Research ArticleBIOPHYSICS

The conduction pathway of potassium channels is water free under physiological conditions

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Science Advances  31 Jul 2019:
Vol. 5, no. 7, eaaw6756
DOI: 10.1126/sciadv.aaw6756
  • Fig. 1 Comparison between water-mediated and direct knock-on ion permeation mechanisms.

    (A) The structure of the SF in NaK2K [Protein Data Bank (PDB) ID: 3ouf], with the ion binding sites (S1 to S4) and amino acid sequence indicated. (B) The two stages of the water-mediated knock-on mechanism. (C) The three stages of the direct knock-on mechanism. K+ ions and water molecules are displayed as purple and small red spheres, respectively. The name of the state is indicated above the structure, showing the occupancy at binding sites S1 to S4 with either K+ (K) or water (W), or unoccupied (0).

  • Fig. 2 Assignment of back-exchanged deuterated NaK2K.

    (A) CP-based NH spectrum of NaK2K, with the peak assignment indicated and with SF residues in purple. (*) T63 and V64 are indicated but could only be assigned on the basis of the spectra recorded after the washing procedure. ppm, parts per million. (B) CP-based NH spectrum of NaK2K, after the washing procedure, with assignments of SF residues indicated. (C) Assigned residues are indicated on the protein sequence in orange (based on back-exchanged deuterated NaK2K) and green (based on back-exchanged deuterated NaK2K after the washing procedure). The structural elements are shown underneath the sequence: the TM1, the pore helix (PH), the SF, and the TM2. (D) The assigned residues are indicated on the crystal structure of NaK2K (PDB ID: 3ouf) in orange and green (based on back-exchanged deuterated NaK2K before and after the washing procedure, respectively). The binding sites (S1 to S4) are indicated as purple spheres, and for clarity, only two opposing subunits are shown.

  • Fig. 3 Spin diffusion buildup from bound water to amide protons in NaK2K.

    2D HN slices with mixing times of 5 ms (A) and 35 ms (B) taken at the chemical shift of bound water (4.86 ppm) from 3D H(H)HN spin diffusion experiments. (C) Close up around the SF in a monomer of the tetrameric NaK2K structure (PDB ID: 3ouf). The magnetization transfer from bound water molecules to amide protons in the SF is indicated as orange spheres, where larger spheres represent faster spin diffusion buildup rates. S1 to S4 represent the binding sites in the SF. (D) Spin diffusion buildup plots from bound water to amide protons for the indicated residues. The error bars are based on the average noise levels in the 3D spectra. A.U., arbitrary unit; SD, spin diffusion.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw6756/DC1

    Fig. S1. Assignments of back-exchanged deuterated NaK2K in the presence of 50 mM K+.

    Fig. S2. 2D 15N-13Cα correlation spectrum of uniformly (13C and 15N)-labeled NaK2K in the presence of 50 mM K+.

    Fig. S3. Predicted H/D exchange pattern based on MD simulations.

    Fig. S4. Occupancies of the main ion binding sites of the NaK2K SF in MD simulations performed without an applied voltage.

    Fig. S5. Comparison of 2D (H)NH spectra and illustrations of the washing procedure for back-exchanged deuterated NaK2K.

    Fig. S6. Comparison of 2D (H)NH spectra of back-exchanged deuterated NaK2K in the presence of K+ using through-bond or through-space transfer.

    Fig. S7. 2D HN slices from 3D H(H)NH spin diffusion experiments with varying mixing times.

    Fig. S8. Spin diffusion buildup plots from bound water (blue circles) to amide protons and between neighboring amide protons (red triangles) in back-exchanged deuterated NaK2K in the presence of 50 mM K+.

    Fig. S9. Spin diffusion buildup plots from bound water (blue circles) to amide protons and between neighboring amide protons (red triangles), recorded after the washing procedure for back-exchanged deuterated NaK2K in the presence of 50 mM K+.

    Fig. S10. The relationship between carbonyl flipping and water presence at S3 in MD simulations under an applied voltage performed with the AMBER force field.

    Fig. S11. The relationship between carbonyl flipping and water presence at S3 in MD simulations under an applied voltage performed with the CHARMM force field.

    Table S1. Chemical shift assignments of deuterated back-exchanged NaK2K, in the presence of 50 mM K+, based on 1H-detected solid-state NMR experiments.

    Table S2. Chemical shift assignments of NaK2K SF residues, in the presence of 50 mM K+, based on 13C-detected solid-state NMR experiments.

    Table S3. Distances (Å) to amide protons of the SF in NaK2K (PDB ID: 3ouf) from the closest water molecule and binding sites (S1 to S4).

    Table S4. Experimental parameters for 3D (H)CANH experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.

    Table S5. Experimental parameters for 3D (H)CONH experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.

    Table S6. Experimental parameters for 3D (H)CACO(N)H and (H)COCA(N)H experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.

    Table S7. Experimental parameters for 3D H(H)NH spin diffusion experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.

    Movie S1. Typical K+ ion permeation process through NaK2K, observed in a 20-ns trajectory segment of MD simulations under an applied voltage.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Assignments of back-exchanged deuterated NaK2K in the presence of 50 mM K+.
    • Fig. S2. 2D 15N-13Cα correlation spectrum of uniformly (13C and 15N)-labeled NaK2K in the presence of 50 mM K+.
    • Fig. S3. Predicted H/D exchange pattern based on MD simulations.
    • Fig. S4. Occupancies of the main ion binding sites of the NaK2K SF in MD simulations performed without an applied voltage.
    • Fig. S5. Comparison of 2D (H)NH spectra and illustrations of the washing procedure for back-exchanged deuterated NaK2K.
    • Fig. S6. Comparison of 2D (H)NH spectra of back-exchanged deuterated NaK2K in the presence of K+ using through-bond or through-space transfer.
    • Fig. S7. 2D HN slices from 3D H(H)NH spin diffusion experiments with varying mixing times.
    • Fig. S8. Spin diffusion buildup plots from bound water (blue circles) to amide protons and between neighboring amide protons (red triangles) in back-exchanged deuterated NaK2K in the presence of 50 mM K+.
    • Fig. S9. Spin diffusion buildup plots from bound water (blue circles) to amide protons and between neighboring amide protons (red triangles), recorded after the washing procedure for back-exchanged deuterated NaK2K in the presence of 50 mM K+.
    • Fig. S10. The relationship between carbonyl flipping and water presence at S3 in MD simulations under an applied voltage performed with the AMBER force field.
    • Fig. S11. The relationship between carbonyl flipping and water presence at S3 in MD simulations under an applied voltage performed with the CHARMM force field.
    • Table S1. Chemical shift assignments of deuterated back-exchanged NaK2K, in the presence of 50 mM K+, based on 1H-detected solid-state NMR experiments.
    • Table S2. Chemical shift assignments of NaK2K SF residues, in the presence of 50 mM K+, based on 13C-detected solid-state NMR experiments.
    • Table S3. Distances (Å) to amide protons of the SF in NaK2K (PDB ID: 3ouf) from the closest water molecule and binding sites (S1 to S4).
    • Table S4. Experimental parameters for 3D (H)CANH experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.
    • Table S5. Experimental parameters for 3D (H)CONH experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.
    • Table S6. Experimental parameters for 3D (H)CACO(N)H and (H)COCA(N)H experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.
    • Table S7. Experimental parameters for 3D H(H)NH spin diffusion experiments acquired at 25°C on 100% H2O back-exchanged uniformly (2H, 13C, and 15N)-labeled NaK2K.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Typical K+ ion permeation process through NaK2K, observed in a 20-ns trajectory segment of MD simulations under an applied voltage.

    Files in this Data Supplement:

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