NMDA Receptor-Mediated Ca2+ Flux Attenuated by the NMDA Receptor/TRPM4 Interface Inhibitor Brophenexin

Transient receptor potential melastatin-4 (TRPM4) forms a complex with N-methyl-D-aspartate receptors (NMDARs) that facilitates NMDAR-mediated neurotoxicity. Here we used pharmacological tools to determine how TRPM4 regulates NMDAR signaling. Brophenexin, a compound that binds to TRPM4 at the NMDAR binding interface, protected hippocampal neurons in culture from NMDA-induced death, consistent with published work. Brophenexin (10 mu M) reduced NMDA-evoked whole-cell currents recorded at 22 degrees C by 87% +/- 14% with intracellular Ca2+ chelated to prevent TRPM4 activation. Brophenexin inhibited NMDA-evoked currents recorded in Na+-free solution by 87% +/- 13%, suggesting that brophenexin and TRPM4 modulate NMDAR function. Incubating cultures in Mg2+-free buffer containing tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, and bicuculline for 30 min inhibited NMDA-evoked increases in intracellular Ca2+ concentration ([Ca2+]i) recorded at 22 degrees C by 50% +/- 18% and prevented inhibition by brophenexin. In the absence of these inhibitors, brophenexin inhibited the NMDA-evoked response by 51% +/- 16%. Treatment with the TRPM4 inhibitor 4-chloro-2-(1-naphthyloxyacetamido)benzoic acid (NBA; 10 mu M) increased NMDA-evoked Ca2+ influx by 90% +/- 15%. Increasing extracellular NaCl to 237 mM, a treatment that activates TRPM4, inhibited the NMDA-evoked increase in [Ca2+]i by a process that occluded the inhibition produced by brophenexin and was prevented by NBA. In recordings performed at 32 degrees C-34 degrees C, brophenexin inhibited the NMDA-evoked [Ca2+]i response by 42% +/- 10% but NBA was without effect. These results are consistent with a model in which TRPM4 interacts with NMDARs to potentiate Ca2+ flux through the NMDAR ion channel and thus provides a potential mechanism for the neuroprotection afforded by NMDAR/TRPM4 interface inhibitors such as brophenexin.