Triheteromeric NMDA Receptors at Hippocampal Synapses

NMDA receptors are composed of two N1 and two N2 subunits. Constituent N2 subunits control pharmacological and kinetic characteristics of the receptor. Although NMDA receptors in hippocampal or cortical neurons are often thought of as diheteromeric, containing only one type of N2 subunit, triheteromeric receptors with more than one type of N2 subunit also have been reported. However, the relative contribution of di- and triheteromeric NMDA receptors at synapses has been difficult to assess. Because wild-type hippocampal principal neurons express N1, N2A and N2B, we used cultured hippocampal principal neurons from N2A and N2B-knockout mice as templates for diheteromeric synaptic receptors. Summation of N1/N2B and N1/N2A excitatory postsynaptic currents (EPSCs) could not account for the deactivation kinetics of wild-type EPSCs however. To make a quantitative estimate of NMDA receptor subtypes at wild-type synapses, we used the deactivation kinetics, as well as the effects of the competitive antagonist NVP-AAM077. Our results indicate that three types of NMDA receptors contribute to the wild-type EPSC, with at least two-thirds being triheteromeric receptors. Functional isolation of synaptic triheteromeric receptors revealed deactivation kinetics and pharmacology distinct from either diheteromeric receptor subtype. Because of differences in open probability, synaptic triheteromeric receptors outnumbered N1/N2A receptors by 5.8 to 1 and N1/N2B receptors by 3.2 to 2 1. Our results suggest that triheteromeric NMDA receptors must be either preferentially assembled or preferentially localized at synapses