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Fig. 1. Structural features of Kir channels and differences in the static conformations of the WT and I223L mutant of chicken Kir2.2. a, b Overall functional tetramer of Kir channels with each subunit in a different color (a) and structural components of each subunit (b). c, d Conformational difference between the crystal structures of the WT (PDB code: 3JYC) and I223L mutant (PDB code: 3SPJ) in the absence of the PIP2; this difference was quantified using residue displacement (c) and is structurally illustrated in CPK and Licorice to highlight the key residues with displacements of more than 1 Å, and the overall channel is shown in newcartoon (d). e, f Conformational adjustments induced by binding of the PIP2 to the WT (black) and I223L mutant (red) (e) and the corresponding structural diagrams (f). g, h Conformational difference between the crystal structures of the WT (PDB code: 3SPI) and I223L mutant (PDB code: 3SPH) in the presence of PIP2 shown as a quantification of the residue displacement (g) and an intuitive presentation of the conformational superposition (h). The residue displacement was defined as the distance between the heavy atom centers of each residue between the two structures after alignment of the two transmembrane helixes
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Fig. 2. Channel-PIP2 interaction difference between the WT and I223L mutant of chicken Kir2.2. Global PIP2 binding energies of the artificially PIP2-added WT (WT-3JYC+) and I223L mutant (I223L-3SPJ+) systems and of the PIP2 co-crystallized WT (WT-3SPI) and I223L mutant (I223L-3SPH) systems are presented in a. Comparisons of the energy distributions among each PIP2-binding site between the WT-3SPI and I223L-3SPH systems, the WT-3JYC+ and WT-3SPI systems, and the I223L-3SPJ+ and I223L-3SPH systems are shown in b, c, and d, respectively. Orientation differences of the residues R78 and R186 between the PIP2-absent (Licorice) and PIP2-present (CPK) crystal structures of the WT-3JYC and I223L-3SPJ mutant are illustrated in e and f, respectively. The interaction energy includes non-covalent van der Waals and electrostatic interactions, and the global energy or distribution of each binding site for each system is presented as the mean ± SD of the tetramer during the last 10 ns of the equilibration simulation. Only a partial subunit of the Kir channel is shown in newcartoon with different colors for clarity
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Fig. 3. Dynamic conformation differences between the PIP2-present WT and I223L mutant of chicken Kir2.2. Distributions of the R78-R186 distance and TMD-CTD distance for systems of artificially PIP2-added WT (WT-3JYC+, black) and I223L mutant (I223L-3SPJ+, white) and of the PIP2-co-crystallized WT (WT-3SPI, gray) and I223L mutant (I223L-3SPH, dark gray) are shown in a and b, respectively. The correlation is shown in c with the means ± SD. The conformational comparisons with emphases on the orientations of R78 and R186 (d, f) and the upward motions of the CTD domains (e, g) for both the WT (d, e) and I223L mutant (f, g) systems are shown in (d–g). The R78-R186 distance was defined as the distance between the geometrical center of the NH1 and NH2 atoms of R78 and that of R186 from the same subunit. The TMD-CTD distance was defined as the distance between the D76-Cα and K220-Cα from the adjacent subunit in the clockwise direction. Only the data from the last 10 ns of equilibration of each system are shown here, and the average in c was obtained from the tetramer after the last 10-ns trajectory average of each subunit. Subunit A in the 50-ns snapshot of each system was used for conformational comparisons in d–g with a presentation similar to that in Fig. 2
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Fig. 4. Difference in the interaction networks of the PIP2-absent WT-3JYC and I223L-3SPJ mutant of chicken Kir2.2. Key amino acid pairs of the intrasubunit interaction with significantly different H bond interactions (a) and the corresponding location (b) are shown. Those of the intersubunit interaction are presented in c, d. The distributions of the sidechain orientation of I223 and L223 are shown in e with a bin size of 15°. The H bond interactions are presented as the mean ± SD of the tetramer after the last 10-ns trajectory average of each subunit. The key amino acids are presented as CPK with labeling of the subunit and the corresponding secondary structures. I(L)223 is shown in Licorice for clarity
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Fig. 5. Evolution of the R78-R186 distance and TMD-CTD distance in PIP2-deleted WT-3SPI− and I223L-3SPH− mutant systems of chicken Kir2.2. The evolutions of the R78-R186 distance (a, b) and TMD-CTD distance (d, e) of the WT (a, d) and I223L mutant (b, e) are presented for each subunit. The corresponding distributions are shown in c and f, respectively. The data of c, f were collected from the tetramer during the last 10 ns of equilibration. References 1 and 2 for the R78-R186 distance (a, b) or TMD-CTD distance (d, e) are the corresponding distances in the crystal structures of the PIP2-absent (Reference_1) and PIP2-present (Reference_2) WT and I223L mutant
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Fig. 6. Differences in the gating kinetics of mouse Kir2.1 and human Kir2.2 induced by the mutual mutation of the Ile/Leu site in the CD loop. Dose-response curves (a, b), PIP2 antibody inhibition trace (d, e), and PIP2-induced gating trace (g, h) of the mouse Kir2.1 WT and L222I mutant (a, d, g) and the human Kir2.2 WT and I223L mutant (b, e, h) were experimentally measured. The corresponding summary data of the peak current, the inhibition time constant τ
off, and the recovery time constant τ
on are provided in c, f, and i, respectively. τ
off represents the time needed for the PIP2 antibody to inhibit the Kir channel current to half of its initial amplitude, and τ
on represents the time needed for PIP2 to increase the Kir current to half of its maximum amplitude. Solid lines are the Hill fitted lines to the data points of the Kir channels. Each data point is the average of five to six cells from at least three independent experiments. The dose-response curve and all summary data are expressed as the mean ± SE, and the inhibition and activation trace data are shown with only the mean values for clarity
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Fig. 7. Comparison of the intrasubunit H222-E304 interaction between the PIP2-absent and PIP2-present Kir2.2 WT and I223L mutant systems. The quantified H bond numbers are presented in a as the mean ± SD of the tetramer after the last 10-ns trajectory average of each subunit, and the visual conformations of the 50-ns snapshot are presented in b–e for the PIP2-absent WT-3JYC (b) and I223L-3SPJ mutant (c) systems and for the PIP2-present WT-3SPI (d) and I223L-3SPH mutant (e) systems, respectively, with blue
newcartoon for the Kir channel subunit and Name CPK for H222 and Licorice for E304
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Fig. 8. Differences in the gating kinetics of mouse Kir2.1 induced by the mutation of H222L in the CD loop. The PIP2 antibody inhibition trace (a) and PIP2-induced gating trace (c) of mouse Kir2.1 WT (squares) and H222L mutant (triangles) were experimentally measured. The corresponding summary data of the inhibition time constant τ
off (b) and recovery time constant τ
on (d) are also provided. The solid lines are the Hill fitted lines to the data points of the Kir channels. Each data point is the average of five to six cells from at least three independent experiments, and the inhibition and activation trace data are shown with only the mean values for clarity. The summary data are expressed as the mean ± SD
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