Dynein binds to the Kv7.4 C terminus. (A) Amino acid alignment of the end of the c-helix of Xenopus Kv7.1 (used for the modeling in B) and human Kv7.1–Kv7.5 channels. (B) Close view of the Kv7 channel c-helix using the structure of Xenopus Kv7.1 suggests that several residues (highlighted in green) in the dynein-binding motifs indeed are accessible to the intracellular environment. Those in red are not exposed to the intracellular environment and are therefore unlikely to be required for dynein recognition. (C, i–iii) Representative whole-cell voltage clamp recordings and respective I-V relations compared between Kv7.4 and Kv7.4-Q580A (i); when cotransfected with p50/dynamitin (ii); or incubation with 3 µM ciliobrevin D (iii). Statistical comparisons were made with a two-way ANOVA, followed by a Bonferroni multiple comparisons test, where P < 0.05, P < 0.01, and P < 0.001 are depicted by *, **, and ***, respectively. (C iv) Mean V_1/2 for steady-state activation was compared for each condition with a one-way ANVOA. (D) Docking simulations performed with SwissDock on Kv7.1, Kv7.4, Kv7.1-Q560A, and Kv7.4-Q580A showing ciliobrevin D binding to both mutant channels but neither WT channel. Each of the four spirals (pink, green, orange, and gold) represent an intercellular C terminus of each of the four Kv7 protein α subunits that multimerize to form a functional channel. Mean values are shown with error bars depicting the SEM.