Cryo–electron microscopy unveils unique structural features of the human Kir2.1 channel

Figures

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  Fig. 1.. Cryo-EM map and atomic structure of the human Kir2.1 channel. (A) Sharpened cryo-EM map of human Kir2.1 obtained at 4.3-Å resolution (bottom, side, and top views). (B) Side view of human Kir2.1 atomic structure fitted in the cryo-EM map (white surface). The chains A and C are in cartoon and highlight the typical structural features of Kir channels: Outer helix (M1, yellow), inner helix (M2, cyan), pore helix (dark blue), slide helix (red), selectivity filter (magenta), extracellular region (light blue), G-loop (black), and helix bundle crossing (HBC) region. The Sr2+ (green) and K+ (purple) ions inside the pore channel are represented as spheres. Chains B and D are in gray ribbons for clarity. Gray horizontal bars indicate the plasma membrane. The TMD and CTD are indicated. (C) Top view of the human Kir2.1 atomic structure fitted in the cryo-EM map. The four chains (A to D) are in a blue cartoon. The cysteine residues (C122 and C154) involved in the intrasubunit disulfide bridge are highlighted as red sticks. Protein dimensions are shown.

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  Fig. 2.. Ions’ location along the pore in the human Kir2.1 channel cryo-EM structure. Cryo-EM densities modeled as Sr2+ (blue mesh at 5σ) and K+ (green mesh at 2.5σ) ions inside the pore of human Kir2.1 cryo-EM atomic structure (blue ribbon). Insets: Detailed views of the regions where the ions are located. The distances between these ions and the negatively charged atoms of the closest residues (yellow sticks) inside the pore channel are indicated.

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  Fig. 3.. Pore radius analysis of human Kir2.1 channel structure and constriction points. (A) Plot of the pore radius between van der Waals surfaces. The constriction points (selectivity filter, I176, M180, and A306) and the position of the two Sr2+ ions are highlighted. (B) Three constriction points below the selectivity filter: I176 (yellow sphere), M180 (orange sphere), and A306 (purple sphere). Chains A and C are in light blue and green cartoons, respectively. Chains B and D are in gray ribbons for clarity. Sr2+ (blue) and K+ (red) ions are represented as spheres. The insets show the zoom of the three constriction points, where the spheres represent the van der Waals surfaces. (C) Comparative analysis of pore radius (stippled surface) between van der Waals surfaces among human Kir2.1, chicken Kir2.2, and mouse Kir3.2 channel structures (light gray ribbons). Along the central ion conduction, red indicates insufficient space for a water molecule, green indicates that one or two water molecules could fit, and blue indicates that many water molecules could fit. Constriction points were considered where the red volume was visualized. Horizontal dashes were placed to compare the constriction…

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  Fig. 4.. Biophysical and functional assays on Kir2.1 WT and R312H mutant. (A and B) Comparative binding kinetics of PIP2 on Kir2.1 WT (A) and R312H mutant (B) immobilized on a CM5 chip. Five sequential injections of PIP2 are shown, 1.25 to 20 μM, at 25 μl/5 min. In color, experimental data; in black, fitted curve. The single-cycle kinetics assays were carried out in triplicate; only one replica is shown. The SPR response is expressed in response units relative to the time in seconds. (C and D) Electrophysiological recordings of Kir2.1 WT (C) and R312H (D) reconstituted in lipid bilayers in the presence of 1% PIP2 at −100 mV. Kir2.1 WT shows an open probability (Po) of 70%, and Kir2.1 R312H has a Po of less than 1%.

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  Fig. 5.. Interaction network between R312, H221, and E303 residues in the human Kir2.1 channel cryo-EM structure. Cartoon representation of the human Kir2.1 channel cryo-EM structure highlighting (inside the circle) the location of the R312 residue. The insets show the intersubunit interaction network of R312 in the WT (top) and that of H312 in the R312H mutant (bottom) between chains A and B. The chains A, B, C, and D are shown in green, blue, gray, and yellow, respectively. Residues R218, K219, H221, E303, and A306 are represented as yellow sticks. The chains are indicated in parenthesis. Salt bridge distances are highlighted in red dashes and the other distances are highlighted in black dashes.

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  Fig. 6.. Description of extension/compression conformational movement of the human Kir2.1 channel using NM analysis. (A) Ribbon representation of the human Kir2.1 cryo-EM structure after displacement of −3.0 Å (green; compacted structure) and +3.0 Å (blue; expanded structure) along mode 5. The longitudinal length and the Cα-Cα distance between residues D78 and K219 (represented by spheres) are indicated in angstroms. (B) Description of the compression movement on the cartoon representation of the human Kir2.1 structure (cyan). Red arrows show Cα displacement from the reference structure toward the −3.0 Å compacted structure along the mode 5. Orange spheres represent D78 and K219. Gray horizontal bars indicate plasma membrane boundaries.

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  Fig. 7.. Description of the opening of the PIP2-binding site of the human Kir2.1 channel using NM analysis. (A and B) Description of the conformational movement on the cartoon representation of the human Kir2.1 structure. Chains A, B, C, and D are in cyan, orange, green, and yellow, respectively. Red arrows show Cα displacement from the reference structure toward the displacement of +3.0 and −3.0 Å along the modes 1 (A) and 2 (B).

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  Fig. 8.. Tethering of K64 and R67 to the membrane displayed in MD simulations. (A) Human Kir2.1 cryo-EM structure and membrane (POPC bilayer) system used in MD simulations. The Kir2.1 channel structure is represented in a green cartoon, and the residues K64 and R67 of chain B are highlighted. (B to E) Trajectory analysis of the K64 and R67 residues along the 200 ns of MD simulations in some representative chains of the three replicates. In each of these panels, snapshots along MD simulations are shown: 0 ns (green), 50 ns (cyan), 100 ns (magenta), 150 ns (yellow), and 200 ns (salmon). K64 and R67 residues are highlighted in sticks. (B) to (E) show the distances between the K64 and R67 residues to the plane of the phosphate groups from the inner membrane. To calculate these distances, the inner membrane was centered at Z = 0. The 0 value represents the membrane level. (B) Chain B, replica 2; (C) chain D, replica 1; (D) chain B, replica 3; (E) chain D, replica 2.