R sensor preparation

4. Björn-Yoshimoto, W. & Underhill, S.M. 2016. Neurochem Int. 98:

According to the Nanion standard procedure “SURFE2R

4–18

Sensor Preparation”. Whole cells were used.

Methods

SURFE2R N1 measurement workflow

Cell Culture

Glutamate and GABA transport are activated byiCell® GlutaNeurons were kindly provided by FUJIFILM
Cellular Dynamics, Inc. (Catalog # R1061 or R1034) andwere cultured and harvested according to Nanion’s
standard procedures for hiPSCs.

providing the respective substrate.

In both cases a

sodium gradient is established prior to substrate addition,and for GAT experiments, an additional chloride gradientis established. Therefore, any 3-buffer Nanion standard
protocol is suitable. Buffers are HEPES based including
NaCl with KCl for EAAT and K-Gluconate for GAT as theElectrophysiology

main salt.

iCell® GlutaNeurons were used on the Patchliner andPlease ask us for further experimental details and for

SyncroPatch 384PE using Nanion’s standard protocols.

more information about buffers and recording solutions.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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