Expression and pharmacology of GluA2-containing AMPA receptors incell lines and stem cell-derived neurons
Alison Obergrussberger, Nina Brinkwirth, Ilka Rinke-Weiß, Nadine Becker, Tom A. Goetze, Sonja Stoelzle-Feix, Patrick
Mumm, Michael George, Andrea Brüggemann, Niels Fertig
Nanion Technologies, Ganghoferstr. 70A, Munich, Germany. Email: info@nanion.deIntroduction
The vast majority of excitatory neurotransmission is mediated by AMPA (α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors1. The functional
receptor exists as a tetramer, either homomers or heteromers, from a repertoireof 4 different subunits, GluA1 – GluA41. It is well known that glutamate is aneurotoxin and it is proposed that overactivation of ionotropic glutamate
receptors may underlie many neurodegenerative disorders such as ischemic
stroke, epilepsy, Parkinson’s and dementia2. Enhancement of AMPA receptor
activation by, for example, BDNF, has been proposed to have beneficial effectsof learning and memory and has potential therapeutic value in the treatment ofdepression, Huntington’s and Parkinson’s diseases1. We have used GluA2receptors expressed in HEK cells on 3 different automated patch clamp systems
recording from either 1, 8 or 384 wells simultaneously. GluA2 was activated byglutamate, inhibited by CNQX and potentiated by LY404187.

Voltage-gated ion channels expressedin hiPSC-derived neurons
iCell® GlutaNeurons wereused on the Patchliner.
Voltage-gatedpotassium
currents were recorded (A)using a 100 ms pre-pulse to-40 mV followed by 200 ms
voltage step increasing in10mV
increments
to+40 mV. The average IV plot
for 6 cells is shown (B).
Voltage-gated Na+ currents
were also recorded from theiCell® GlutaNeurons (C). The
average IV plot for 12 cells isshown in (D). A Boltzmann
equation revealed a Vhalf of-26 mV. The NaV wasblocked by TTX in the nMrange (E, F), IC50 = 12.7 ± 1.5
nM (n = 4) indicating a TTXsensitive channel. The IC50for TTX agrees well with theIC50 obtained for iCell®
Neurons7.

In addition to GluA2 expressed in HEK cells, we also recorded glutamatergicenriched cortical neurons derived from induced pluripotent stem cells on an
automated patch clamp platform. In these neurons we recorded various
voltage-gated channels. In addition, glutamate responses were recorded whichwere potentiated by LY404187.

Activation of GluA2 expressed in HEK cells

Port-a-Patch

Patchliner

SyncroPatch 384PE

GluA2 expressed in HEK cells
was recorded on the Port-aPatch(left),Patchliner(middle) and SyncroPatch
384PE (right) where 1, 8 or 384
wells are recorded in parallel,
respectively.
GluA2 was activated byglutamate in a concentrationdependent manner with EC50
similar across all 3 platformsand in agreement with therange found in the literature3,4(lower panel). Cumulative
concentration
response
curves were performed oneach cell. The data wasnormalized to the highest
concentration (1 mM) andthe concentration response
curves for all 3 platforms isshown here overlaid.
Cells

kindlyprovidedbySB

Cells kindly provided
Dynamics International.

Cellular

Activation of glutamate channels in iCell
GlutaNeurons
The presence of glutamate
receptors was investigatediniCell®
GlutaNeurons.
Although only a minimal
current could be observed
upon
applicationofglutamate, the response
was enhanced by theAMPA-specificPAM,
LY404187. This indicates thepresence of AMPA receptors
intheseneurons.
The
presence of other ligandgated ion channels, e.g.
NMDA was not yet fully
investigated.

Drug

Discovery.

Inhibition and potentiation of GluA2 expressed in
HEK cells

byConclusions

GluA2 expressed in HEK cells wasactivated by glutamate and inhibitedby CNQX on the Patchliner in aconcentration-dependent
manner.
Interestingly, the IC50 of CNQX wasdependent
ontheglutamate
concentration (A). A similar IC50 valuewasalso
obtained
using
theSyncroPatch 384PE (588 nM, n = 128)
using 100 µM glutamate, in good
agreement with the literature5.

• GluA2-mediated responses expressed in HEK cells could be reliably recordedon the Port-a-Patch, Patchliner and SyncroPatch384PE with similar EC50
values for glutamate.

The positive allosteric modulator(PAM), LY404187, was pre-incubatedandco-applied
with
100µM
glutamate. LY404187 enhanced theglutamate response with an EC50 of679 ± 57 nM (n = 5) on the Patchliner(B) and 443 nM (n = 367) on theSyncroPatch 384PE (not shown), ingood agreement with the literature6.

• AMPA receptors are expressed in iCell® GlutaNeurons (CDI), a response toglutamate was measured in the presence of LY404187.

• GluA2 expressed in HEK cells was blocked by CNQX and the IC50 wasdependent on glutamate concentration.
• GluA2 expressed in HEK cells was potentiated by the AMPA-specific PAM,
LY404187.
• Voltage-gated Na+ and K+ currents were recorded in glutamatergic corticalneurons derived from iPSCs, iCell® GlutaNeuronsReferences
1. Traynelis, S.F., et al. (2010) Pharmacol. Rev. 62: 405-496
2. Dingledine, R., et al. (1999) Pharmacol. Rev. 51: 7-61
3. Coquelle, T., et al. (2000) Neuroreport. 11(12): 2643- 2648
4. Zhang, W., et al. (2006) Biophys. J. 91: 1336–1346
5. Stein, E., et al. (1992) Mol. Pharm. 42: 864-871
6. Miu, P., et al. (2001) Neuropharm. 40: 976-983
7. Haythornthwaite et al. (2012) JBS. 17(9): 1264-1272