<p>Excitotoxic signaling mediated by <i>N</i>-methyl-D-aspartate receptors (NMDARs) is inhibited by NMDAR/TRPM4 complex inhibitors such as brophenexin (BPN). We used rat hippocampal neurons grown in culture to determine the effects of BPN on NMDAR and TRPM4 function. NMDA evoked concentration-dependent increases in intracellular Ca²⁺ that were inhibited by 10 µM BPN in a non-competitive manner. In contrast, the TRPM4 inhibitor 4-chloro-2-(2-(naphthalene-1-yloxy) acetamido) benzoic acid (NBA) increased the potency of NMDA at 22&#xa0;°C. BPN inhibition of NMDAR-mediated increases in Ca²⁺ was fully reversible and recovered by rapid (30&#xa0;s) and slow (90&#xa0;min) processes. The rapid phase of recovery from BPN inhibition was mediated by trafficking through recycling endosomes as indicated by blockade of this phase with bafilomycin A1 an agent that prevents endosomal acidification. The full recovery of NMDAR function observed 90&#xa0;min after washout of BPN was not affected by this treatment. Immunocytochemistry experiments suggested that BPN did not directly alter NMDAR trafficking but instead changes in surface and internal GluN2B immunoreactivity were likely homeostatic responses to inhibition of NMDAR function by BPN. In the presence of MK-801 to block NMDARs, 10 µM NBA inhibited spontaneous network-driven Ca²⁺ spiking by 74 ± 11% whereas 10 µM BPN reduced activity by 49 ± 6%. Thus, BPN inhibits TRPM4-dependent activity. In summary, BPN is a non-competitive, fully reversible inhibitor of NMDAR-mediated Ca²⁺ influx and produces a modest inhibition of TRPM4 function. Functional inhibition of these ion channels likely contributes to the neuroprotective properties of NMDAR/TRPM4 interface inhibitors.</p> Graphical Abstract <p></p>