Wu161 binding to the S3/S4 and the S4/pore sites. (A) Zoomed-in side view of the S3/S4 site in the presence of Wu161. The channel is shown as gray ribbons, the Wu161 compounds are displayed in CPK representation, each atom type colored differently (yellow: S; black: C; red: O). Apolar, polar, positively charged, and aromatic residues are represented as sticks and depicted in shades of gray, orange, pink, and green, respectively. (B) Distance between closest heavy atoms of Wu161 and I360 along a 500-ns-long MD simulation of the WT Shaker K_V channel system. Each of four subunits is depicted in a different color. (C) Zoomed-in side view of the S4/pore site in the presence of Wu161. Representations are the same as in A. (D) Distance between closest heavy atoms of Wu161 and R362 along a 500-ns-long MD simulation of the WT Shaker K_V channel system. Each of four subunits is depicted in a different color. (E and F)G(V) curves before (black) and after (red) application of 100 µM Wu161 to the channels. G(V) shifts = −10.4 mV and −4.7 mV, respectively, pH = 7.4. (G) Correlation between G(V) shifts for Wu50 and Wu161 (100 µM) on different Shaker mutants. Error bars represent mean ± SEM (n = 4). The solid black line marks an equal G(V) shift for Wu50 and Wu161. Norm., normalized.