An integrated approach to evaluate the functional effects of disease-associated NMDA receptor variants

The NMDA receptor (NMDAR) is a ubiquitously expressed glutamate-gated ion channel that plays key roles in brain development and function. Not surprisingly, a variety of disease-associated variants have been identified in genes encoding NMDAR subunits. A critical first step to assess whether these variants contribute to their associated disorder is to characterize their effect on receptor function. However, the complexity of NMDAR function makes this challenging, with many variants typically altering multiple functional properties. At synapses, NMDARs encode pre- and postsynaptic activity to carry a charge transfer that alters membrane excitability and a Ca²⁺ influx that has both short- and long-term signaling actions. Here, we characterized epilepsy-associated variants in GluN1 and GluN2A subunits with various phenotypic severity in HEK293 cells. To capture the complexity of NMDAR gating, we applied 10 glutamate pulses at 10 Hz to derive a charge integral. This assay is advantageous since it incorporates multiple gating parameters - activation, deactivation, and desensitization - into a single value. We then integrated this gating parameter with Mg²⁺ block and Ca²⁺ influx using fractional Ca²⁺ currents to generate indices of charge transfer and Ca²⁺ transfer over wide voltage ranges. This approach yields consolidated parameters that can be used as a reference to normalize channel block and allosteric modulation to better define potential patient treatment. This is especially true for variants in the transmembrane domain that affect not only receptor gating but also often Mg²⁺ block and Ca²⁺ permeation.