Voltage gating and 4-aminopyridine inhibition in the Shaker Kv channel revealed by a closed-state model

Figures

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  Figure 1. Obtaining a closed state model for the Shaker channel. (A) Plot of R4 α carbon displacement versus pLDDT for AF2 generated models of Shaker VSD (6000 models). Points colored according to the predicted template modeling (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: 7sip (22)) 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 versus pLDDT for AF2 generated models (400 models per plot) of Shaker tetrameric channel (residues 215–495) 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 and 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…

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  Figure 2. Conformational changes from open to 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 and C) Intramembrane view of two opposed subunits of the R4up open state (B) and R4down closed state (C). Residues I470, V474, and V478 side chains are shown in ball and stick representation. 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 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 and 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) G-V relationship for the WT channel (black), M393A (red), and M393P (blue) mutants. (D) Q-V relationship for the WT channel (black), M393A (red), and M393P (blue) mutants. (E) G-V relationship for ILT (black), ILT-M393A (red), and ILT-M393P (blue) mutants. (F) Q-V relationship for ILT (black), ILT-M393A (red), and ILT-M393P (blue) mutants. Results shown as mean ± SEM, n ≥ 4. Continuous lines correspond to a two-state fit (Eq. 1) in the GV curves and an interpolation between points in the Q-V curves.

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  Figure 4. Identification of the 4-aminopyridine 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, 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, half inhibitory concentration [IC50] = 170 μM), L399A (red squares, IC50 = 32 μM), and V467I (blue triangles, IC50 = 28 mM). Results shown as mean ± SE, n ≥ 4.

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  Figure 5. Proposed gating mechanism and 4-aminopyridine 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) shows 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. Intracellular 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.