BackgroundConstitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization – homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions.ResultsIn electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions.ConclusionThese data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0167-1) contains supplementary material, which is available to authorized users.

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A chronoamperometric method, based on the oxidation of reduced nicotinamide adenine dinucleotide (NADH) on a glassy carbon electrode, for assaying glutamate dehydrogenase activity is described. Once the reaction was initiated by addition of enzyme into a well stirred assay mixture containing NADH, the concentration of NADH and the steady-state limiting current of NADH oxidation on the electrode decreased linearly in a short time range from the start of the reaction. The rate of the enzyme-catalysed reaction can be calculated from the decrease in the current. This method is reliable for the accurate determination of enzyme activity in purified extracts from liver and crude extracts from cucumber root. The major advantage of this method is the real-time and continuous measurement of the change in coenzyme concentration, so the real initial reaction rate, VO, can be determined. This method has been used to detect the effect of La3+, Eu3+, Yb3+ and Zn2+ ions on the enzyme activity. The results suggest that lanthanide ions can increase the activity of the enzyme in glutamate synthesis, and Zn2+ acts as a potent inhibitor of the enzyme.