Fig. 6.

Thr/Val and Ala/Leu mutations reduce cell surface expression of GluK2 and GluK3 subunit proteins. a Transiently transfected HEK293 cells expressing wild type (GluK2-WT, GluK3-WT) or mutant subunits with impaired ligand binding sites (GluK2-T659V, GluK2-A487L, GluK3-T661V; red) or conversion mutants with functional ligand binding sites (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue) were surface biotinylated 24 h after transfection. Following the solubilisation of membranes (total; T), biotin-labelled (cell surface exposed; S) proteins were separated from non-biotinylated (intracellular; I) proteins using streptavidin coated beads. The different subunit populations were analysed using immunoblotting with a GluK2/3 specific antibody. The β-actin content of the samples was analysed to confirm that biotinylation only labelled cell surface exposed proteins in transfected HEK293 cells. Due to differences in total expression levels of various KAR subunit mutants (Fig. 4), immunodetection of bands were optimised for the quantitative comparison of biotinylated and non-biotinylated (intracellular) bands for each construct. b Bar diagrams compare the biotinylated (surface) fractions of subunits expressed as % of total. Mutants with impaired LBDs (GluK2-T659V, GluK2-A487L and GluK3-T661V; red bars) show reduced cell surface expression (biotinylation) than the corresponding WT subunits (white bars). In contrast, LBD conversion mutants of GluK2 and GluK3 that retain ligand binding (GluK2-S689N/N690S, GluK2-A487T, GluK3-N691S; blue bars) show no significant change in cell-surface biotinylation compare to WT. Data are mean ± SEM (n = 3), *p < 0.05, Student’s t test. (Color figure online)