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Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca2+ affecting several downstream signaling pathways that involve Ca2+/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca2+ responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIα translocation was observed. In cells solely expressing Ca2+-impermeable NMDARs containing NR1N598R-mutant subunits, prolonged NMDA application elevated internal Ca2+ to the same degree as in wild-type controls, yet failed to translocate CaMKIIα. Brief local NMDA application evoked smaller Ca2+ transients in dendritic spines of mutant compared to wild-type cells. CaMKIIα mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1N598R cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca2+ entry directly through NMDARs, rather than other Ca2+ sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event. [ABSTRACT FROM AUTHOR]

NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laeris oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose-response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30- to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D (T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype. [ABSTRACT FROM AUTHOR]

Presents a study that introduced the N598R mutation in the native NR1 gene to test the role of NMDA receptor signaling in the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem of rodents. Materials and methods; Generation of NR1 mutant genotypes; Neuroanatomy of N598R mutant mice.