![Figure 7. A homology model for ΔK2PØ channels shows bilateral symmetry with a fourfold symmetric selectivity filter. Model building, refinement, and electrostatic calculations are described in Materials and methods. One subunit is colored in magenta, and the second is colored in blue. The P1 pore helices are in yellow, and P2 pore helices are in green. A file with the main chain coordinates is included in the supplemental material (available at http://www.jgp.org/cgi/content/full/jgp.200910235/DC1). (A) The model of K2PØ from the extracellular side reveals overall symmetry like a parallelogram. The model includes residues 1–276 without the TM1-P1 loop (residues 30–91), TM2–TM3 linker (residues 153–173), and TM2-P2 loop (residues 225–238). (B) Model of K2PØ viewed from the cytoplasm. (C) Side view of domain I of both subunits. Pro131 in TM2 is indicated. (D) Side view of domain II of both subunits. Proline residues in TM3 are indicated. (E) Illustration of electrostatic calculations to ascertain the effect of charged mutations on ion permeation (static field contribution). (top) ΔΔG for T216K ΔK2PØ: ΔG for the T216K channel (solid line) and ΔG for the WT channel (dashed line) are shown along the pore axis. The membrane bilayer spans z = −12.5 to z = 12.5; ΔΔG = max[|ΔGT216K − ΔGWT|]. This definition of ΔΔG provides a measure of the dominant free energy barrier for ion conduction relative to the WT channel. The arrow indicates where ΔΔG is evaluated. (middle and bottom) Same calculations for T216K–I260D (no rescue) and T216K–M261D (rescue), respectively. (F) Electrostatic ΔΔG (static field contribution) for T216K ΔK2PØ channels with second site alterations to aspartate (131–139 in TM2 or 255–263 in TM4). Black shading indicates 7 of 18 mutations that restore current. The highest ΔΔG where complementation was observed is marked with a dotted line. The red arrow notes G139D. (G) Section of C showing a disulfide bridge between residues E28C and T115C; the S–S distance in the two subunits (Cβ–Cβ) are 5.0 and 5.1 Å. (H) Section of D showing a disulfide bridge between residues F199C and F239C; the S–S distance in the two subunits (Cβ–Cβ) are 6.7 and 6.9 Å. (I) Effectiveness of complementation as a function of distance for T216K ΔK2PØ channels. Rescue percentage was determined at −100 mV as (IMut − IT216K)/(IWT − IT216K). Distances were measured as in Table I. Data were fit to f(x) = a (eb/x − 1).](https://cdn.rupress.org/rup/content_public/journal/jgp/134/1/10.1085_jgp.200910235/6/jgp_200910235_rgb_fig7.jpeg?Expires=1773511831&Signature=T7bI2XPGMNpd0Y7FMOxIs8PDjewzFq3ob8BgsPjOLzuoQ6n-CbT~JcTi86qQeez7x0FzzEtAJFLm2kBYgeLIBQm7Yv3d2aJ2OIDQol0b~8UmsetweJjcYchowrfKgjZ0HtSv~28sxtE5ORKu9I3gpojHfwl8nmzYurJNFhS7jJWiNg6~iW1Zxz5PkFQjGYJdr0FH3UVCAz1Bl7u82tnsN48YvEpLn9~JkYWhkE7Rbs1GKeku4lZneZKELVvsE-BQyB6YW~-OW21~CkRw8a14W83x5fLCuVkD708Qc4P1kQyrSqPQo9phod9g3p~~0p31z7Xo4iyBOayRI66e9OrcqQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
A homology model for ΔK2PØ channels shows bilateral symmetry with a fourfold symmetric selectivity filter. Model building, refinement, and electrostatic calculations are described in Materials and methods. One subunit is colored in magenta, and the second is colored in blue. The P1 pore helices are in yellow, and P2 pore helices are in green. A file with the main chain coordinates is included in the supplemental material . (A) The model of K2PØ from the extracellular side reveals overall symmetry like a parallelogram. The model includes residues 1–276 without the TM1-P1 loop (residues 30–91), TM2–TM3 linker (residues 153–173), and TM2-P2 loop (residues 225–238). (B) Model of K2PØ viewed from the cytoplasm. (C) Side view of domain I of both subunits. Pro131 in TM2 is indicated. (D) Side view of domain II of both subunits. Proline residues in TM3 are indicated. (E) Illustration of electrostatic calculations to ascertain the effect of charged mutations on ion permeation (static field contribution). (top) ΔΔG for T216K ΔK2PØ: ΔG for the T216K channel (solid line) and ΔG for the WT channel (dashed line) are shown along the pore axis. The membrane bilayer spans z = −12.5 to z = 12.5; ΔΔG = max[|ΔGT216K − ΔGWT|]. This definition of ΔΔG provides a measure of the dominant free energy barrier for ion conduction relative to the WT channel. The arrow indicates where ΔΔG is evaluated. (middle and bottom) Same calculations for T216K–I260D (no rescue) and T216K–M261D (rescue), respectively. (F) Electrostatic ΔΔG (static field contribution) for T216K ΔK2PØ channels with second site alterations to aspartate (131–139 in TM2 or 255–263 in TM4). Black shading indicates 7 of 18 mutations that restore current. The highest ΔΔG where complementation was observed is marked with a dotted line. The red arrow notes G139D. (G) Section of C showing a disulfide bridge between residues E28C and T115C; the S–S distance in the two subunits (Cβ–Cβ) are 5.0 and 5.1 Å. (H) Section of D showing a disulfide bridge between residues F199C and F239C; the S–S distance in the two subunits (Cβ–Cβ) are 6.7 and 6.9 Å. (I) Effectiveness of complementation as a function of distance for T216K ΔK2PØ channels. Rescue percentage was determined at −100 mV as (IMut − IT216K)/(IWT − IT216K). Distances were measured as in Table I. Data were fit to f(x) = a (eb/x − 1).
