Deriving the Ca 2+ Integral as a Single Index of the Clinical Outcome of NMDA Receptor Disease‐Associated Variants

Abstract

Channelopathies are clinical disorders arising from alterations in ion channels. Genetic variants underlying these channelopathies potentially drive neurological disorders such as epilepsy. The NMDA receptor (NMDAR) is a ubiquitously expressed glutamate-gated ion channel that plays key roles in numerous brain functions. 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. Current characterization is not ideal since it often misses the complexity of ion channel function and the complex effects of variants, which rarely just affect one biophysical property. The central signaling mechanism of NMDARs is to regulate Ca2+ influx at synapses with the magnitude of this Ca2+ influx modulated by their diverse biophysical properties. Here, we characterized a set of epilepsy-associated variants in NMDAR subunits which have diverse effects on receptor function. To capture the dynamics of NMDARs at synapses, we applied 10 glutamate pulses at 10 Hz to derive a charge integral, which encompasses multiple gating parameters into a single value. We then developed a means to produce a single easily comparable index using the charge integral, Mg2+ block, and fractional calcium data. We hypothesize that this final parameter – the Ca2+ integral – will be correlated across clinical phenotype severity and provide a value for determining the contribution of variants to disease. This protocol has the potential to speed up future bench to bedside methods of investigating variants to determine patient treatment.