Research ArticleNEUROSCIENCE

A drug pocket at the lipid bilayer–potassium channel interface

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Science Advances  25 Oct 2017:
Vol. 3, no. 10, e1701099
DOI: 10.1126/sciadv.1701099
  • Fig. 1 Wu32 keeps the Shaker KV channel in the open state.

    (A) Cartoons of a voltage-gated ion channel. a to c are principal drug-binding sites described in the text. Negatively charged polyunsaturated fatty acids (PUFAs) (in red) attract the gating charges to open the channel. (B) Molecules studied. (C to F and H) Channels as indicated: black, control; red, 100 μM Wu32 (pH = 7.4). Holding voltage (VH) = −80 mV. (C) Representative normalized tail currents at −70 mV, following a step to +50 mV. (D) Representative G(V) shift = −16 mV. (E) Representative G(V) shift = −29 mV. (F) Time constants as indicated. Closed symbols measured from the opening and open symbols measured from the closing. (G) Kinetic scheme for the Shaker KV channel. C, closed states; O, open state; Q, large gating change; q, small gating charge. (H) ILT-434F (curves to the left): No shift of gating currents [VH = −100 mV; mean ± SEM (n = 3)]. ILT (curves to the right): G(V) shift = −63.9 ± 3.1 mV (n = 5). Norm., normalized.

  • Fig. 2 Effects of S3 mutations on compound-induced G(V) shifts.

    (A) Effects on I325C (gray bar) and modifications of the positively charged MTSEA+ (red bar) and the negatively charged MTSES (blue bar), respectively. Data are means ± SEM (n = 4 to 5). *P < 0.05; ***P < 0.001 [one-way analysis of variance (ANOVA) together with Bonferroni’s multiple comparison test]. (B) G(V) shifts of 100 μM Wu32 (pH 7.4) on S3 mutations. Data are means ± SEM (n = 4 to 10). G(V) shifts are compared with WT (white bar, black line): gray bars, not different from WT; red bars, significantly different from WT. *P < 0.05, **P < 0.01, ***P < 0.001(one-way ANOVA together with Dunnett’s multiple comparison test). (C) Mutated residues as space filled in one VSD of the Shaker KV channel in the open state (side view) (23). Gating charges, blue sticks. Color coding as in (B). (D) Effects of P322 mutations on DHAA- and Wu32-induced G(V) shifts as indicated. Data are means ± SEM (n = 4 to 10). Color coding as in (B). *P < 0.05, ***P < 0.001(one-way ANOVA together with Bonferroni’s multiple comparison test). ns, not significant.

  • Fig. 3 Electrostatic interactions between S4 and the DHAA derivatives.

    All compounds 100 μM (pH 7.4 if not otherwise stated). (A) pH dependence for G(V) shifts. Data are means ± SEM (n = 4 to 8). (B) Molecular structure of the amine (Wu165) in (A). (C) Black, control; red, Wu32. G(V) shift = −33 mV. (D) Wu32-induced G(V) shifts for arginine mutants. Data are means ± SEM (n = 4 to 10). G(V) shifts are compared with R362Q (white bar, black line): gray bars, not different from R362Q; blue bars, significantly smaller than R362Q; red bars, significantly larger than R362Q. *P < 0.05, ***P < 0.001 (one-way ANOVA together with Dunnett’s multiple comparison test). (E) Mutated residues as space fill in one VSD of the Shaker KV channel in the open state. Coloring as in (D). (F) Schematic illustration of S4 rotation during last (opening) step. The red symbol denotes a position consistent with data. (G) Effects of glutamate mutations (red bars) on Wu32-induced shifts, compared to arginine mutations (blue bars) from (D). Data are means ± SEM (n = 4 to 6). *P < 0.05, **P < 0.01, ***P < 0.001(t test). (H) Wu32-induced G(V) shifts from (D) versus DHAA-induced G(V) shifts. Data are means ± SEM (n = 4 to 10), slope = 5.7. Wu32 data in (A), WT data in (D) and (H) from the study of Ottosson et al. (19).

  • Fig. 4 Resin acid effects on kinetics.

    (A and B) Effects on opening and closing kinetics of the 3R Shaker KV channel. Black, control; red, 100 μM DHAA (pH 7.4). (A) Normalized currents. Opening at +40 mV (0 to 80 ms) and closing at −20 mV (80 to 95 ms). (B) Effects on time constants. Closed symbols measured from the opening and open symbols measured from the closing. (C) Relative closing time constants (τcompoundcontrol) at −20 mV, following 100 ms at +70 mV. Black line, linear regression from fig. S5. Dashed lines, confidence interval. Red symbols, DHAA derivatives with G(V) shift >|–20| mV. Date are means ± SEM (n = 4 to 11). (D) Molecular structure of Wu122.

  • Fig. 5 Molecular dynamics identifies an interaction site between S3, S4, and the lipid bilayer.

    (A) Predicted binding pose of Wu122 on the WT Shaker KV channel. Different colors denote different subunits. (B) Top view and (C) side view of one VSD. *, C7 side chain.

  • Fig. 6 Small side chain alterations have large effects compound-induced G(V) shifts.

    (A and B) G(V) shifts for 100 μM compound (pH 7.4). (A) Effect of chirality in the nitro group. Dashed line, molecule lacking the nitro group (19). Data are means ± SEM (n = 5 to 6). ***P < 0.0001 (t test). (B) Effects of chirality of the carboxyl group. Data for DHAA (19). Data are means ± SEM (n = 4 to 11). ***P < 0.0001 (t test).

  • Fig. 7 Log P values for all compounds.

    G(V) shifts at a concentration of 100 μM and pH 7.4 versus calculated log P values for all tested 125 compounds (bin size = 0.5; table S1).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/10/e1701099/DC1

    Supplementary Methods

    fig. S1. Wu32 affects the closure step.

    fig. S2. The charge of the resin acid is required for G(V) shifting effects.

    fig. S3. Wu32 affects closure but not opening.

    fig. S4. Small effects of DHA and DHAA on kinetics.

    fig. S5. Molecular dynamics identifies an interaction site between S3, S4, and the lipid bilayer.

    fig. S6. Stability of VSD-Wu122 complexes.

    fig. S7. The negatively charged cluster EEED333-336 is not important for compound-induced G(V) shifts.

    fig. S8. Wu170 affects closing but not opening.

    fig. S9. Correlation analysis for 3R Shaker KV channel.

    fig. S10. The method to calculate conductance (or permeability) does not affect the estimated G(V) shift.

    table S1. Properties of synthesized compounds.

    References (4751)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • fig. S1. Wu32 affects the closure step.
    • fig. S2. The charge of the resin acid is required for G(V) shifting effects.
    • fig. S3. Wu32 affects closure but not opening.
    • fig. S4. Small effects of DHA and DHAA on kinetics.
    • fig. S5. Molecular dynamics identifies an interaction site between S3, S4, and the lipid bilayer.
    • fig. S6. Stability of VSD-Wu122 complexes.
    • fig. S7. The negatively charged cluster EEED333-336 is not important for compound-induced G(V) shifts.
    • fig. S8. Wu170 affects closing but not opening.
    • fig. S9. Correlation analysis for 3R Shaker KV channel.
    • fig. S10. The method to calculate conductance (or permeability) does not affect the estimated G(V) shift.
    • table S1. Properties of synthesized compounds.
    • References (47–51)

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