Role of protein domains in trafficking and localization of the voltage-gated sodium channel β2 subunit

Figure 1. Lack of the cytoplasmic tail or extracellular loop disruption impairs complex N-glycosylation of β2 needed for its efficient surface delivery.A, representative confocal XY sections of polarized cells taken at the apical plasma membrane, or deeper into the nuclear level (for 181X only), show strong overlap of transiently transfected β2-YFP (green) WT with apically located gp114 (red). On the contrary, the 181X mutant remains in dense intracellular aggregates (arrowheads). Normal pattern of the tight junction marker ZO-1 further confirms proper cell polarity (blue). Horizontal yellow lines in the corresponding z-axis reconstructions (XZ) mark the cell sections shown by XY. In merged images, nuclear staining by DAPI is in grey. Scale bars are 1 μm. B and C, representative Western blot after cell surface biotinylation showing plasma membrane levels of various β2 mutants compared to the WT in transiently transfected cells. C, as irrelevant controls, cells were transfected to express APP-YFP or simply GFP. Surface markers, gp114 (B) and E-cadherin (C), were blotted to normalize quantifications. D, band quantitation of mutants whose surface localization was visibly affected confirms…

Figure 2. Shortening of the β2 transmembrane domain affects glycosylation and leads to its accumulation in the endoplasmic reticulum.A and B, representative western blots after cell surface protein biotinylation of polarized cells stably expressing β2-YFP show apical localization also of TMD mutants. However, whereas their immature, still unprocessed, band is apparent in lysates, it does not reach the cell surface. Note that the TMC mutant expresses proportionally much lower levels of mature, fully glycosylated β2, than the WT, or even than the other mutants; this is the only form reaching efficiently the cell surface. Evidence of polarity is shown by proper surface localization of basolateral (B) Na/K-ATPase (A) or apical (A) gp114 (B) in pulldowns (Pull); Lys indicates lysate. Note that apparent traces of gp114 in the basal region (marked by an asterisk) come from a previous blot signal to Na/K-ATPase that could not be completely stripped off. C, representative western blot showing full cleavage (deglycosylation) by Endo H in all TMD mutants of the immature form, which is the main constituent of β2 TMC. D, representative confocal XY sections and corresponding z-axis reconstructions (XZ)…

Figure 3. β2 oligomerizes through Cys-55.A, representative western blots of lysates from cells transiently expressing WT or mutant β2-YFP, analyzed in reducing (DTT) or non-reducing conditions (No DTT), show that the C55A (in red fonts) is the only mutant unable to dimerize. Oligomerization is manifested by the presence of high molecular weight bands displaying a pattern coincident with that of dimers fully glycosylated and others with immature glycosylation. High-order oligomers, likely tetramers, are also observed in C50A and C127A mutants. In all Cys mutants shown here, this residue was replaced by Ala (A). Upper and lower bands seen in reducing conditions are, respectively, fully glycosylated and immature monomeric β2. The blot to α-spectrin-1 (SPTAN1) serves as a loading control. B, representative western blots after cell surface protein biotinylation of transiently transfected cells show apical localization (Api) of β2 C55A. Evidence of polarity is confirmed by proper apical localization of gp114; Bas indicates basolateral and Lys indicates lysate.

Figure 4. Mutation on both Cys-55 and Cys-182 does not affect β2 apical localization.A, representative Western blots after cell surface protein biotinylation of polarized cells transiently expressing β2-YFP show apical localization of the mutant C55A/C182S in a manner indistinguishable from the WT. Markers gp114 and Na/K-ATPase remain at their apical (Api) and basolateral (Bas) domains in pulldowns, respectively, confirming proper cell polarity. B and C, representative confocal XY sections and corresponding z-axis reconstructions (XZ) of polarized cells transiently expressing β2-YFP show that both WT and the C55A/C182S mutant (green) are exclusively located at the apical section, merging with gp114 (C), but not with Na/K-ATPase (B), the latter, well visible at the nuclear level (both surface markers are in red). The normal pattern of the tight junction marker ZO-1 further confirms proper cell polarity (blue). The two parallel yellow lines in XZ mark the cell sections shown by XY, either apical or nuclear. In merged images, the nuclear staining by DAPI is in grey. Scale bars are 10 μm.

Figure 5. The TMD mutant TMC displays a dimerization defect. Representative western blots of lysates from cells stably expressing β2-YFP, analyzed in reducing (DTT) or non-reducing conditions (No DTT), show that the WT, as well as the TMA and TMB mutants, display a considerable proportion of β2 as a mature (fully glycosylated) dimer. On the contrary, the TMC mutant exhibits a proportionally smaller fraction in comparison with levels of its immature (ER-glycosylated) monomeric form; the bands at around 120 kDa and above likely correspond to a minor portion of dimerized TMC mutant displaying different degrees of glycosylation (band pattern marked by arrowheads). Note that the mutant TMA, while expressed at comparatively lower levels than the other β2 forms, displays a considerable portion of the mature dimer. Although inefficiently solubilized in the absence of DTT, actin serves as a loading control.

Figure 6. Effects on NaV1.5 localization to the plasma membrane in the presence of mutated β2. Representative Western blots after cell surface protein biotinylation of parental cells (A–C: −), or stably expressing NaV1.5-YFP (A–D: NaV1.5), and transiently expressing WT or mutant β2-YFP, reveal slight reductions in plasma membrane levels of NaV1.5 when β2 was mutated; this effect could be estimated, confirming a decrease by all mutants except for β2 181X (E, p values are shown). Conversely, glycosylation pattern and surface distribution of the various β2 forms (marked by arrowheads) appears unaltered; dimers are referred on the image as oligomers. As irrelevant controls, cells were transfected to express APP-YFP, GFP (D), or left untransfected (A: Utf). A blot to E-cadherin is included as loading control to adjust for fluctuation in solubilized material. Non-specific band(s) above the 180 kDa marker in lanes of parental cells are labeled with an asterisk. While the same amount of plasmid DNA was used for all transfections, band intensity may vary depending on the stability of each mutated β2; note for instance that the C127A mutant (B, 127) tends to be poorly expressed, but behaves…

Figure 7. All β2 variants display a comparable homodimerization pattern in the presence of NaV1.5. Representative western blots of lysates from parental cells (−), or stably expressing NaV1.5-YFP (NaV1.5), and transiently expressing WT or mutant β2-YFP, were analyzed in reducing (+DTT) or non-reducing conditions (No DTT), showing a comparable oligomerization pattern of the various β2 forms with and without NaV1.5. The band pattern is marked by arrowheads, with oligomers also referring to higher-order forms, likely tetramers, such as the band above the 245 kDa marker manifested in the C50A mutant (A, 50). As irrelevant controls, cells were transfected to express GFP (D), or left untransfected (A: Utf). A blot to E-cadherin is included as loading control to adjust for fluctuation in solubilized material. While the same amount of plasmid DNA was used for all transfections, band intensity may vary depending on the stability of each mutated β2; note for instance that the C127A mutant (A, 127) consistently tends to be poorly expressed, yet displaying a pattern comparable to that of β2 C50A. Note that, in reducing conditions, a pale pattern of nonspecific bands can be seen above β2-YFP in the…

Figure 8. The tail-minus mutant does not undergo endocytosis. Representative western blots of polarized cells stably expressing β2-YFP after cell surface biotinylation show the absence of WT and the 181X mutant in pulldowns after 60 min (60′) from glutathione treatment (+), denoting lack of apical endocytosis. Pulldowns at t = 0 min (0′), before glutathione treatment (−), indicate total apically biotinylated β2. As a control, note absence of biotinylated protein in pulldowns of cells treated with glutathione (+) immediately after labeling (0′). As expected, apical gp114 does not get endocytosed either.

Figure 9. Cytoplasmic tail, extracellular loop, and transmembrane domain all contribute to proper β2 trafficking and localization. Cartoon highlighting the portions of YFP-tagged β2 determinant for its proper trafficking to the cell surface and polarized localization. Cytoplasmic tail, shown as the ICD; extracellular loop, where the major intramolecular disulfide bond forms the Ig loop; and TMD, with a tendency to locate in cholesterol-rich raft domains, are highlighted as important regions for β2 trafficking and localization. Mutations found for cardiac-associated arrhythmias, i.e., atrial fibrillation (AF), on Arg-28 (56), and Brugada Syndrome (BrS), on Asp-211 (57), are also shown. See additional details in the text.

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