Development and validation of NMDA ligand-gated ion channel assays using the Qube 384 automated electrophysiology platform

Ligand-gated ion channels are of particular interest to the pharmaceutical industry for the treatment of diseases from a variety of therapeutic areas including CNS disorders, respiratory disease and chronic pain. Ligand-gated ion channels have historically been investigated using fluorescence-based and low throughput patch-clamp techniques. However, the development of the Qube 384 automated patch-clamp system has allowed rapid exchange of liquid and direct measurement of ion channel currents on a millisecond timescale, making it possible to run HTS campaigns and support SAR with a functional readout.

Here, we have used the Qube platform to develop an assay against the NR1/NR2A receptor, which is part of the N-methyl-D-aspartate (NMDA) glutamate receptor family. For this assay, we utilized stacked liquid addition which enabled us to assess the open state kinetics of the channel and to investigate the effects of antagonists with multiple modes of actions.

We observed stable agonist responses for both NMDA and glycine, with EC₅₀ values comparable to literature values. The assay format was stable over multiple agonist applications and wash periods, which meant it was suitable for compound testing and sensitive enough to detect antagonists with multiple modes of action. D-AP-5, a competitive antagonist, showed stable NR1/NR2A receptor over six consecutive agonist/antagonist applications. The potency of ketamine, a use-dependent inhibitor, increased over the six agonist/antagonist applications.

By utilizing of stacked liquid additions in a 384 automated electrophysiology platform we have created an assay against NMDA receptors which is suitable for compound testing and sensitive enough to detect different modes of actions.