Fig 2: Effects of 14-3-3 inhibition on Kir2.1-Nav1.5 channel complexes. (A,B) IKir2.1 density at -120 mV (A) and peak INav1.5 (B) recorded in CHO cells expressing the constructs indicated, cotransfected or not with 14-3-3DN. Each bar represents the mean±SEM of “n” cells of = 4 preparations. *P < 0.05 vs. cells transfected with Kir2.1 or Nav1.5 channels alone; #P < 0.05 vs. cells transfected with Kir2.1+Nav1.5. For clarity the results of other statistical comparisons were not shown. (C,D) Normalized IKir2.1 measured at -120 mV (C) and peak INav1.5 (D) measured in the absence and presence of anti-Nav1.5 and anti-Kir2.1 antibodies, respectively, in cells cotransfected with Kir2.1, Nav1.5, and 14-3-3DN plotted as a function of time. Each point represents the mean ± SEM of 4 experiments of =3 preparations in each group.

Fig 3: Schematic diagram summarizing the main findings of the present study. Nav1.5 and Kir2.1 channels are represented by green and golden cylinders, respectively. ER, endoplasmic reticulum; P, phosphate; Ub, ubiquitin chains.

Fig 4: Kir2.1 and Nav1.5 channels are in close proximity at the membrane of cardiac myocytes. (A) Confocal microscopy images of HEK293 cells expressing Kir2.1-CpVenus alone (left) or in the presence of Nav1.5 (right). The insets show the intensity of the signal emitted by CpVenus throughout the cell region selected (yellow line). (B) Peak mean signal corresponding to the cell membrane in cells expressing Kir2.1-CpVenus with or without Nav1.5 channels. (C) Confocal microscopy images of rat ventricular myocytes processed using Duolink® PLA. The green signal demonstrates a physical interaction between Nav1.5 and Kir2.1. Cell nuclei were visible by DAPI staining (blue). (D) Mean intensity of the green signal in the negative control and in the presence of Nav1.5+Kir2.1 antibodies. In (B,D), each bar represents the mean ± SEM of “n” cells of “N” preparations. *P < 0.05 vs. Kir2.1-CpVenus alone. **P < 0.01 vs. negative control.

Fig 5: Kir2.1 and Nav1.5 channels closely interact at the cell membrane. (A–D) IKir2.1 (A,C) and INav1.5 (B,D) traces recorded by applying the protocols shown at the top in CHO cells expressing Kir2.1 or Nav1.5 channels alone (A,B) or together (C,D) in control conditions (solid lines) or after dialyzing the cell with the antibodies indicated (dashed lines). (E–G) Normalized IKir2.1 (measured at -120 mV) (E,G) and peak INav1.5 (F,G) measured in the absence and presence of anti-Kir2.1 or anti-Nav1.5 antibodies in cells transfected with Kir2.1 or Nav1.5 channels alone (E,F) or cotransfected with both (G) plotted as a function of time. (H) Bar graphs showing the mean normalized current density values measured in each experimental group at the end of the recordings. Each point/bar represents the mean ± SEM of (n) cells of = 3 preparations. In (H), *P < 0.05 vs. IKir2.1 or INav1.5 generated by Kir2.1 or Nav1.5 channels alone in the presence of anti-Nav1.5 or anti-Kir2.1 antibodies. For clarity the results of other statistical comparisons were not shown.