Mg2+ block properties of triheteromeric GluN1-GluN2B-GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurones

Key points NMDAR receptors (NMDARs) are tetrameric cation channels permeable to calcium and blocked by Mg2+. Voltage-dependent Mg2+ block of NMDARs is crucial to several forms of synaptic plasticity and to the integration of synaptic activity with neuronal activity. Although diheteromeric GluN1-GluN2A or GluN1-GluN2B NMDARs display stronger voltage-dependent Mg2+ block than GluN1-GluN2C or GluN1-GluN2D NMDARs, the extracellular Mg2+ block properties for triheteromeric NMDARs are still elusive. Here, we show that in dopaminergic neurones the voltage dependence of Mg2+ block is less steep than previously observed in hippocampus or cortex, consistent with the presence of triheteromeric GluN1-GluN2B-GluN2D NMDARs. These results may help to understand the role of triheteromeric NMDARs in dopaminergic neurone synaptic plasticity and to inform simulations of dopaminergic neurone physiology. Native NMDA receptors (NMDARs) are tetrameric channels formed by two GluN1 and two GluN2 subunits. So far, seven NMDARs subunits have been identified and they can form diheteromeric or triheteromeric NMDARs (more than one type of GluN2 subunit). Extracellular Mg2+ is an important regulator of NMDARs, and particularly the voltage dependence of Mg2+ block is crucial to the roles of NMDARs in synaptic plasticity and the integration of synaptic activity with neuronal activity. Although the Mg2+ block properties of diheteromeric NMDARs are fully investigated, properties of triheteromeric NMDARs are still not clear. Our previous data suggested that dopaminergic neurones expressed triheteromeric GluN1-GluN2B-GluN2D NMDARs. Here, using NMDARs in dopaminergic neurones from postnatal day 7 (P7) rats as a model system, we characterize the voltage-dependent Mg2+ block properties of triheteromeric NMDARs. In control conditions, external Mg2+ significantly inhibits the whole cell NMDA-evoked current in a voltage-dependent manner with IC50 values of 20.9 mu m, 53.3 mu m and 173 mu m at -90mV, -70mV and -50mV, respectively. When the GluN2B-selective antagonist ifenprodil was applied, the Mg2+ sensitivity of the residual NMDA-mediated currents (which is mainly carried by GluN1-GluN2B-GluN2D NMDARs) is reduced to IC50 values of 45.9 mu m (-90mV), 104 mu m (-70mV) and 276 mu m (-50mV), suggesting that triheteromeric GluN1-GluN2B-GluN2D NMDARs have less affinity for external Mg2+ than GluN1-GluN2B receptors. In addition, fitting I-NMDA-V curves with a trapping Mg2+ block model shows the triheteromeric GluN1-GluN2B-GluN2D NMDARs have weaker voltage-dependent Mg2+ block (delta=0.56) than GluN1-GluN2B NMDARs. Finally, our concentration jump and single channel recordings suggest that GluN1-GluN2B-GluN2D rather than GluN1-GluN2D NMDARs are present. These data provide information relevant to Mg2+ block characteristics of triheteromeric NMDARs and may help to better understand synaptic plasticity, which is dependent on these triheteromeric NMDARs.