Figure 2.

Elimination of the positive charge at residue 663 in the M3–S2 linker reduced plasma membrane localization of myc-GluR6a receptors. A, Plasma membrane expression of myc-GluR6a, E662, and R663 mutants were quantitated by cell ELISA assays in transfected COS-7 cells. Wild-type myc-GluR6a receptors showed 85.6 ± 2.7% (n = 6) plasma membrane localization relative to total protein expression. Myc-GluR6a(E662) mutants were localized to the plasma membrane at near-wild-type levels, from 63.4 to 94.0% (n = 3–4), whereas all myc-GluR6a(R663) mutants, with the exception of R663K, showed significantly lower cell surface expression (3.6–37.8%; n = 3 for all mutants; ***p < 0.001 compared with wild-type receptors; individual values given in supplemental Table 1, available at www.jneurosci.org as supplemental material). B, Confocal images of myc-GluR6a, R663E, and R663K receptors in transfected hippocampal neurons. Total expression of myc-GluR6a receptors was detected after permeabilization (left column, green). Plasma membrane-localized receptors were detected under nonpermeabilized conditions (middle column, red). GluR6a(R663E) was mostly absent from the plasma membrane. These results and data from other receptor mutants were quantitated by calculating the ratio of red to green fluorescence intensity; E662D, E662R, and R663K mutants exhibited the same level of plasma membrane expression as myc-GluR6a receptors (0.96–1.21; n = 3–4). In contrast, other R663 substitutions caused profound reductions in neuronal membrane receptors (0.04–0.59; n = 3–8; **p < 0.01; ***p < 0.001 compared with wild-type receptors; individual values given in supplemental Table 1, available at www.jneurosci.org as supplemental material). Data represent the mean ± SEM.