Structural Basis for Voltage Gating and Dalfampridine Binding in the Shaker Potassium Channel

Figures

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  Figure 1:. Obtaining a closed state model for the Shaker channel. A) Plot of R4 alpha carbon displacement vs pLDDT for AF2 generated models of Shaker VSD (6000 models). Points colored according to pTM score. MSA subsampling parameters used was 4:8 (“max_seq”: “max_extra_seq”). Circle indicates the selected VSD model, dotted red line indicates the displacement in the WT structure. B) Comparison between the VSD from the Shaker WT structure (R4up, PDB: 7sip17) and the selected VSD model (R4down), showing position of residues F290 and R4. S3 was removed for clarity. C-E) Plot of quadrilateral area at the level of V474 vs pLDDT for AF2 generated models (400 models per plot) of Shaker tetrameric channel (residues 115–495) using R4down template (C), no template (D) or R4up template (E). Dotted line indicates the area in the WT structure. MSA subsampling parameters used was 16:32. Circle in C indicates the selected model. Side plots show the kernel density estimates distribution for each axis. F,G) Intracellular view of the tetrameric Shaker channel in the closed (R4down) (F) and open R4up (G) conformations, each subunit is shown in a different color. Surface representation is shown for residues of…

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  Figure 2:. Conformational changes from the open to the closed state of Shaker. A) Superposition of the S4 to S6 region of a single subunit in the open (R4up, blue) and closed (R4down, red) states of the Shaker channel. Arrows indicate two major conformational changes between states: the movement of the S4 helix (shown as the displacement of R4) and the S6 C-terminus. B,C) Intra-membrane view of two opposed subunits of the R4up open state (B) and R4down closed state (C). Residues I470, V474, and V478 sidechains are shown in purple. The pore diameter obtained through HOLE calculations is shown in gray. D) Pore radius analysis for the open (blue line) and closed (red line) states. The y-axis represents the position along the pore axis aligned with the structures in B and C. The position of key residues I470, V474, and V478 are indicated, highlighting the region of major constriction in the closed state. Dashed red and green lines indicate radii of 1.5 Å and 3.3 Å corresponding to the ionic and hydrated radius of potassium respectively.

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  Figure 3:. S4-S5 linker movement during opening involves breakage of a backbone hydrogen bond. A,B) S4-S5 linker position relative to the S5 in the R4up open conformation (A) and the R4down closed conformation (B). The dashed line shows the distance between K390 carbonyl group and M393 amide group. The cartoon exemplifies the movement of the S4-S5 linker relative to the S5. C) Steady-state G-V (blue) and Q-V (green) curves for the WT channel (continuous line), M393A (triangles) and M393P (circles) mutants. D) Steady-state G-V (blue) and Q-V (green) curves for ILT (continuous line), ILT-M393A (triangles) and ILT-M393P (circles) mutants. Results shown as mean ± SEM, n ≥ 4.

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  Figure 4:. Identification of the dalfampridine (4-AP) binding pocket. A) Docking analysis results showing the hydrophobic cavity where 4-AP binds, formed by residues in the S5 (L399, I400, L403) and S6 (V467, L468, T469, L472, P473). Scale bar 1 nm. B) Inhibition of ionic currents by 0.4 mM 4-AP in WT, L399A, and V467I mutant channels. C) Dose-response curves for the WT channel (green circles, IC50 = 170 μM), L399A (red squares, IC50 = 32 μM) and V467I (blue triangles, IC50 = 28 mM). Results shown as mean ± SEM, n ≥ 4.

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  Figure 5:. Proposed gating mechanism and dalfampridine binding for the Shaker channel family. Top (R4up) Shaker channel in the active open state, where the VSD S4 segments (purple) are in the “R4up” conformation, and the channel pore is open (green). Bottom (R4down): Channel in its partially activated closed state, where the VSD S4 segments are in the “R4down” position. The downward movement of the S4 segments triggers a downward and radial displacement of the S4-S5 linker (orange) that produces a contraction and rotation of the S6 C-terminal helices, closing the pore and allowing for the binding of 4-AP. Side view (left) show two opposed subunits from a view parallel to the membrane, residues F290, R377 (R4) in the VSD and K390 and M393 in the S4-S5 linker hinge are shown. Bottom view (right) shows S4-S5 linker (orange), S5 and S6 (green) from the intracellular side. Residues I470 (yellow), V474 (gray) and V478 (pale blue) are shown. Created with BioRender.com