Waking the sleeping beauty: A novel calcium-flux assay for the study of NMDA receptor
Fred Yeboah1, Hongqiu Guo1, L. Miguel Camargo1, , Mary Ellen Digan1, Honglin Niu1, Yue Pan1, Stephan Reiling1, Gilberto Soler-Llavina1, Wilhelm A. Weihofen1, Hao-Ran Wang1, Y. Gopi Shanker1,Travis Stams1, and Anke Bill*1
1Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139
*Corresponding author: email@example.com
N-methyl-D-aspartate-receptors (NMDARs) are ionotropic glutamate receptors that function in synaptic transmission, plasticity and cognition. Malfunction of NMDARs has been implicated in a variety of nervous system disorders, including schizophrenia, epilepsy and pain, making them attractive therapeutic targets. NMDARs require membrane depolarization as well as binding of glycine/D-serine and glutamate for their activation. Overexpression of functional receptor in non-neuronal cells results in cell death by excitotoxicity, hindering the development of flexible, robust, and high throughput cell-based assays for NMDAR drug discovery. Here we report a novel, plate-based, high-throughput approach to study NMDAR function that overcomes many limitations of previously available approaches.
Our assay enables the functional study of NMDARs with different subunit composition after activation by glycine/D-serine or glutamate and hence presents the first plate-based, high throughput assay that allows for the measurement of NMDAR function in glycine/D-serine and/or glutamate sensitive modes. Our approach leverages the use of weak antagonists to mitigate cellular toxicity and - after antagonist wash out - to free up glycine/D-serine and glutamate binding sites. This allows to assay the effect of small molecule modulators on the activation of NMDARs at different concentrations or combinations of the co-ligands. The reported assay system faithfully replicates the pharmacology of the receptor in response to known agonists, antagonists, positive and negative allosteric modulators, as well as the receptor's sensitivity to magnesium and zinc. We believe that the ability to study the biology of NMDARs rapidly and in large scale screens will enable the identification of novel therapeutics whose discovery has otherwise been hindered by the limitations of existing cell based approaches.