Assembly and intracellular distribution of kainate receptors is determined by RNA editing and subunit composition

Abstract

J. Neurochem. (2010) 114, 1805–1818. Abstract Kainate receptors (KARs) modulate neuronal network activity. The molecular mechanisms that control the assembly and trafficking of KARs are unclear. Here, we examined the role of Q/R editing and subunit composition on KAR subunit assembly and subcellular distribution. The majority of GluK2 subunits undergo editing at the Q/R site in the channel pore loop. Cell surface biotinylation, cross‐linking, Endoglycosidase‐H analysis and gradient separation of KAR subunit assembly states revealed that Q/R editing reduces oligomerization, endoplasmic reticulum (ER) export, plasma membrane expression and stability of homomeric GluK2‐containing KARs. These results indicate that Q/R editing of GluK2 may orchestrate channel subunit composition during KAR assembly in the ER. GluK2/GluK5 heteromers are the most abundant KAR subtype in the brain. While subcellular fractionation of brain tissue confirmed that both GluK2/3 and GluK5 are present in synaptosomes and tightly associated with post‐synaptic density fractions, biochemical analysis revealed that endogenous GluK2/3 subunits show less complete assembly and trafficking compared with GluK5. In transgenic mice, the loss of the key assembly partner GluK2 leads to dramatic reduction in GluK5 expression. These results support the idea that the assembly and intracellular distribution of KARs is determined by RNA editing at the Q/R site and subunit composition.

AMPA Receptor Assembly: Atomic Determinants and Built-In Modulators

“…11.1c and 11.4b). An analogous case has been described recently for GluK2 (Ball et al 2010) and most likely results from unfavourable electrostatics from the approximation of four arginines during pore formation (Fig. 11.4b).…”

Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310

“…Therefore, EndoH sensitivity is routinely used to monitor the maturation state of glycoproteins during their biosynthesis and trafficking through the secretory pathway. Our previous study established that EndoH resistance directly correlates to the cell surface expression of KAR subunit proteins (Ball et al, 2010). After EndoH treatment, both GluK2 and GluK3 yielded double bands in immunoblots (Fig.…”

“…Cell lysates were cleared of cellular debris by centrifugation at 28,000g for 15 min at 4°C. Equal aliquots of samples were incubated with either endoglycosidase H (EndoH; 5 mU/100 l of lysate) or endoglycasidase F (EndoF; 1 U/100 l of lysate) (Roche Diagnostics) overnight at 37°C as described previously (Molná r et al, 1995;Ball et al, 2010) and analyzed by immunoblotting.…”

“…Protein samples were separated using 7% (w/v) SDS-polyacrylamide gel electrophoresis gels and transferred electrophoretically onto polyvinylidene difluoride membranes as described previously (Gallyas et al, 2003). Rabbit polyclonal antibodies to GluK1 (20 g/ml) and GluK2/3 (1:2000 dilution; Millipore, Watford, UK) with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilution; Sigma) were used to identify KAR subunit proteins as described previously (Gallyas et al, 2003;Ball et al, 2010). A series of antigen concentrations, primary and secondary antibody dilutions, and enhanced chemiluminescence (Roche Diagnostics) exposure times were used to optimize our experimental conditions for the linear sensitivity range of the autoradiography film (HyperfilmTM MP; GE Healthcare).…”

Kainate receptor trafficking

“…The majority of GluK2 subunits undergo editing at the Q/R site (Figure 1), which reduces receptor oligomerization and ER egress, highlighting editing as another regulatory mechanism for KAR assembly and exit from the ER. 88 However, another group have reported no effect of Q/R editing on the release of KARs from the ER, 89 suggesting further work will be required to determine how Q/R editing effects the surface expression of KARs.…”