Subtype-specific modulation of human Kv7 channels by the anticonvulsant cannabidiol through a membrane-facing pore-domain site

Cannabidiol (CBD) is used clinically as an anticonvulsant. However, its precise mechanism of action has remained elusive. CBD was recently demonstrated to enhance the activity of the neuronal K_V7.2/7.3 channel, which may be one important contributor to how CBD reduces neuronal excitability. Curiously, CBD has also been shown to inhibit the cardiac K_V7.1/KCNE1 channel. However, whether and how CBD affects other K_V7 subtypes remains unstudied, and the CBD interaction sites mediating these diverse effects remain unknown. Here, we have used electrophysiology combined with molecular dynamics simulations, molecular docking, and site-directed mutagenesis to address these open questions. We found that CBD modulates the activity of all human K_V7 subtypes and that the effects are subtype-dependent. CBD enhanced the activity of K_V7.2-7.5 subtypes, seen as a V₅₀ shift towards more negative voltages or increased maximum conductance. In contrast, CBD inhibited the K_V7.1 and K_V7.1/KCNE1 channels, seen as a V₅₀ shift towards more positive voltages and reduced conductance. In K_V7.2 and K_V7.4, we propose a CBD interaction site at the subunit interface in the pore domain which 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_V7.1, with a non-conserved phenylalanine being important. In conclusion, we identify novel targets of CBD, contributing to a better understanding of CBD’s clinical effects, and provide mechanistic insights into how CBD modulates different K_V7 subtypes.