Oligomerization and Spatial Distribution of Kvβ1.1 and Kvβ2.1 Regulatory Subunits

Figures

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  FIGURE 1. Kvβ1 and Kvβ2 expression in the brain and leukocytes. Total lysates from rat brain and different leukocytes were immunoblotted against Kvβ1 and Kvβ2. Mouse dendritic CY15, human Jurkat T cells, human CD4+ lymphocytes, and murine BMDM samples were obtained and processed as described in the methods. Top panel, Kvβ1; middle panel, Kvβ2; bottom panel, β-actin as a control. Note that representative western blots are shown only for qualitative purposes. No comparison among groups due to species, cell type and sample processing is intended.

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  FIGURE 2. Kvβ1.1 and Kvβ2.1 form oligomers. HEK 293 cells were transfected with Kvβ1.1 and Kvβ2.1. (A) Coimmunoprecipitation assay against Kvβ1.1CFP in the presence of Kvβ1.1. (B) Coimmunoprecipitation assay against Kvβ2.1CFP in the presence of Kvβ2.1. (C) Coimmunoprecipitation assay against Kvβ1.1CFP in the presence of Kvβ2.1. (C). Top panels: immunoprecipitation (IP) of CFP and immunoblot (IB) against CFP. Bottom panels: IB against Kvβ1.1 (A) and Kvβ2.1 (B,C). SM: starting material. SN+: supernatant in the presence of the antibody. IP+: Immunoprecipitation in the presence of the antibody. SN-: supernatant in the absence of the antibody. IP-: immunoprecipitation in the absence of the antibody. (D) Kvβ1.1 and Kvβ2.1 form homo- and hetero-oligomers. Representative image of Kvβ1.1 and Kvβ2.1 cotransfection. (Da) Donor, Kvb1.1CFP; (Db) Acceptor, Kvb2.1 YFP; (Dc) merge, yellow indicates colocalization; (Dd) FRET image obtained from the relationship between the donor prebleach versus postbleach after acceptor photobleaching. The white square highlights the bleached section. The bar represents 20 μm. (E) FRET efficiencies (%) calculated from the acceptor photobleaching experiments. Values are…

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  FIGURE 3. Kvβ1.1 and Kvβ2.1 exhibit high FRET efficiencies for homo- and hetero-oligomerization with a basic unit of two peptides per complex. (A) Kvβ1.1CFP/Kvβ1.1YFP. (B) Kvβ2.1CFP/Kvβ2.1YFP. (C) Kvβ1.1YFP/Kvβ2.1CFP. (D) Kvβ1.1CFP/Kvβ2.1YFP. Left panels, schematic representation of possible interactions upon cotransfection. Right panels, plot of EfD (blue circles), EfD fitting (solid line), EfA (green circles), EfA fitting (dashed line) vs. the donor molar ratio (xD). (E) Values obtained from lux-FRET experiments performed on Kvβ combinations represented in orange squares in (A–D). E, FRET efficiency (%); N, number of units per complex; ROI, number of ROIs analyzed. Green shapes, Kvβ1.1; purple shapes, Kvβ2.1. Yellow represents YFP; blue represents CFP.

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  FIGURE 4. Kvβ1.1 and Kvβ2.1 can form homo- and heteromeric complexes with similar affinities. (A) Model of the interaction between donor and acceptor proteins. (B) Model of Kvβ1.1 and Kvβ2.1 oligomerization. (C). The calculated plot was based on the formula developed in the MATLAB program to find the affinity constants. Plot of the different parameters [DA], [DD], [AA], [D], [A], fD and fA depending on the donor molar fraction when a model presents the same affinity for forming three different complexes. [β2.1–β1.1], concentration of donor–acceptor complexes. [β2.1–β2.1], concentration of donor–donor complexes. [β1.1–β1.1], concentration of acceptor–acceptor complexes. [β2.1], donor concentration, [β1.1], acceptor concentration. fβ2.1, apparent donor efficiency. fβ1.1, apparent acceptor efficiency.

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  FIGURE 5. Kvβ1.1 and Kvβ2.1 form dimeric and tetrameric structures. (A) Model representing the two putative options for Kvβ oligomerization. Kvβ forms dimers, and Kvβ forms tetrameric structures by association. The affinity constant (k) for every step would be the same in all conditions. (B–D) HEK 293 cells were transfected with YFP, Kv1.3YFP, Kvβ1.1YFP and Kvβ2.1YFP. Total lysates were analyzed in semidenaturating conditions. (B) YFP, (C) Kv1.3YFP, (D) Kvβ1.1YFP and Kvβ2.1YFP. Immunoblots (IB) were performed with an anti-GFP antibody. Blots were split into two for low- and high-molecular-weight forms for better visualization. The black arrow indicates monomeric forms. The green arrow indicates dimeric forms. The red arrow highlights tetrameric forms.

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  FIGURE 6. Kvβ1.1 and Kvβ2.1 form heteromeric complexes at the plasma membrane. HEK 293 cells were transfected with Kv1.3YFP-Kv1.3CFP, Kvβ1.1YFP-Kvβ1.1CFP and Kvβ2.1YFP-Kvβ2.1CFP. After transfection, CUPs were purified, and FRET was analyzed. (A–C) CFP-YFP-transfected cells were used as negative controls. Note that CFP-YFP was analyzed in entire cells. (D–F) Kv1.3CFP-Kv1.3YFP. (G–I) Kvβ1.1CFP-Kvβ1.1YFP. (J–L) Kvβ2.1CFP-Kvβ2.1YFP. (M–O) Kvβ1.1CFP-Kvβ2.1YFP. Red panels, CFP; green panels, YFP; merged panels, yellow indicates colocalization. (P) FRET efficiency (%). **, p < 0.01; ***, p < 0.001 versus CFP-YFP (Student’s t test). Values are the mean of 20–30 cells. Scale bars represent 10 µm.

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  FIGURE 7. Kvβ1.1 interaction shifts out Kvβ2.1 from lipid raft domains. HEK 293 cells were transfected with Kvβ1.1CFP, Kvβ2.1CFP and Kvβ1.1CFP-Kvβ2.1. Lipid rafts were isolated as described in the methods. One-milliliter fractions were sequentially collected from the top (1, high buoyancy) to the bottom (12, low buoyancy) of the tube. (A) Western blotting of lipid raft fractions of Kvβ1.1CFP. (B) Western blot analysis of lipid raft fractions of Kvβ2.1. (C) Western blotting of lipid raft fractions of Kvβ1.1CFP and Kvβ2.1 cotransfection. While caveolin identifies floating lipid rafts, clathrin indicates nonlipid raft fractions.

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  FIGURE 8. Scheme representing the homo- and heteromeric Kvβ fate. While the tetrameric Kv1.3 channel is assembled early at the ER, soluble Kvβ is synthesized in the cytoplasm. Kvβ would form homo- and heterodimers. Kvβ tetramers would be formed by dimerization of dimers. Kvβ tetramers, especially Kvβ2, interact early with Kv1.3 to target lipid rafts via the Golgi apparatus. The extent to which Kv1.3 associated with Kvβ1.1 or Kvβ1.1/Kvβ2.1 tetramers follows different destinies outside lipid rafts is uncertain. In the absence of a channel, Kvβ2.1 tetramers target lipid rafts in a palmitoylation-dependent manner. However, Kvβ2.1 could also interact with Kvβ1.1, forming heterotetramers. In this context, not only homo-Kvβ1.1 but also Kvβ1.1–Kvβ2.1 heterotetramers would be displaced from lipid rafts targeting other plasma membrane surface locations. By this mechanism, Kvβ1.1 fine-tunes the Kvβ2.1-dependent physiological mechanisms at specific cell surface spots. Blue shape, Kv1.3; green balls, Kvβ2.1; yellow balls, Kvβ1.1. Kvβ palmitoylation (red sparkline).