Abstract
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.