Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

Fig. 1. Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1WT, Kir2.1Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1WT/Δ314-315 SpC ratio “R ”, we classified the interactors in three categories: 75 Kir2.1WT-preferred interactors, 66 Kir2.1Δ314-315-preferred interactors and 77 Kir2.1WT/Δ314-315-neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

Fig. 2. Kir2.1WTversus Kir2.1Δ314-315 interactome profiling. Variations in the Kir2.1WT and Kir2.1Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log2-transformed, normalized SpC counts for both the Kir2.1WT (y axis) and Kir2.1Δ314-315 (x axis) bait proteins. The Kir2.1 bait, Kir2.1WT-preferred interactors, Kir2.1Δ314-315-preferred interactors and Kir2.1WT/Δ314-315-neutral interactors are represented as blue, green, red and gray dots, respectively.

Fig. 3. Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1WT-preferred interactors, Kir2.1Δ314-315-preferred interactors and Kir2.1WT/Δ314-315-neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1WT/Δ314-315 SpC ratio “R” value. The size of each circle represents the average SpC counts observed in either the Kir2.1WT or Kir2.1Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

Fig. 4. Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 (A, red), Actinin (B, green) and PKP4 (C, light blue) in a freshly isolated rat adult ventricular myocytes. (D) Merge image. (E) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in (D) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. (F) Differential interference contrast (DIC) image of the myocyte. (G–J) Zoomed in images of the intercalated disks. Scale bars: 10 μm. ID: intercalated disk.

Fig. 5. PKP4 is a positive regulator of IKir2.1. Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4, i.e. (A) upon overexpression of PKP4 and (B) upon CRISPR/Cas9-mediated depletion of PKP4. *P < 0.05 versus control. N: number of transfected cell batches; n: number of cells; OE: overexpression; KD: knock-down.