Figure 8.
Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.