Molecular Requirements Controlling CluN2 Subunit Specific Rules for NMDA Receptor Synaptic Incorporation

NMDA receptors are unique ionotropic glutamate receptors that serve as coincident detectors of pre and post synaptic activity and initiate some forms of synaptic plasticity at excitatory synapses in the brain. The predominant forms of the NMDA receptor in the mammalian forebrain and hippocampus are heterodimers consisting of two GluN1 subunits and two GluN2A or GluN2B subunits. Many of the NMDA receptor's physiological properties are controlled by the GluN2 subunit including incorporation of the receptor into the synapse. Currently, research suggests that GluN2B containing receptors are expressed prenatally, inserted into synapses in a constitutive manner, and are gradually replaced by GluN2A containing receptors in activity-dependent manner. However, what is not understood are the molecular differences between the GluN2A and GluN2B subunits that control constitutive and activity-dependent forms of synaptic incorporation and if the replacement of GluN2B with GluN2A at the synapse first requires the removal of GluN2B. Using biolistic transfection of cultured organotypic hippocampal slices with electrophysiologically-tagged recombinant NMDA receptors containing genetically modified chimeras of the GluN2 subunit, experimental evidence was obtained suggesting that a single predicted glycosylation site found at the amino acid N675 in the GluN2B but not the GluN2A subunit was sufficient for changing the mode of synaptic incorporation. In addition, using molecular, pharmacological, and electrophysiological techniques evidence was acquired to directly show that the synaptic incorporation of GluN2A requires calcium influx and GluN2B removal. Interestingly, and somewhat surprisingly, incorporation of GluN2A seems linked to an increase in AMPA receptor mediated synaptic transmission. These data provide fundamental new insights into how the subunit composition of NMDA receptors is regulated by experience and synaptic activity.