Fig. 2.

Evidence that GPI-anchoring of α₂δ-3 is required for cell-surface localization and function. (A Left) Immunoblot profile of GAS-WKW α₂δ-3 (Upper) and CGG-WKW α₂δ-3 (Lower) in DRM fractions from transfected tsA-201 cells using α₂-3 (71–90) Ab. (Right) Bar chart showing the average proportion of the total α₂δ-3 found to be in DRM (sucrose gradient fractions 4–6) for WT α₂δ-3 (black bar), CGG-WKW α₂δ-3 (blue bar), and GAS-WKW α₂δ-3 (red bar) from two independent experiments each. (B) Mean I-V relationship for Ca_V2.2/β1b coexpressed with WT α₂δ-3 (▪ n = 12), with GAS-WKW α₂δ-3 (red • n = 25), or without α₂δ (△ n = 23) in tsA-201 cells. All recordings are in 5 mM Ba²⁺. (C) Peak I_Ba (mean ± SEM) measured at +15 mV was determined from I-V relationships including those in A. Ca_V2.2/β1b is shown without α₂δ (white bar; n = 36), with WT α₂δ-3 (black bar; n = 16), with CGG-WKW α₂δ-3 (blue bar; n = 22), or with GAS-WKW α₂δ-3 (red bar; n = 25). Data were pooled from several experiments and normalized to the respective control (+WT α₂δ-3) in each experiment. The statistical significances of the differences compared to +WT α₂δ-3 were determined by ANOVA and post hoc Bonferroni test. **, P < 0.001. Example I_Ba currents (from −30 mV to +15 mV from a holding potential of −90 mV) are shown above the bars for no α₂δ (gray), WT α₂δ-3 (black), CGG-WKW α₂δ-3 (blue), and GAS-WKW α₂δ-3 (red). (D Upper) Cell-surface biotinylation of α₂δ-3 (WT), CGG-WKW α₂δ-3 and GAS-WKW α₂δ-3. The left three lanes show WCL, and the right six lanes show WT and mutant α₂δ-3 after cell-surface biotinylation, before and after deglycosylation with EndoF as indicated. Open arrow, full-length α₂δ-3; closed arrow, cleaved α₂-3. Lower shows lack of biotinylation of cytoplasmic Akt. (E) Quantification of relative amounts of full-length α₂δ-3 and cleaved α₂-3 for the CGG-WKW α₂δ-3 (blue) and GAS-WKW α₂δ-3 (red) mutants at the cell surface from two experiments, including the data shown in C, that were normalized to the amount in the WCL. (F) Confocal microscopic images showing membrane localization of α₂δ-3 using the δ-3 Ab (Left, red) for WT (Upper) and CGG-WKW α₂δ-3 (Lower) when coexpressed with green fluorescent protein Ca_V2.2 (Middle) and β1b in nonpermeabilized Cos-7 cells. (Right) Merged images show nuclear staining with DAPI (blue). Note that cell-surface staining in nonpermeabilized Cos-7 cells is seen, not as a fine ring but as a wide annulus, because of the flattened geometry (see also Fig. S3A). (Scale bar: 50 μm on merged images.) (G) Quantification of cell-surface immunofluorescence using δ-3 Ab for WT α₂δ-3 (black bars; n = 68), CGG-WKW α₂δ-3 (blue bars; n = 38), and GAS-WKW α₂δ-3 (red bars; n = 38). Statistical significance is P < 0.001 for one-way ANOVA and Tukey’s post hoc tests.