Ion Channels:

Application Note

hGluA2Cells:
HEK293 cells

Activation, potentiation and inhibition ofAMPA receptors on the Patchliner

Tools:Patchliner

Nanion Technologies GmbH, Munich

Summary
AMPA receptors are cation-permeable ionotropic glutamate

reported in the literature4,5. CNQX inhibited and LY404187

receptors of the non-NMDA receptor sub-family . To date fourenhanced GluA2-mediated responses.

1subunits, GluA1-4, have been identified1-3 which are of similar
size (approx. 900 kDa) and share 68-73% amino acid sequence
identity3. The human GluA2 subunit is encoded by the GRIA2
gene located on the 4q32-33 chromosome2. Each of the 4subunits has four distinct domains: an extracellular aminoacid terminal domain (ATD); the extracellular ligand binding
domain (LBD); the transmembrane domain (TMD) with 3transmembrane segments (M1, M3 and M4) and 1 cytoplasmic
facing re-entrant loop (M2); and an intracellular carboxyterminal domain1,2. The functional receptor exists as a tetramer,Results
hGluA2 expressed in HEK293 cells was activated using
increasing concentrations of glutamate (Figure 1). hGluA2
started to activate at a glutamate concentration of 10 µM.
At the highest concentration tested (3 mM) a rise time of 12.5
ms was estimated (Figure 1, inset). The EC50 for glutamate wascalculated to be 36.1 ± 6.4 µM (n = 4) in good agreement withthe range found in the literature for GluA24,5.

either as homomers or heteromers (GluA1 and GluA4)1.

The application speed of glutamate (100 µM) had a large in-The vast majority of excitatory fast synaptic transmission

fluence on activation and deactivation kinetics (Fig. 1C), at ain the mammalian central nervous system is mediated byslower speed of application, the initial fast peak was not ob-AMPA receptors of differing subunit combinations1. It is wellserved. NB: Multi-hole chips (4 holes per well) were used.

known that glutamate is a neurotoxin and it is proposedthat overactivation of ionotropic glutamate receptors may

hGluA2 expressed in HEK293 cells was reproducibly activatedunderlie many neurodegenerative disorders such as ischemic(Figure 2A) using repetitive (at least 4 x) applications of a gluta-stroke, epilepsy, Parkinson’s and dementia, amongst others2.

mate concentration close to the EC50 (30 µM).

Enhancement of AMPA receptor activation by, for example,BDNF, has been proposed to have beneficial effects onlearning and memory and has potential therapeutic value
in the treatment of depression, Huntington’s and Parkinson’sAB

Glutamatediseases1.
Here we present data collected on an 8-channel Patchliner
showing recordings of GluA2-mediated currents. Glutamate
activated GluA2 receptors with an EC50 similar to those0.5 nA
500 msNormalized Integral

1.0010 µM
30 µM
100 µM
300 µM
1 mM
3 mM

0.8Glutamate (n = 4)
EC50 = 36.1 ± 6.4 µM
Hill = 1.1 ± 0.2

0.6
0.4
0.20.0C

1 µM

100 µM[Glutamate]

10 mM

12.5 ms

29 µl/s
57 µl/s
86 µl/s
114 µl/s

1 nA

Figure 1: A Example traces showing activationof GluA2 by Glutamate. A rise time of 12.5 ms
was calculated at 3 mM (inset). B Concentration
response curve for an average of n = 4 wells. C
Application speed greatly affects activation anddeactivation kinetics of GluA2.

5 ms
1 nA

500 msA0 µM

1st30 µM

2nd30 µM

3rd30 µM

Figure 2: A GluA2 could be repetitively activated

4th30 µM

at least 4 times in the cell with similar peak
amplitudes (currents shown on the same scale).
B GluA2 was inhibited by CNQX, the IC50 was

100 pA
2sB

CIC50 CNQX:
30 µM glutamate = 378 ± 51 nM (n = 3)
100 µM glutamate = 519 ± 89 nM (n = 4)300 µM glutamate = 1.9 ± 0.3 µM (n = 7)CNQX

Control
300 nM CNQX
1 µM CNQX
3 µM CNQX
20 µM CNQX
1 mM CNQX

Normalized block

0.80.60.4

1.0

100 µM glutamate

Normalized response

1.0200 pA
1s

0.20.0

1nM

100nM
10µM
[CNQX]

1mM

dependent on glutamate concentration. C GluA2
LY404187 / 100 µM glutamate
EC50 = 679 ± 57 nM (n = 5)
Hill = 0.95 ± 0.1

a concentration-dependent manner.

LY404187
100 µM glutamate
Control
100 nM LY404187
300 nM LY404187
1 µM LY404187
10 µM LY404187
30 µM LY404187

0.80.60.4

was potentiated by LY404187of GluA2 responses in

5 nA
1s0.20.0
10 nM

1 µM
[LY404187]

100 µM

The GluA2 response was blocked by CNQX in a concentration dependent manner with an IC50 within the range foundin the literature for GluA2-containing receptors6. The potencyof block was dependent on glutamate concentration (Figure3A). The positive allosteric modulator (PAM) LY404187 potentiated the glutamate response (100 µM glutamate) with an EC50of 679 ± 57 nM (n = 5) in good agreement with the literature forGluA27. The potency was not affected by glutamate concentration (data not shown).
In conclusion, fast exchange of the external solution on thePatchliner can be used to reliably activate GluA2 receptors.
Studies were undertaken to investigate modulators of GluA2
receptors, including inhibitors or PAMs. In this way, newcompounds with potential for treating, for example, epilepsy,
Parkinson’s and Huntington’s diseases can be identified.

Methods
Cells
HEK cells expressing hGluA2 were kindly provided by SB Drug
Discovery.
Electrophysiology measurements
Cells were cultured and harvested according to Nanion’s
standard cell culture protocol. Cells were induced to express
hGluA2 by incubation in 1 µg/ml tetracycline 18 - 24 hours
prior to experiments. Cells were resuspended in external
recording solution and stored in the CellHotel of the Patchliner
before being dispensed into each well of the NPC-16 chip.
Internal and external solution compositions are available uponrequest. Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for the Patchliner.
Cells were held at a holding potential of -80 mV. To achieve
short exposure times, solutions were stacked in the robotic

References
1. Traynelis, S.F., et al. (2010) Pharmacol. Rev. 62: 405-496
2. Dingledine, R., et al. (1999) Pharmacol. Rev. 51: 7-61

pipettor. First, wash solution was aspirated followed by 40 µlof the agonist-containing solution and then applied to thecell at a speed of 57 µl/s (or at the other speeds indicated).
Cells were pre-incubated in CNQX or LY404187 prior to coapplication with glutamate.

3. Hollmann, M., & Heinemann, S. (1994) Annu. Rev. Neurosci.
17: 31-108
4. Coquelle, T., et al. (2000) Neuroreport. 11(12): 2643-2648
5. Zhang, W., et al. (2006) Biophys. J. 91: 1336–1346
6. Stein, E., et al. (1992) Mol. Pharm. 42: 864-871
7. Miu, P., et al. (2001) Neuropharm. 40: 976-983

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