Application Note

Channel:Cells:
Tools:

hGluA2
HEK293
SyncroPatch 384PE

Activation, potentiation and inhibition ofAMPA receptors on the SyncroPatch 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by SB Drug Discovery.

SummaryResults
ionotropic

hGluA2 expressed in HEK293 cells was activated using

glutamate receptors of the non-NMDA receptor sub-increasing concentrations of glutamate (Fig. 1). hGluA2

family1. To date four subunits, GluA1-4, have been iden-

started to activate at a glutamate concentration of 10 μM.

tified1-3 which are of similar size (approx. 900 kDa) andAt the highest concentration tested (1 mM) a rise timeshare 68-73% amino acid sequence identity . The humanof 34 ms was estimated (Fig. 1 inset). The EC50 for gluta-AMPA

receptors

arecation-permeable

3

GluA2 subunit is encoded by the GRIA2 gene located onmate was estimated to be 71.1± 2.1 μM (n = 222) in goodthe 4q32-33 chromosome . Each of the 4 subunits has four

agreement with the range found in the literature for

distinct domains: an extracellular amino acid terminal

GluA24,5 and with the value obtained for the Patchliner

domain (ATD); the extracellular ligand binding domain(see App Note).

(LBD); the transmembrane domain (TMD) with 3 transmembrane segments (M1, M3 and M4) and 1 cytoplasmic

ABGlutamate

facing re-entrant loop (M2); and an intracellular carboxy0 Glutamate
1 µM Glutamate
10 µM Glutamate
100 µM Glutamate
1 mM Glutamate

tetramer, either as homomers or heteromers (GluA1 andGluA4)1. The vast majority of excitatory fast synaptictransmission in the mammalian central nervous system ismediated by AMPA receptors of differing subunit
combinations1. It is well known that glutamate is a200 ms

C1 µM

10 µM

100 µM 1 mM0-100-200-300-40000.2 nA
Rise Time 34 ms

00.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6

AUC (pA/s)

terminal domain1,2. The functional receptor exists as a

Ipeak (nA)

2200 400 600 800 1000 1200
Time (s)neurotoxin and it is proposed that overactivation ofionotropic glutamate receptors may underlie manyneurodegenerative

disordersstroke,Parkinson’sepilepsy,

suchandasischemic
dementia,

amongst others2. Enhancement of AMPA receptor
activationby,forexample,BDNF,

hasbeenproposed to have beneficial effects on learning andmemory and has potential therapeutic value in the treatment of depression, Huntington’s and Parkinson’s diseases1.
Here we present data collected on the SyncroPatch
384PE showing recordings of GluA2-mediated currents.
Glutamate activated GluA2 receptors with an EC50 similar

to those reported in the literature4,5. CNQX inhibited andLY404187 enhanced GluA2-mediated responses.
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0.2 nA
10 ms

Figure 1: Activation of hGluA2 expressed in HEK293 cells on the SyncroPatch
384PE. A Glutamate was applied in increasing concentrations on eachcell for approximately 500 ms. A rise time of 34 ms was calculated at thehighest concentration of glutamate (1 mM; inset). B Online analysis values
Ipeak and area under the curve (AUC) versus time for well shown in A.
C A concentration response curve for an average of n = 222 wells wasconstructed revealing an EC50 = 71.1 ± 2.1 μM (Mean ± S.E.M of individual
EC50s). NB: Each well contained 8 holes.

Application NoteCurrents mediated by GluA2 could be reliably recorded

The GluA2 response was blocked by CNQX in a concentra-on the SyncroPatch 384PE. Figure 2 shows the current

tion-dependent manner with an IC50 within the range foundtraces and online analysis for a selection of 16 wells.

in the literature for GluA2-containing receptors6. The positive allosteric modulator, LY404187, potentiated the gluta-mate response in a concentration-dependent mannerwith an EC50 within the range found in the literature7.
LY404187 was pre-incubated prior to co-application withglutamate.

Normalized response

1.00.8CNQX
IC50 = 588 nM
Hill = 0.7
n = 128

100 µM Glutamate/0 µM CNQX
100 µM Glutamate/10 µM CNQX
Glutamate/CNQX

0.60.40.2

200 pA
200 ms0.0
10-9C0100 µM

100 µM

100 µM

100 µM

1 mM

0Ipeak (nA)

Normalized response

D-1400600Time (s)80010-2

1 nA
200 ms

105LY404187
EC50 = 443 nM
Hill = 1.2n = 367

0-320010-3

1 mM Glutamate
1 mM Glutamate/300 nM LY404187-2010-4151s
100 µM

10-610-5[CNQX (M)]1 mM

0.5 nA

100 µM

10-7Figure 3: Inhibition of GluA2 by CNQX. Currents in the presence of theinhibitor were normalized to control and the concentration response curve
for CNQX calculated across the plate (experiment was conducted over halfa plate). The curve was fitted with a Hill equation and the IC50 calculated tobe 588 nM (n = 128) in good agreement with the literature6.

20010-81000

Figure 2: Stability of GluA2 recorded on the SyncroPatch 384PE. A Raw
current traces from a selection of 16 cells showing current activation
by a concentration of glutamate repeated 3 times in the same cell.
Concentration is indicated. B Online analysis plots showing peak amplitude
versus time for the same 16 cells shown in A. A concentration response curve
was constructed across the whole plate. C Example traces in response to100 µM glutamate from one cell are shown. At the end of the experiment1 mM glutamate was added. D Online analysis plot of Ipeak versus time forthe cell shown in C. GluA2 was repetitively activated at least 3 times in thecell and the peak amplitude was highly reproducible.

10-9

10-8

10-7
10-6
[LY404187 (M)]

10-5

10-4

Figure 4: Positive allosteric modulation of GluA2 by LY404187. Currents inthe presence of the PAM were normalized to the response generated withglutamate and the concentration response curve for LY404187 calculated
across the whole plate The curve was fitted with a Hill equation and the EC50
calculated to be 443 nM (n = 367), within the range found in the literature7.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application NoteFigure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot of depiction of raw data traces ofGluA2-expressing HEK cells as recorded on one NPC-384 patch clamp chip. A single concentration for 5 different compounds was applied across the plate,with 4 columns receiving each compound and 4 columns receiving no compound as control. Compounds and concentrations are indicated. Compounds
were pre-incubated and then co-applied with 100 µM glutamate. Multi-hole chips were used where 8 holes were present per well. The data of the 384 wellplate representation in the upper left part are color-coded for easy assessment of data. Depending on the seal resistance, pictures are green (Rmemb >50MΩ) or blue (Rmemb = 5 – 50 MΩ). One highlighted experiment is displayed at the bottom, 16 selected experiments are displayed on the right. Graphs
show raw data traces of GluA2 channels following activation first by glutamate (blue trace) followed by co-application with compound (black trace).

Exp end and chipunload
15 - 20 min

5 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 1- 3 control applications(glutamate), 1 - 5 compound co-applications plus 1 max
dose (glutamate)

Analysis

Figure 6: Timeline of an experiment on the SyncroPatch 384PE. The completion of 1 experiment on the SyncroPatch 384 patch clamp chip (384 wells) for a
1 - 5 point concentration response curve on GluA2-mediated currents took approximately 22 - 27 min.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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

Application Note
Figure 5 shows a screenshot of the SyncroPatch 384

tive concentration response of glutamate. Finally, asoftware during an experiment. A color-coded over-max concentration of glutamate (2 mM) was typicallyview (based on seal resistance in this case) of allapplied). If required, compounds were pre-incubat-

384 wells gives the user a good impression of theed and then co-applied with glutamate. The GluA2-success rate of the experiment. The user can easily

mediated response was inhibited by CNQX in a concen-

toggle between raw traces and online analysis. In thetration-dependent manner as expected6 and potentiat-

example shown, raw traces are chosen and theed by LY404187 in a concentration-dependent mannergraphs show control responses to glutamate followedwith an EC50 within the range found in the literature7.

by potentiation or inhibition of the response by coapplication with the compound indicated. The ex-The SyncroPatch 384PE is a high throughput and highly

periment shows that a range of different compounds

reliable

automatedcan be tested within 1 plate. An individual well can becording

GluA2

highlighted to monitor progression of the experiment andfriendly

software,is shown enlarged at the bottom of the screen.

additionsorpatchclamp

devicereceptor-mediatedexcellent

multiple

success

additionsoffor

re-currents.

User-rates,

singlecompoundtoeach cell and easy analysis result in reliable highIn conclusion, GluA2 expressed in HEK cells can bequality data at an increased throughput with anreliably and reproducibly recorded on the SyncroPa-

economical cost per data point.

tch 384PE. The timeline of each experiment was about
22 - 27 minutes (start – end) and included 1 - 3 control
applications with glutamate followed by co-application
with 1 - 5 concentrations of a modulator or a cumula-

References

Electrophysiology

1. Traynelis, S.F., et al. (2010) Pharmacol. Rev. 62: 405-496

Whole cell patch clamp recordings were conducted

2. Dingledine, R., et al. (1999) Pharmacol. Rev. 51: 7-61

according to Nanion’s standard procedure for the

3. Hollmann, M., & Heinemann, S. (1994) Annu. Rev. Neu-SyncroPatch 384PE using multi-hole (8 or 9 holes per well)rosci. 17: 31-108

chips. Cells were held at -80 mV for the duration of the

4. Coquelle, T., et al. (2000) Neuroreport. 11(12): 2643-experiment. For pharmacology experiments, cells were2648

pre-incubated in compound and then compound was

5. Zhang, W., et al. (2006) Biophys. J. 91: 1336–1346

co-applied with glutamate. The peak amplitude or area

6. Stein, E., et al. (1992) Mol. Pharm. 42: 864-871

under the curve in the presence of the PAM or inhibitor

7. Miu, P., et al. (2001) Neuropharm. 40: 976-983

was normalized to the current amplitude in glutamate
alone. Single point concentration response curves wereMethods

performed and the concentration response curves

Cells

calculated across the whole plate. For glutamate

HEK293 cells expressing GluA2 kindly provided by SB Drugconcentration

Discovery.

concentrations were added to each well and the data

response

curves,cumulative

normalized to the response elicited with the highest
Cell culture

concentration.

Cells were cultured and harvested according toNanion’s standard cell culture protocol. Cells were induced to express hGluA2 by incubation in 1 µg/ml tetracycline 18 - 24 hours prior to experiments.

Nanion Technologies GmbHphone +
 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

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