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  • br Experimental section br Introduction NMDA receptor induce

    2023-09-28


    Experimental section
    Introduction NMDA receptor-induced increases in AMPAR trafficking to the synaptic surface mediates changes in synaptic efficacy in a number of oligomycin regions (Shi et al., 1999, Sun et al., 2005, Frenkel et al., 2006). While this process has been linked to the regulated insertion of GluR1-containing AMPARs, subsequent insertion of GluR2-containing receptors is also critical to maintaining synaptic potentiation (Hayashi et al., 2000, Passafaro et al., 2001, Plant et al., 2006). However, evidence for a role or mechanism of regulated GluR2-containing AMPARs insertion is limited. We have previously found that elevated NMDAR activation unclusters dendritic PICK1 and results in the release of GluR2-containing AMPARs (GluR2-AMPARs) to the membrane surface (Sossa et al., 2006). This PICK1-dependent increase in surface AMPARs suggests that GluR2 localization with and retention by PICK1 in intracellular pools serves as a site of regulation for receptor-induced changes in synaptic transmission. To provide additional insight into the mechanisms of such NMDAR-induced plasticity, further examination of the mechanisms that regulate PICK1 clustering is required. The process of modulating GluR2-AMPAR levels at synapses is regulated by a number of AMPA receptor interacting proteins (Braithwaite et al., 2000, Kim and Sheng, 2004), one of which is N-ethylmaleimide sensitive factor (NSF). NSF chaperones GluR2-AMPARs to the membrane surface and disassembles PICK1 from GluR2-AMPARs (Hanley et al., 2002), perhaps freeing pools of intracellularly anchored AMPARs to return to the membrane surface. Activation of NSF by nitric oxide (NO)-dependent S-nitrosylation increases the binding of NSF to GluR2 and enhances the surface expression of AMPARs (Huang et al., 2005). We have, therefore, investigated whether the PICK1-dependent delivery of AMPARs following NMDAR activation is mediated by NSF and its modification by NO. We find that dendritic PICK1 unclustering and the subsequent delivery of AMPARs to the membrane surface occurs via NMDAR-mediated S-nitrosylation of NSF. NMDAR stimulation increases NO production and S-nitrosylation of NSF. Consistent with the possibility that S-nitrosylation increases NSF function, we find that NMDA/low Mg2+ treatment increases NSF-GluR2 association and surface AMPAR levels. NO donors mimic the NMDA/low Mg2+-induced decrease in dendritic PICK1 levels, a result blocked by NO scavengers. We show that the NMDA/low Mg2+-induced loss in PICK1 labeling is an NO-dependent process, which leads to enhanced NSF function and subsequent increase in surface AMPAR expression.
    Material and methods
    Results
    Discussion This study shows that NO can affect the plasma membrane expression of a population of receptors regulated by PICK1. Our data further elucidates a mechanism underlying the surface delivery of AMPARs following NMDAR activation. NMDAR stimulation enhances nitric oxide levels in hippocampal neurons and induces an increase in the S-nitrosylation of NSF. Consistent with work by Huang et al. (2005), this modification of NSF is accompanied by an increase in the binding of NSF to GluR2. We now find that these events are accompanied by an unclustering of PICK1, presumably from endosomes in neuronal dendrites (Sossa et al., 2006). A link between NO generation and the effects on PICK1 was confirmed by NO donors, which alone induce the unclustering of dendritic PICK1 and the surface delivery of AMPARs. As PICK1 is also known to be involved in AMPAR endocytosis, it is possible that the increase in surface AMPARs with PICK1 unclustering reflects not increased delivery, but rather a decrease in the constitutive endocytosis of AMPARs. This would lead to an accumulation of cycling AMPARs at the membrane surface. However, we find that NO-induced unclustering of PICK1 does not reduce the rate of constitutive endocytosis for GluR2 AMPARs, suggesting that the changes in PICK1 are influencing the overall delivery of surface AMPARs (Supplementary Fig. 2).