Abstract
Palmitoylation of NMDARs occurs at two distinct cysteine clusters in the carboxyl-terminus of GluN2A and GluN2B subunits that differentially regulates retention in the Golgi apparatus and surface expression of NMDARs. Mutations of palmitoylatable cysteine residues in the membrane-proximal cluster to non-palmitoylatable serines leads to a reduction in the surface expression of recombinant NMDARs via enhanced internalization of the receptors. Mutations in a cluster of cysteines in the middle of the carboxyl-terminus of GluN2A and GluN2B, leads to an increase in the surface expression of NMDARs via an increase in post-Golgi trafficking. Using a quantitative electrophysiological assay, we investigated whether palmitoylation of GluN2 subunits and the differential regulation of surface expression affect functional synaptic incorporation of NMDARs. We show that a reduction in surface expression due to mutations in the membrane-proximal cluster translates to a reduction in synaptic expression of NMDARs. However, increased surface expression induced by mutations in the cluster of cysteines that regulates post-Golgi trafficking of NMDARs does not increase the synaptic pool of NMDA receptors, indicating that the number of synaptic receptors is tightly regulated.
Highlights
The activation of NMDA-type glutamate receptors (NMDARs) promotes a wide range of signaling pathways that underlie the development, maturation, plasticity, and elimination of synapses in the central nervous system [1,2]
Transfected cells expressing etag GluN1 and GFP-GluN2 subunits exhibit evoked excitatory post synaptic currents (EPSCs) at hyperpolarized potentials (270 mV) with a fast component due to the activation of endogenous AMPA-type glutamate receptors (AMPARs) and a slow component that reflects the activation of recombinant NMDARs (Fig. 1, Insets)
Because endogenous NMDARs are blocked by magnesium at 270 mV, the late component of evoked EPSCs at 270 mV in transfected neurons is measured to monitor whether recombinant NMDARs have been incorporated into synapses
Summary
The activation of NMDA-type glutamate receptors (NMDARs) promotes a wide range of signaling pathways that underlie the development, maturation, plasticity, and elimination of synapses in the central nervous system [1,2]. All glutamate receptor subunits are composed of four domains: extracellular amino-terminal domain (NTD), extracellular ligand binding domain (LBD), trans-membrane domain (TMD), and intracellular carboxyl-terminal domain (CTD) [3]. The subunit composition of NMDARs determines the biophysical and pharmacological properties of the receptor. Glutamate receptors are synthesized in the somatic endoplasmic reticulum and processed in the Golgi apparatus [5,6]. They undergo several forms of post-translational modifications, phosphorylation being the most studied. NMDARs are synthesized in the somatic ER but sorted away from AMPA-type glutamate receptors (AMPARs), bypassing the somatic Golgi, and transported to dendritic Golgi outposts before being inserted into the plasma membrane [9]
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