A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

Figures

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  Fig. 1. C-type inactivation and selectivity filter gating. a Topology diagram of voltage-gated K+ (Kv) channels. Helices S1-S4 form the voltage-sensor, while helices S5-S6 form the pore comprising the selectivity filter. b Slow C-type inactivation renders the selectivity filter of K+ channels non-conductive. c The bacterial KcsA channel is a model for the pore of Kv channels. d, e The selectivity filter (T74-G79 in KcsA) and residues W67, D80 are conserved while the residue corresponding to E71 in KcsA is substituted by a valine in Kv channels.

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  Fig. 2. X-ray structures of WT and E71V KcsA. a The conductive selectivity filters of WT and E71V KcsA, both with closed lower gates. b Comparison of the constricted selectivity filter of WT KcsA (with a closed lower gate) and the narrowed selectivity filter of the open-gate structure of E71V KcsA. Both structures were obtained in the absence of K+. The Cα-Cα distances between residues of opposing protomers are shown. The closed-state and open-state E71V KcsA structures were obtained with 5 mM and 0 mM K+, respectively. The green mesh shows the difference density map, the orange mesh the anomalous density. The difference (green) map is for the K+ ions and contoured at 8 σ. The orange density map (anomalous) is contoured at 5 σ. An ion cannot be clearly assigned to the density due to the weak signal. c The W67 sidechain flips in the E71V open-state structure, breaking the hydrogen bond with the D80 sidechain that is important for inactivation in Kv channels12,19,44,45.

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  Fig. 3. Comparing inactivation gating by ssNMR. a Superposition of 2D NH ssNMR spectra of (a) closed-conductive (red) and open-inactivated (cyan) WT KcsA channels acquired in membranes. Arrows indicate major signal shifts of key residues. b 2D NH spectra of E71V KcsA in closed-conductive (yellow) and open-inactivated (pink) states. The E71V KcsA filter shows two conformations A and B, highlighted with blue labels, under inactivation conditions. V76, G77, and Y78 of the canonical conductive filter (1K4C) are labeled in brown for clarity. c Schematics of the changes that accompany inactivation in WT and E71V. The inactivated WT filter constricts at sites S2 and S3, accompanied by a rupture of the E71–Y78 interaction and the influx of water into a cavity behind the filter. NMR data also reveal major structural changes at the S1 site and the pore mouth. The inactivated E71V filter does not constrict like KcsA, does not feature structural water in the cavity, and shows two conformations. d Upper panel: 2D NH and 2D CC spectra of the WT filter showing large chemical shift perturbations (CSPs) of key residue Y78 upon constriction. Lower panel: HN and Cα CSPs of the open-inactivated state in…

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  Fig. 4. X-ray structures of barium-bound WT and E71V KcsA. a Closed-gate and b open-gate structures of WT KcsA with Ba2+ bound at the S4 position of the selectivity filter. The filter is sharply constricted at the level of G77 in both the closed-gate and in the open-gate structures. c Closed-gate and d open-gate structures of E71V KcsA with Ba2+ bound at the S3 position of the selectivity filter. The filter is conductive in the closed-gate structure and narrows in the open-gate structure. Cα-Cα distances between like-residues in opposing protomers are indicated.

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  Fig. 5. ssNMR barium-binding studies. 2D NH spectra of a closed-conductive WT KcsA (red), b open-inactivated WT (cyan), c closed-conductive E71V KcsA (yellow), and d open-inactivated E71V (pink). Superimposed (in gray) are 2D NH spectra after equilibration with 5 mM Ba2+ solution of the same pH as the original sample. Major Ba2+-caused signal changes are indicated by arrows. The two conformations A and B of the E71V filter under inactivating conditions are highlighted by blue labels. Blue lines show that Ba2+ binds the E71V filter with a conformation similar to conformation B at neutral and acidic pH. e, f Schematics of Ba2+ binding to the WT and E71V selectivity filters. Barium binds at the S4 site of the conductive, however not the constricted WT filter. Barium binds the conductive and the inactivated E71V filter at the S3 or S2 site. g 2D CC spectra show the effect of Barium binding to key residues T75 and G77 of the S2-S4 filter sites. h Backbone carbonyl CSPs in reference to the conductive E71V filter show that the Ba2+-bound states of E71V at pH7 and pH3 closely resemble state B of the inactivated E71V filter. Source data are provided as a Source Data file.

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  Fig. 6. Dynamics measurements in different gating modes. a 15N rotating-frame ssNMR relaxation rates (R1rho) that report on slow molecular motions in closed-conductive (cc) and open-inactivated (oi) WT KcsA (red, cyan) and E71V (yellow, pink), measured at 700 MHz and 60 kHz MAS. The error bars show the standard error of the fit. Source data are provided as a Source Data file. b R1rho signal decay curves for selected filter residues. Symbols mark data points and lines represent best fits. c The valine behind the E71V filter is in direct contact with the hydrophobic V76 and M96 sidechains. d Plots of the differences in the dynamics between the closed-conductive (cc) states of E71V and WT, and for each WT and E71V the dynamics differences between their open-inactivated (oi) and closed-conductive (cc) states. Source data are provided as a Source Data file. e Illustration of the site-resolved selectivity filter dynamics. The size of the green spheres represents the R1rho relaxation rates.

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  Fig. 7. MD simulations for the structures of the KcsA E71V mutant in different conditions. a Open-gate and K+ bound, b open-gate and Na+ bound, c open-gate and Ba2+ bound, d closed-gate and Ba2+ bound, and e closed-gate and K+ bound. The first and last snapshots of the selectivity filter for each MD trajectory are respectively shown (right and left panel) in licorice representation for backbone and in van der Waals representations for K+ (purple), Na+ (orange), and Ba2+ (pink) ions. In the middle panel is the time series of the cross-subunit distance (r) between the Cα atoms of G77 of diagonally opposed monomers A and C (green), and B and D (red). Two levels representing conductive and constricted states are illustrated in black dashed line. The z-coordinate of ions in the selectivity filter are shown as solid dots (with the same colors as the conformation images). The z-coordinates of carbonyl oxygens of G79, Y78, G77, V76, and T75 are respectively shown in red lines to indicate the position of ion binding sites.

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  Fig. 8. The dynamics of the W67 and D80 side chains in MD simulations. a Open-gate and K+ bound, b open-gate and Na+ bound, c open-gate and Ba2+ bound, d closed-gate and Ba2+ bound, and e closed-gate and K+ bound. The crystal structures are shown as reference (labeled with their PDB ID) in licorice representation and carbon atoms in white with multiple snapshots from simulations. The side-chain conformations are shown in red licorice, and these snapshots are selected for every 100 ns in all four subunits.

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  Fig. 9. Free energy landscapes to assess the conformational preferences of the selectivity filter of WT and the E71V KcsA mutant as a function of intracellular gate opening. a 2D PMF depending on two coordinates. The horizontal reaction coordinate r describes the width of the selectivity filter and is defined as the average cross-subunit pinching distance between the Cα atoms of G77. The vertical reaction coordinate z indicates the position of the external K+ ion along Z axis relative to the center of the selectivity filter. Extensions of previous calculations29,32. b 1D PMF projected along the horizontal reaction coordinate r (after Boltzmann integration of the vertical reaction coordinate z).