Interaction of Transient Receptor Potential Melastatin-7 Ion Channels with N-Methyl-D-Aspartate Receptor GluN2B Subunits and its Implication in Stroke

Ischemic stroke is one of the leading causes of neurological disability and mortality worldwide. It places a huge burden on the health care system. However, the pathological mechanisms of ischemic stroke are not fully understood and few effective therapies are currently available. We have previously reported two key signaling pathways involved in calcium-mediated oxidative stress during ischemic neuronal death. The first is the formation of nitric oxide, which is facilitated by the coupling of the ionotropic, N-methyl-D-aspartate glutamate receptors (NMDARs), to neuronal nitric oxide synthase (nNOS) by the intermediary postsynaptic density 95 protein (PSD-95). The second is the formation of reactive species mediated by the transient receptor potential cation channel, subfamily M, member 7 (TRPM7). Previously our lab has shown that another TRPM family member TRPM2 can influence NMDAR expression in neurons to modulate excitability and vulnerability to ischemic insults. Therefore in the current study we investigated whether TRPM7 and NMDARs, the two key signaling pathways of ischemic injury, also interact in neurons. Here we show that TRPM7 channels and NMDARs are not only expressed in the same neurons, but co-associate directly, largely through interactions of TRPM7 with GluN2B NMDAR subunits. This was demonstrated by membrane fractionation and co-immunoprecipitation (co-IP) analyses from rat brain tissue and from HEK293 cells that co-expressed TRPM7 and NMDARs, and then validated with bimolecular fluorescence complementation (BiFC) assays that demonstrated the preferential association of TRPM7 channels with GluN2B over GluN1 subunits of NMDARs. Furthermore, NMDAR and TRPM7 are also functionally interconnected because inhibiting TRPM7 channels also inhibited NMDA-mediated excitotoxicity in neurons. This is the first demonstration that TRPM7 and NMDAR channels, two key modulators of ischemic cell death, co-associate in the same protein complex in neurons and are capable of a direct interaction. These findings provide mechanistic insights into the cross talk between these important signaling pathways of anoxic cell death.Ph.D.2021-07-21 00:00:0