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Figure 1. Mutations in Kir2.1 associated with Andersen–Tawil syndrome. (A) DNA sequencing of two patients revealed mutations in Kir2.1. (B) Representative ECG from the carrier of V77E variant showing bidirectional ventricular tachycardia, prolonged QT interval with ST and T wave abnormalities. (C) Left, cartoon presentation of two subunits (blue and gray) out of four, of the inwardly rectifying potassium channel protein (Kir2.2 with PIP2, PDB ID: 3SPI (Hansen et al., 2011)). PIP2 is in ball-and-stick (atom code: oxygen, red; carbon, green; phosphorous, orange). The N-terminal slide helix is in cyan and the cytoplasmic gate G-loop is in yellow. Side chains as sticks of V75 and M308 in Kir2.2, homologous to V77 and M307 in Kir2.1, are in green and red, respectively. The plasma membrane is in light gray. Right, zoom on the mutated residues’ location. (D) Kir channels phylogenetic tree. Amino acid sequence alignments of 15 human Kir proteins, in addition to mouse and Zebrafish Kir2.1.
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Figure 2. Mutations V77E and M307V induce Kir2.1 loss-of-function. Net Ba2+-sensitive currents recorded from HEK cells expressing Kir2.1-GFP (WT, V77E, M307V, M307L) elicited by a voltage-steps protocol. (A) Representative currents. (B) Average current density (WT, n = 12; V77E, n = 10; M307V, n = 6; M307L, n = 8). Statistical analysis in the voltage step to −140 mV using one-way ANOVA (Dunnett’s test, comparison to WT). M307L, not significant; V77E and M307V, ***p < 0.001.
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Figure 3. Loss-of-function Kir2.1 mutations express on the cell surface. (A) Immunostaining of HL-1 cells transfected with Kir2.1-GFP with and without an extracellular HA-tag. Top, an illustration of the Kir2.1 subunit constructs. Cylinders: Kir2.1 transmembrane domains; blue: mutation location; green: GFP; Red: HA-tag. Bottom, HA-tag was immunostained (red), expressed GFP was detected (green) and the nucleus was stained with DAPI (blue). The three optical channels are superimposed (upper panel) and the red channel is also shown separately (lower panel). Arrows point to the cell surface signal. (B) Biotinylation of HEK cells transfected with Kir2.1-GFP monomers and Kir2.1 dimers (WT and mutant) as illustrated above and detected with the HA-tag antibody. Left, representative blots from one experiment. Right, normalized signals. a. Total Kir2.1 protein level. Total expression was normalized to WT in the same experiment. Low film exposure was used to quantify the difference in Kir2.1 monomers’ level within a dynamic range (upper panel, N = 4). Statistical analysis: one-way ANOVA. The differences between Kir2.1 monomers and dimers were shown in high film exposure of the blot, where Kir2.1 monomer signal was saturated (lower panel, N = 3). Statistical analysis: one-way ANOVA (Dunnett’s test, comparison to WT Group). b. biotin-labeled Kir2.1 level. As a control for non-specific signals, Kir2.1-GFP WT and WT dimer expressing groups were not incubated with biotin. The amount of protein on the plasma membrane relative to total was quantified by a division of the biotinylated with input level in high exposure (N = 3). Statistical analysis: one-way ANOVA.
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Figure 4. Functional properties of Kir2.1 dimers. (A) Normalized current densities of Kir2.1-GFP WT monomer and WT dimers, recorded in HEK cells transfected with equal amounts of DNA. Normalization was performed to the mean current density of Kir2.1-GFP from the same experiment (N = 3). Statistical analysis: t-test. (B) Normalized currents (left) elicited by a ramp protocol (right). Every 10th recorded point is presented. This is a summary of all cells from different experiments (N = 7 experiments for Kir2.1-GFP WT monomer and N = 12 for WT dimer). Inset, zoom on the reversal potential and the outward currents. Statistical analysis: two-way ANOVA with repeated measures, p < 0.001. (C) Left, representative currents elicited by ramp protocol of HEK cells expressing Kir2.1 dimers. Middle, current densities at −120 mV. Right, current density at −120 mV normalized to mean current density from cells expressing Kir2.1 WT dimers from the same experiment (WT-WT was compared to WT-V77E in N = 7, to WT-M307V in N = 4 and to WT-R67W in N = 6 experiments). Statistical analysis: one-way ANOVA (Dunn’s test, comparison to WT Group). (D) Kir2.1 dimers RNA (1 ng/oocyte) was injected in Xenopus oocytes and recorded in 24 mM K+ bath solution. a. Top, representative evoked currents by a voltage ramp protocol. Bottom, average current amplitudes at −80 mV. Statistical analysis: t-test compared to the WT-WT group. N = 1 experiment. b. Reversal potentials (Vrev) and rectification index calculated from the current elicited by voltage ramps, n = 10–11, N = 2 experiments. Statistical analysis: one-way ANOVA.
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Figure 5. The calibrated VSP activation protocol testing Kir2.1 sensitivity to PIP2 depletion by VSP in HEK cells. (A) A representative four sweeps (I–IV)-protocol used to quantify Kir2.1 current amplitude reduction following VSP activation by depolarization. Each sweep started with a voltage ramp, followed by a 100-mV voltage step (for 1, 3, 5, or 7 s) and ended with a second voltage ramp. An example of currents elicited in a HEK cell transfected with Kir2.1 WT dimer and VSP in 1:0.16 DNA ratio. (B–D) Summary of the maximal amplitude ratio in each sweep after/before VSP activation (left Y-axis), also displayed as % VSP effect (right Y-axis, in pink). Voltage-protocol on the right. (B) At DNA ratio Kir2.1:VSP 1:0 (△), n = 10, 6, 4, 3; 1:0.16 (▼), n = 63, 63, 52, 44; 1:0.5 (•), n = 26, 22, 19, 18; 1:2 (○), n = 13, 10, 9, 8 for VSP activation at 100 mV for 1, 3, 5, and 7 s, respectively. Statistical analysis: one-way ANOVA at 5-s VSP activation [comparison to 1:0.16 (▼) group]. (C) VSP activation by steps to increasing voltages in each sweep (right). Currents amplitude ratio and the corresponding VSP effect from HEK cells transfected with WT dimer and VSP in 1:0.5 DNA ratio (left). n = 8, 8, 7, 6 for 5-s VSP activation at 50, 70, 90, and 110 mV, respectively. (D) Currents amplitude ratio and the corresponding VSP effect in Kir2.1-GFP monomers. At DNA ratio 1:0.16 of Kir2.1-GFP M307L:VSP (△), n = 5, 5, 4, 3; Kir2.1-GFP WT : VSP 1:0.16 (▼), n = 8, 9, 4, 4; Kir2.1-GFP WT : VSP 1:0.5 (•), n = 22, 19, 14, 9 for 1, 3, 5, and 7 s, respectively. Statistical analysis: t-test at 5-s VSP activation of the WT expressing groups, p = 0.004.
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Figure 6. Increased sensitivity of mutant dimers to PIP2 depletion using the calibrated VSP activation protocol. (A–C) Currents in HEK cells expressing Kir2.1 dimers and VSP. (A) Representative ramp currents before (black) and after (red) VSP activation by a 3-s step to 100 mV. Traces of the WT dimer are the same as sweep II in Figure 5A. (B) Currents amplitude ratio and the corresponding VSP effect recorded from cells transfected with Kir2.1 dimer:VSP DNA ratio of 1:0.16. WT-WT is as summarized in Figure 5B (n = 63, 63, 52, 44). WT-V77E, n = 20, 20, 22, 17; WT-M307V, n = 16, 15, 12, 7; WT-R67W, n = 26, 24, 20, 15 for VSP activation by 100 mV step for 1, 3, 5, and 7 s, respectively. Statistical analysis: one-way ANOVA (Dunn’s test, comparison to WT-WT group). At 5-s VSP activation: WT-V77E and WT-R67W, p < 0.001; WT-M307V, p = 0.003. The dashed magenta lines in panel B illustrate the normalizing value for the VSP effect on panel C. (C) Normalized VSP effect at a 5-s VSP activation compared to WT dimer. Statistical analysis: one-way ANOVA (Dunn’s test, comparison to WT-WT Group). (D–H) Currents in Xenopus oocytes expressing Kir2.1 dimers without (D, E) and with VSP (F–H). Representative currents in naïve and Kir2.1 dimers expressing oocytes (D), and the average current amplitude ratio and the corresponding VSP effect (left, n = 7–8; right, n = 3–4) (E). (F) Representative currents elicited in oocytes using the VSP activation protocol, and following subtraction of the averaged current from naïve cells. I1 and I2 of sweep I are in black, I2 of the sweeps II in red, III in blue, and IV in purple. Current amplitude ratio and the corresponding VSP effect (G) and the normalized VSP effect per experiment, at 5-s VSP activation (H). N = 2 experiments. Statistical analysis at 3- and 5-s VSP activation was one-way ANOVA (Dunnett’s test, comparison to WT-WT group). In panel G, at 5-s VSP activation, WT-R67W, p = 0.005. At 7-s VSP activation: statistical analysis was using Kruskal–Wallis one-way ANOVA. In panel H, statistical analysis was using Kruskal–Wallis one-way ANOVA. WT-R67W, p = 0.003. The dashed magenta lines in panel G illustrate the normalizing value for the VSP effect in panel (H).
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Figure 7. BGP-15 reduces the sensitivity of mutant dimers to PIP2 depletion using the calibrated VSP activation protocol. (A) Left, averaged current densities at −120 mV from HEK cells expressing Kir2.1 WT dimers with and without BGP-15 treatment. Top, current densities, bottom, current densities in cells that were incubated with BGP-15 and normalized to the mean current density in non-treated cells from the same experiment. Statistical analysis: Mann–Whitney rank-sum test. Right, ramp current waveform normalized to the current at −120 mV, from non-treated cells (same as WT dimer in Figure 4B), and BGP-15 treated cells. Statistical analysis: two-way ANOVA with repeated measures, p < 0.001. (B) Representative currents elicited in non-treated and 50 µM BGP-15 incubated HEK cells. Example in cells transfected with Kir2.1 WT dimer and VSP in 1:0.5 DNA ratio. (C) Average current amplitude ratio and the corresponding VSP effect of WT dimer and VSP expressing cells, following BGP-15 treatment. Top, n = 13, 12, 12, 11 for BGP-15-treated and n = 25, 21, 19, 15 for non-treated cells. N = 3 experiments. Middle, n = 16, 13, 13, 13 for BGP-15-treated and n = 17, 14, 11, 10 for non-treated cells. N = 2 experiments. Bottom, n = 12, 11, 11, 5 for BGP-15-treated and n = 13, 10, 9, 7 for non-treated cells. N = 2 experiments. Statistical analysis: t-test comparing treated with non-treated groups in the same protocol. (D) Average current amplitude ratio and the corresponding VSP effect of mutant dimer and VSP expressing cells with and without BGP-15 treatment. Kir2.1:VSP DNA ratio was 1:0.16. VSP effect from non-treated mutant dimers is the same as in Figure 6B. For BGP-15 treated groups, WT-V77E n = 18,16,13,7 from N = 3 experiments, WT-M307V n = 5,7,8,3 from N = 1 experiment, WT-R67W n = 7, 8, 6, 6 from N = 2 experiments. (E) Normalized VSP effect on Kir2.1 currents following a 5-s depolarization step to 100 mV. The mean VSP effect in non-treated cells (represented by dashed magenta lines in (C, D), of each experiment, is the normalizing value for the VSP effect in panel (E). Statistical analysis: t-test.
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Figure 8. Structural predictions of ATS-associated Kir2.1 variants. (A) Left, wheel plot illustration of the residues in Kir2.1 N-terminal slide helix. Polar residues in magenta, non-polar residues in cyan. Mutations in the underlined residues are disease-associated (Table 1). Arrow points on the ATS-associated residue in this report. Middle, polar (magenta) and non-polar (cyan) residues in chicken Kir2.2 + PIP2 structure (PDB ID:3SPI). V75 in Kir2.2 (green) is the homolog of V77 in Kir2.1. Side chains of K183, K188 (light blue) and R186 (orange) are illustrated. Right, V75E mutation was introduced in Kir2.2 + PIP2 structure and underwent a structural refinement. Putative interaction with R186 is shown in yellow. (B) Functional properties of Kir2.1 variants in position 307.
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