Atomic-level investigation of KCNJ2 mutations associated with ventricular arrhythmic syndrome phenotypes

Figures

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  Fig. 1. Functional assessment of Kir2.1-WT and clinical mutation. (a) Representative whole cell current traces are obtained by stepping the membrane from a holding of -50 mV to voltages ranging from -120 to +50 mV. (b) Mean Steady-state current-voltage relationship for WT Kir2.1 with inset highlighting the outward current at physiologic voltages. Currents are calculated following barium subtraction. The error bar represents the standard error of the mean (SEM) (c) Mean outward IK1 current measured at -50mV, and all three mutants showing no current at baseline compared to WT. Data expressed as mean ± SEM. Mutants show significantly reduced current compared to WT. Statistical significance determined by one-way ANOVA with Dunnett’s multiple comparisons test (*** p < 0.001 for all mutants vs. WT) (d) Western blot of lysates from HEK stable cell lines expressing each of the clinical Kir2.1 mutations along with ß–actin loading control.

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  Fig. 2. Kir2.1 full-length 3D structure and pore analysis. (a) Full-length Kir2.1 structure in open conformation with complete N and C-terminal regions (b) Pore radius of open and closed conformation also showing gating residues/construction points. Upper gates are indicating the helix bundle crossing region. The lower gate is by the G-loop region (c) Open channel surface with enhanced structural feature Selectivity filter (SF) using ball-and-stick representation with carbonyl oxygen atoms (red balls). Black lines represent the distances between backbone oxygen of SF residues (141-147). Five distinct positions of K+ in the SF, sites S0-S4 are labeled. The upper gate residue inset showing I176, G177 and M180 forms the gating structure. G-loop inset showing the lower gating residues A306 and M307 (d) Location and amino acid change (inset) for the KCNJ2 mutations studied in this article.

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  Fig. 3. Site-directed mutation analysis of R to Q change at location 67 in open and closed state of Kir2.1. (a) Interaction profile of R67 with neighboring residues in closed conformation state highlighting the hydrogen bond interaction (b) Overlay of WT structure and change in residue from R to Q and change in hydrogen bond interactions (c) Interaction profile of R67 with neighboring residues in open conformation state highlighting the hydrogen bond interaction (d) Overlay of WT structure and change in residue from R to Q and change in hydrogen bond interactions in open conformation state of the channel.

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  Fig. 4. Site-directed mutation analysis of R to L change at location 218 in open and closed state of Kir2.1. (a) Interaction profile of R at location 218 with the neighboring residues in closed conformation state highlighting the hydrogen bond interaction (b) Overlay of WT structure and change in residue from R to L and change in hydrogen bond interactions (c) Interaction profile of R at location 67 with the neighboring residues in open conformation state highlighting the hydrogen bond interaction (d) Overlay of WT structure and change in residue from arginine to L and change in hydrogen bond interactions in open conformation state of the channel.

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  Fig. 5. Site-directed mutation analysis of G to D change at location 300 in open and closed state of Kir2.1. (a) Interaction profile of G at location 300 with the neighboring residues in closed conformation state highlighting the hydrogen bond interaction (b) Overlay of WT structure and change in residue from G to D and change in hydrogen bond interactions (c) Interaction profile of G at location 300 with the neighboring residues in open conformation state highlighting the hydrogen bond interaction (d) Overlay of WT structure and change in residue from G to D and change in hydrogen bond interactions in open conformation state of the channel.

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  Fig. 6. RMSD and RMSF plots of Kir2.1-WT and clinical mutants (R67Q, R218L, G300D) in opened and closed conformation state. (a) Combined RMSD plot of Kir2.1-WT and mutants overall protein in open conformation state (b) Combined RMSD plot of Kir2.1-WT and mutants overall protein in closed conformation state (c) showing movement of ion through selectivity filter in open state of the channel during simulation time (d) RMSF plots for each Kir2.1-WT open state showing all four chains the x-axis showing residue number and Y-axis is labeled as RMSD in Å that showing residue fluctuation throughout simulation time from reference structure RMSF plots for each Kir2.1-WT and Mutants showing 4 chains and x-axis are labeled with residue number and Y-axis is labeled as RMSD in Å that showing residue fluctuation throughout simulation time from reference structure. (e) RMSF plot of Kir2.1-WT in closed state (f) RMSF plot of R67Q mutation in closed state (g) (g) RMSF plot of R67Q mutation in open state (h) RMSF plot of R218L mutation in closed state (i) RMSF plot of R218L mutation in open state (j) RMSF plot of G300D mutation in closed state k) RMSF plot of G300D mutation in open state.

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  Fig. 7. PCA analysis of MD trajectories of Kir2.1-WT and mutants in open and closed conformation the inset underneath each PCA plot shows the representative frame from local minima and explains the change with respect to reference structure. (a) PCA plot explains the projection of molecular dynamics trajectories onto first two principal components (PC1 and PC2). Each point represents a snapshot from the trajectory, and clusters indicate regions of the conformational space where the protein spends significant time, corresponding to energy minima. The color density indicates the population density of the snapshots, with yellow representing higher density and purple representing lower density. for Kir2.1-WT in closed state (b) PCA plot of R67Q in closed state (c) PCA plot of R218L in closed state (d) PCA plot of G300D in closed conformation state. (e) PCA plot of R67Q in open state (f) PCA plot of R218L in open state (g) PCA plot of G300D in closed conformation state. The structure underneath each plot is showing the represented frame structure overlay on refence frame in open conformation state.

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  Fig. 8. PCA based NM analysis of Kir2.1-WT and ATS-mutations (R67Q, R218L, G300D) in open and closed state. The porcupine plots illustrate the likely modes of large-scale collective motion, direction, and magnitude of atomic displacements along the principal normal modes derived from PCA for (a) Kir2.1-WT closed and (b) R67Q closed, (c) R218L closed, and (d) G300D in the closed state. The second row shows (e) Kir2.1-WT open state, (f) R67Q open, (g) R218L open, and (h) G300D in open conformations state. Each green arrow represents the direction and relative magnitude of motion for Ca atoms of each residue.