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

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