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 most variants typically altering multiple functional properties. At synapses, NMDARs encode presynaptic 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. To capture the dynamics of NMDAR encoding, we applied 10 glutamate pulses at 10 Hz to derive a charge integral. This encoding assay is advantageous since it incorporates multiple gating parameters - activation, deactivation, and desensitization - into a single value. We then integrated this encoding 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 allosteric modulation and has the potential to speed up future bench to bedside methods of investigating variants to determine patient treatment.