Contact analysis for inner leaflet LIN and STE in CG simulations. (A) Histogram of total interaction time of each residue in KCNQ1 for intracellular LIN. The gray highlighted boxes in the background indicate the position of the transmembrane helices. The residues identified as interacting with the LIN head group (i.e., R239 and R249) are colored according to E and F onto the histograms. (B) Same as in A but for longest lifetime of each residue. (C and D) Same as in A and B but for STE. (E and F) Side view of a LIN binder interacting with residues R249 and R239 located on opposite ends of the S4–S5 linker. (G) Experimental mutation of predicted binder residues does not impair the effect of LIN. Data shown as mean ± SEM; n = 5 per data point. Concentration-response curves were fitted using Eq. 2 with the Hill coefficient constrained to −1 (see Materials and methods for details). ΔV_50,max was constrained to shared values between WT and mutant to make the fits more robust, and data are shown as normalized effect (100% was defined as the maximal effect from the fit). For 70 µM LIN, ΔV₅₀ and ΔG_max were within ±1.2 mV and ±9%, respectively (P > 0.05 with one-way ANOVA followed by Dunnett’s multiple comparisons test to compare with WT). Rel., relative.