Chen_2020_Neurobiol.Dis__104850

Mutations in synaptic NMDA receptors (NMDARs) are associated with epilepsy and neurodevelopmental disorders. The effects of several such mutations have been investigated in recombinantly-expressed NMDARs under conditions of steady-state activation. Such experiments provide only limited insight into how mutations affect NMDAR-mediated excitatory synaptic currents (EPSCs). The present study aimed to characterize the effects of the GluN2A(N615K), GluN2B(N615I) and GluN2B(V618G) gain-of-function mutations on EPSCs mediated by diheteromeric GluN1/2A and GluN1/2B receptors and triheteromeric GluN1/2A/2B receptors, as these are the most abundant synaptic NMDARs in vivo. Subunit composition was controlled by studying 'artificial' synapses formed between cultured neurons (which provide presynaptic terminals) and HEK293 cells that express the NMDAR subunits of interest plus the synapse-promoting molecule, neuroligin-1B. When incorporated into diheteromeric receptors, all three mutations ablated voltage-dependent Mg(2+) block of EPSCs, as previously shown. In addition, we were surprised to find that increasing external Mg(2+) from 0 to 1mM strongly enhanced the magnitude of EPSCs mediated by mutant diheteromers. In contrast, triheteromeric receptors exhibited normal voltage-dependent Mg(2+) block. The GluN2A(N615K) mutation also slowed the decay of GluN1/2A/2B- but not GluN1/2A-mediated EPSCs. The GluN2B(N615I) mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs. The GluN2B(V618G) mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs, although these effects were partly compensated by a faster EPSC decay rate. The mutations also diminished the potency of the anti-epileptic pore-blocker, memantine, thus explaining the lack of memantine efficacy in patients with GluN2B(N615I) or GluN2B(V618G) mutations. Given these effects, the three mutations would be expected to enhance the cation influx rate and thereby contribute to epilepsy phenotypes.