Application Note

Channel:Cells:
Tools:NMDA (NR1/NR2B)
HEK293
SyncroPatch 384PE

Activation and Inhibition of human NMDA
Channels on Nanion’s SyncroPatch 384PE
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Chantest, a Charles River Company.

Summary

Results

N-Methyl-D-aspartate (NMDA) receptors are a member

For the evaluation of the performance of HEK-NR1/of the ionotropic glutamate receptor family, ligand-NR2B cells, seal resistance was determined from one

gated ion channels that mediate the majority ofexperiment (Fig. 1) using single-hole chips. At the start ofexcitatory neurotransmission in the mammalian CNS.

the experiment, 66% of cells had a seal resistance (RSeal)

They are expressed primarily in the CNS but also in> 1 GΩ, with a further 26% with a RSeal > 500 MΩ giving aperipheral locations such as pancreatic islet cells,success rate of 92% for cells with RSeal > 500 MΩ. This was

1sensory nerve terminals in skin and cardiac ganglia .

only marginally reduced by the end of the experiment

Seven subunits of the NMDA receptor have been(89% of cells had RSeal > 500 MΩ, experiment time ~ 20

identified, NR1, NR2A-D and NR3A-B2, they assemble asmin).

a tetramer consisting of two NR1 subunits and either two
270

NR2 subunits or a combination of NR2 and NR3 subunits3.

60

Activation of NMDA receptors requires the simultaneous

50

neuropathic pain, major depression and Parkinson’s
disease .
2Herewepresent

highquality

data

ata

high

throughput collected on the SyncroPatch 384PE showing
activation and inhibition of NMDA NR1/NR2B expressedin HEK cells. Stable recordings of NMDA receptor wereachieved and modulation of the response by spermine
and ketamine is shown.

Download more Application Notes from www.nanion.dehm
OOGG>1000050

antagonists may also be targets for the treatment of

10

treatment of moderate to severe Alzheimer’s2. NMDA

<1NMDA antagonist which has been approved for theMO

epilepsy and Alzheimer’s1. Indeed, memantine is anhm

0-1

play a role in a number of neurological diseases such as

10hm

such as learning and memory1. It is also proposed to20hm

plasticity and is proposed to underlie higher processes

30

OsynapticOandM

developmentMin-500

role

40hm

important% Cells

binding of glutamate and glycine3.
Calcium entry through NMDA receptors plays anstart of experimentend of experiment

Seal Resistance

Figure 1: Statistics of NR1/NR2B expressing HEK293 cells recorded on one
NPC-384 chip on the SyncroPatch 384PE. Success rate (seal resistance) ofindividual HEK cells on the SyncroPatch 384PE. Shown is a bar graph ofseal resistances at the start (light blue) and end of the experiment (darkblue).

Application Note
Currents mediated by NR1/NR2B could be reliably
recorded on the SyncroPatch 384PE with a high successrate. Figure 2 shows a screenshot of the data acquisition
and analysis software of the SyncroPatch 384PE during anexperiment recording the current response of NR1/NR2B
to 3 applications of 3 µM glutamate plus 10 µM glycine.
In this experiment, multi-hole chips containing 4 holes perwell were used.

Figure 3: Positive allosteric modulation of NR1/NR2B by spermine. Currents
in the presence of the PAM were normalized to the response generated
with glutamate/glycine and the concentration response curve for spermine
calculated across the whole plate as shown in Figure 5. The curve was fittedwith a Hill equation and the EC50 calculated to be 134 µM (n = 290).

Ketamine is an NMDA receptor blocker used clinically as apediatric and veterinary anesthetic. It is also an important
tool in schizophrenia research as it is used to generate
Figure 2: Typical recording from NR1/NR2B expressed in HEK cells on theSyncroPatch 384PE. The screenshot shows the data acquisition and analysissoftware used on the SyncroPatch 384PE. Three hundred and eighty-four
small color-coded pictures shown in the upper left part from A1 to P24
display 384 recordings. Wells are color-coded based on seal resistance(green: Rmemb > 100 MΩ, blue: Rmemb = 25 - 100 MΩ, light blue or grey:
Rmemb < 25 MΩ or disabled). One highlighted experiment is displayed
at the bottom, 16 selected experiments are displayed on the right. On
the far right, the online analysis view showing peak current amplitude isprovided. Vertical lines and grey shaded areas show application of solution
containing 3 µM glutamate plus 10 µM glycine and white shows incubationin wash solution. The experiment shows current activation in response to
glutamate/glycine repeated 3 times (with wash between applications) toshow reproducibility of the signal. Holding potential was -80 mV. Note thatmulti-hole chips (4 holes per well) were used for this experiment.

NR1/NR2B-mediatedresponses

couldanimal models of the disease. (±)-Ketamine was coappliedwith glutamate and glycine and cumulative

concentration response curves were performed, i.e.
multiple concentrations applied to each cell. Figure 4
shows the concentration response curve for (±)-ketamine
for an average of 321 cells. Multi-hole chips were used (4holes per well). The IC50 was 2.26 ± 0.38 μM (n = 321) which
is within the range found in the literature6,7 although a direct comparison with the literature is difficult due toexperimental variables (e.g. Mg2+ concentration, glycineconcentration, racemic ketamine) which affect the IC50.

bereliably and reproducibly recorded on the SyncroPatch384PE

asshowninFigure2.

The

current responses to multiple applications of 3 µM
glutamate plus 10 µM glycine could be exactly overlaid.
Figure 3 shows the response curve to increasing
concentrations of spermine. Spermine has been shown
to potentiate NMDA responses3,4,5 but does not activate
NMDA currents alone3. Cells were activated by application of 10 µM glutamate/10 µM glycine followed by coapplication with different concentrations of spermine ina double-stacked application approach. The concentration response curve was calculated across the wholeplate and revealed an EC50 = 134 μM (n = 290). Multi-holechips (9 holes per well) were used for the experiments.

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Figure 4: Average concentration response curve for (±)-ketamine block of
NR1/NR2B. Cumulative concentration response curves were peformed oneach cell. The SyncroPatch 384PE analysis software (DataControl 384) wasused to calculate the average concentration response curve, normalized
to maximum block and fitted with a standard Hill-equation. The IC50 for(±)-ketamine block of NR1/NR2B was 2.26 ± 0.38 µM (n = 321), within therange found in the literature6,7.

Application Note

Figure 5: Graphical user interface of the screening and data analysis software used on the SyncroPatch 384PE. Screenshot of depiction of raw data traces of
NR1/NR2B-expressing HEK cells as recorded on one NPC-384 patch clamp chip. A single compound (in this case spermine) was co-applied with glutamate
and glycine to individual wells at varying concentrations (including control wells) across the plate. Multi-hole chips were used where 4 holes were presentper well. The data of the 384 well plate representation in the upper left part are color-coded for easy assessment of data. Depending on the seal resistance,
pictures are green (Rmemb > 100 MΩ), blue (Rmemb = 3.2 – 100 MΩ), light blue or grey (Rmemb < 3.2 MΩ or cells disabled). One highlighted experimentis displayed at the bottom, 16 selected experiments are displayed on the right. Graphs show raw data traces of NR1/NR2B channels following activation
first by glutamate and glycine (red cursor), followed by co-application with spermine (green cursor) at the concentrations indicated. Spermine potentiates
and glutamate/glycine response.

Exp end and chipunload

5 min

10 - 12 min

2 min
1 min

Chip loadand priming

Patch clamp experiment: 3 control applications(glutamate) plus 1 - 4 compound co-applications

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 - 4 point concentration response curve on NMDA-mediated currents took approximately 17-19 min.

Download more Application Notes from www.nanion.deApplication Note
Figure 5 shows a screenshot of the SyncroPatch 384

mediated response could be potentiated by spermine

software during an experiment. A color-coded over-in a concentration-dependent manner as expected3,4,5

view (based on seal resistance in this case) of all

and blocked by ketamine with an IC50 within the range

384 wells gives the user a good impression of thefound in the literature6,7.

success rate of the experiment. The user can easily
toggle between raw traces and online analysis. In theThe SyncroPatch 384PE is a high throughput and highly

example shown, raw traces are chosen and the graphs

reliable

automatedshow control responses to glutamate plus glycine

cording

NMDA

followed by potentiation of the response by co-friendly

software,application of spermine. An individual well can beadditions

highlighted to monitor progression of the experiment andeach cell and easy analysis result in reliable highis shown enlarged at the bottom of the screen.

quality data at an increased throughput with anorpatch

clamp

devicereceptor-mediatedexcellent

multipleofre-User-

rates,single

success

additionsforcurrents.

compoundto

economical cost per data point.
In conclusion, NMDA receptors with the subunit combination NR1/NR2B expressed in HEK cells can berecorded on the SyncroPatch 384PE with high successrates for completed experiments (typically >75%). The
timeline of each experiment was about 17-19 minutes(start – end) and included 3 control applications withglutmate plus glycine followed by co-application with
1 - 4 concentrations of a modulator.

The NR1/NR2B-Electrophysiology

References
1. Dingledine, R., et al., 1999. Pharmacol. Rev. 51(1): 7-61
2. Traynelis, S.F., et al., 2010. Pharmacol. Rev. 62(3): 405496
3. McGurk, J.F., et al., 1990. PNAS. 87: 9971-9974
4. Brackley, P., et al., 1990. Neurosci. Lett. 114: 51-56
5. Han, X., et al., 2008. J. Neurochem. 107: 1566–1577
6. Liu, H-T., et al., 2001. Anesth. Analg. 92: 1173–81
7. Kotermanski, S.E. & Johnson, J.W. 2009. J. Neurosci.
29(9): 2774 –2779

Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch 384PE using single- or multi-hole (4 or 9 holes
per well) chips. Cells were held at -80 mV for the durationof the experiment. For potentiator concentration response
curves, a double-stacked solutions approach was used
for the pipette. In this way, the cells were first activatedby glutamate/glycine and then spermine was coapplied. The peak amplitude in the presence of the PAM
was normalized to the current amplitude in glutamate/Methods

glycine alone. Single point concentration response curves
were performed and the concentration response curves

Cells

HEK293 cells expressing NR1/NR2B kindly provided byChanTest (a Charles River company).

calculated
experiments,

acrossthewhole(±)-ketamine

plate.

wasForinhibitor

co-appliedwithglutamate/glycine after 3 control applications without
inhibitor. Data was normalized to the maximum peak

Cell culture
Cells were cultured and harvested according toNanion’s standard cell culture protocol.

amplitude without inhibitor. Cumulative concentration
response curves were performed. For all experiments,
Mg2+-free external solution was used.

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fax80339 Munich, Germany

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