Selective Reduction of Ca 2+ Entry Through the Human NMDA Receptor: a Quantitative Study by Simultaneous Ca 2+ and Na + Imaging.

Excessive Ca 2+ 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 Ca 2+ entry, leaving unaltered the Na + current, could represent a valid neuroprotective strategy. We developed a new two-fluorophore approach to efficiently assess the Ca 2+ permeability of ligand-gated ion channels, including NMDARs, in different conditions. This technique was able to discriminate differential Ca 2+ /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 Ca 2+ permeability. Furthermore, we measured for the first time the fractional Ca 2+ current (P f , i.e. the percentage of the total current carried by Ca 2+ 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 Ca 2+ entry in human neurons derived from induced pluripotent stem cells. This last effect was stronger in the absence of extracellular Mg 2+ , but still significant in its presence, supporting the hypothesis to use NMDA-selective allosteric modulators to lower Ca 2+ influx in human neurons, to prevent Ca 2+ -dependent excitotoxicity and consequent neurodegeneration.