Three putative resin acid–binding sites act on activation via differential stabilization of S4 in various activation states. Top views of the motion of S4 during the last steps of activation occurring in one subunit (N.B. the pore domain and VSD are from two different subunits) in the WT Shaker K_V channel (left) and the 2R mutant (right), from the C1 closed state (top row) to the open O state (middle row) to the putative open O⁺ state (bottom row). The arrows show the transitions occurring upon depolarization (indicated by depol.) and upon hyperpolarization (indicated by hyperpol.). Binding to the S3/S4 site is represented as a red triangle, to the S4/pore site as a purple triangle, and to the top-VSD site as a yellow triangle. The charged residues are shown as blue filled circles. In the last intermediate (C1) closed state, both for the WT and the 2R channel, binding to the S3/S4 site is weak (light red triangle), binding to the S4/pore site is weak (light purple triangle), and binding to the top-VSD site is strong (yellow triangle). In the open O state, binding to the S3/S4 site is weak (light red triangle) in the WT but strong (bright red triangle) in 2R due to the proximity of R₋₁, binding to the S4/pore site is strong (bright purple triangle) in the WT due to the proximity of R₁ but presumably weak in the 2R (light purple triangle), and binding to the top-VSD site is strong (yellow triangle) in both the WT and the 2R mutants. In the hyperactivated/open O⁺ state, binding to the S3/S4 site is weak (light red triangle) in the WT but presumably strong (bright red triangle) in 2R due to the proximity of R₀, binding to the S4/pore site is weak (light purple triangle) in both the WT and the 2R mutants, and binding to the top-VSD site is strong (yellow triangle) in both the WT and the 2R mutants.