Selective Reduction of Ca²⁺ Entry Through the Human NMDA Receptor: a Quantitative Study by Simultaneous Ca²⁺ and Na⁺...

Excessive Ca²⁺ influx through N-methyl-D-aspartate type glutamate receptors (NMDAR) is associated with excitotoxicity and neuronal death, but the inhibition of this receptor-channel causes severe adverse effects. Thus, a selective reduction of NMDA-mediated Ca²⁺ entry, leaving unaltered the Na⁺ current, could represent a valid neuroprotective strategy. We developed a new two-fluorophore approach to efficiently assess the Ca²⁺ permeability of ligand-gated ion channels, including NMDARs, in different conditions. This technique was able to discriminate differential Ca²⁺/Na⁺ permeation ratio through different receptor channels, and through the same channel in different conditions. With this method, we confirmed that EU1794-4, a negative allosteric modulator of NMDARs, decreased their Ca²⁺ permeability. Furthermore, we measured for the first time the fractional Ca²⁺ current (P_f, i.e. the percentage of the total current carried by Ca²⁺ ions) of human NMDARs in the presence of EU1794-4, exhibiting a 40% reduction in comparison to control conditions. EU1794-4 was also able to reduce NMDA-mediated Ca²⁺ entry in human neurons derived from induced pluripotent stem cells. This last effect was stronger in the absence of extracellular Mg²⁺, but still significant in its presence, supporting the hypothesis to use NMDA-selective allosteric modulators to lower Ca²⁺ influx in human neurons, to prevent Ca²⁺-dependent excitotoxicity and consequent neurodegeneration.