Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors

Abstract

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.

Nmda Receptor Internalization Down-Regulates Nmda Receptor-Mediated Synaptic Responses Through the Inhibition of Remaining (Non-Internalized) Surface Nmda Receptors

“…Our data showed that bath application of either DHPG or NMDA/glycine not only caused significant reductions of NMDARs expressed on neuronal surface but also inhibited NMDAR activity recorded in cell-attached patches [25] . Application of dynamin inhibitory peptide (DIP), Myr-4- …”

“…To address this question we conducted investigations by using a combination of electrophysiological, biochemical, and genetic approaches. Our data demonstrate that through activating PKD1, NMDAR endocytosis down-regulates remaining surface NMDARs and thereby inhibits NMDAR-mediated synaptic transmissions [25] .…”

“…Furthermore, immobilization of NMDARs outside of the membrane patch successfully prevented the reduction of the activity of recorded NMDARs which were within the membrane patches and not immobilized. Thus, we conclude that following bath application of either DHPG or high NMDA/glycine, the inhibition of recorded NMDARs which remained on the neuronal surface is induced by endocytosis of remote NMDARs outside of the membrane patch [25] .…”

“…Regulated internalization of NMDARs inhibits the activity of remaining (non-internalized) surface NMDARs [25] . NMDAR-mediated single-channel activity evoked with NMDA (10 µM) and glycine (3 µM) added into the patch pipette was recorded in cell-attached patches on cultured hippocampal neurons (Fig.…”

“…In this recording configuration (Fig. 1), the patch electrode isolates the recorded receptor/channels from the extracellular bath environment and therefore prevents the direct stimulation by bath-applied lipophobic agents such as NMDA and/or glycine [23,24,25] . The regulated endocytosis of NMDARs, which were located outside the membrane patches, was induced by bath application of the group 1 mGluR agonist, (S)-3,5-Dihydroxyphenylglycine (DHPG, 50 µM) [14,15] or high concentration of NMDA (1 mM) and glycine (100 µM) (NMDA/glycine) [13] .…”