Pharmacological Characterization of theNMDA A-B-C by Automated Patch Clamp
Ilka Rinke1, Søren Friis2, Claudia Haarmann1, Alison Obergrussberger1, Michael George1, Andrea Brüggemann1,Niels Fertig1
1Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany, contact: Info@nanion.deNMDAR Kinetics on the SyncroPatch 384PE

Introduction
N-Methyl-D-Aspartate receptors (NMDAR) are one of the key players in basic
and complex excitatory neurotransmission. Seven subunits of NMDAR havebeen identiﬁed: GluN1, GluN2A-D and GluN3A-B1. Assembled as a tetramer1,
they consist of two NR1 subunits and either two NR2 subunits or a combinationof NR2 and NR3 subunits².
Unique properties of NMDAR are the activation of the channel bysimultaneous binding of glutamate and glycine, a voltage-dependent blockby magnesium ions, a relatively slow current kinetic and a predominantly
calcium carried conductance. The exceptional calcium entry through open
NMDARs triggers on the one hand a number of regulatory processes importantin development and synaptic plasticity processes of learning and memory3.
On the other hand NMDARs are proposed to play a pathophysiological role ina number of neurological disorders such as epilepsy and Alzheimer’s3. Given
the importance in the treatment of such neurological diseases, it is of greatinterest developing clinically relevant NMDAR antagonists that would block
excitotoxic NMDAR activation, without interfering with NMDAR functionneeded for normal synaptic transmission and plasticity.
This study focuses on the basic biophysical properties of different NMDAR
subtypes and the pharmacological relevance of modulation of NMDARs.
Among others, we show activation kinetics of GluN2A, GluN2B, GluN2C andGluN2D subunit containing NMDARs. Moreover, we present data from thetarget screen of the positive allosteric modulator CIQ and blockers whichwere recorded with high throughput. All experiments were performed on theSyncroPatch384PE, the only APC device, which is able to stack the solutions
inside a pipette and rapidly apply it to the cell, allowing for brief and accurate
solution exchange (<10ms) and exposure times (<200ms). NMDAR expressing
cell lines were kindly provided by Chantest.

0.01

0.03

0.1

0.3

NR2A

NR2BNR2C1.0

Cumulative dose response inhibition by the anestesia ketamine. Data werenormalized to maximum block and fitted with a standard Hill-equation. The IC50for (±)-ketamine block of NR1/NR2A was 2.45 ± 0.38 μM (n = 348).
no0 300 3 10 30 100 [µM]Mg2+
no Mg2+Mg2+ Mg2+

NR2D

GluN1/GluN2A, GluN2B, GluN2C and GluN2D expressed in HEK cells
simulaneously recorded on the SyncroPatch® 384PE. The experiment shows
current activation and decay kinetics in response to glutamate/glycine
repeated 3 times (with wash between applications) to show reproducibility ofthe signal. Holding potential was -80 mV. Note that multi-hole chips (4 holes perwell) were used for this experiment.

Positive Allosteric Modulation of NMDA A-B-C-D

Spermine (mM)

Flexible Experimental Design

control
0.01
0.3

0.03
1.0

0.1
control

NMDAR modulation by spermine (GluN2B), pregnenolone sulfate (GluN2A) and CIQ (GluN2C and GluN2D). Left graph shows raw data traces of GluN2B channels followingactivation first by glutamate and glycine (red cursor), followed by co-application with spermine (green cursor) at the concentrations indicated. Spermine potentiates andglutamate/glycine response. Currents in the presence of the individual PAM were normalized to the response generated by glutamate/glycine. Right graphs show dose response curves calculated across the plate. Each curve was fitted with a Hill equation and EC50s were calculated to be for pregnenolone sulfate 39.6 μM (n = 284), spermine
260 µM (n = 303), CIQ modulating GluN2C 4.33 µM (n = 169) and CIQ modulating GluN2D 5.48 µM (n = 149).

Single point and coincident activation experiment. Left: Recordings were performed in solution without Mg2+. The IC50 is within the range found in cumulative
dose response experiments. Right: Recordings under physiological Mg2+ block
conditions. Current response generated by activation with glutamate/glycine
triggered to a depolarizing pulse protocol to overcome the natural Mg2+ blockand to potentiate the open probability of the receptors. Currents were subsequently blocked by Ketamine.

SummaryThe data collected on the SyncroPatch 384 PE relaibly show the differences incurrent kinetics of NMDA receptors containing different subunit compositions.
GluN1/GluN2A containing receptors show a higher open probability and fasterglutamate deactivation kinetics than GluN1/GluN2B or GluN1/GluN2C and
GluN1/GluN2D receptors, two characteristics which have been previously
described in literature1,6.
Current responses could be shown to be highly reproducible when recorded on
the SyncroPatch 384PE providing the basis for accurate pharmacology. IC50 ofketamine and EC50s of the modulator experiments on all four GluN2 subunit
containing receptors were in agreement with the literature2,4,5.
Brief ligand application mimicking synaptic transmission and the ability to
precisely time the ligand addition to a voltage pulse mimicking membrane
depoalrisation make the SyncroPatch 384PE predestinated to record NMDA
receptors under physiological synaptical conditions.
In conclusion, the SyncroPatch 384 PE is highly suitable to study NMDA receptors
and their role in synaptic plasticity, which includes mechnisms underlying
learning and memory.
References
1
Traynelis, S.F., et al., 2010. Pharmacol. Rev. 62(3): 405-4962McGurk, J.F., et al., 1990. PNAS. 87: 9971-997
3Dingledine, R., et al., 1999. Pharmacol. Rev. 51(1): 7-61
4Liu, H-T., et al., 2001. Anesth. Analg. 92: 1173–81
5Kotermanski, S.E. & Johnson, J.W. 2009. J. Neurosci. 29(9): 2774 –2779
6Paoletti, P. 2011. Eur. J. Neurosci. 33, 1351–1365.