Sara I. Liin

Background and Purpose Cannabidiol (CBD) is used clinically as an anticonvulsant. Its precise mechanism of action has remained unclear. CBD was recently demonstrated to enhance the activity of the neuronal K V 7.2/7.3 channel, which may be one important contributor to CBD anticonvulsant effect. Curiously, CBD inhibits the closely related cardiac K V 7.1/KCNE1 channel. Whether and how CBD affects other K V 7 subtypes remains uninvestigated and the CBD interaction sites mediating these diverse effects remain unknown. Experimental Approach Here, we used electrophysiology, molecular dynamics simulations, molecular docking and site‐directed mutagenesis to address these questions. Key Results We found that CBD modulates the activity of all human K V 7 subtypes and that the effects are subtype dependent. CBD enhanced the activity of K V 7.2–7.5 subtypes, seen as a V 50 shift towards more negative voltages or increased maximum conductance. In contrast, CBD inhibited the K V 7.1 and K V 7.1/KCNE1 channels, seen as a V 50 shift towards more positive voltages and reduced conductance. In K V 7.2 and K V 7.4, we propose a CBD interaction site at the subunit interface in the pore domain that overlaps with the interaction site of other compounds, notably the anticonvulsant retigabine. However, CBD relies on other residues for its effects than the conserved tryptophan that is critical for retigabine effects. We propose a similar, though not identical CBD site in K V 7.1, with a non‐conserved phenylalanine being important. Conclusions and Implications We identify novel targets of CBD, contributing to a better understanding of CBD clinical effects and provide mechanistic insights into how CBD modulates different K V 7 subtypes.