Background and PurposeCannabidiol (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 ApproachHere, we used electrophysiology, molecular dynamics simulations, molecular docking and site-directed mutagenesis to address these questions. Key ResultsWe 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 ImplicationsWe 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.

Several essential physiological systems express voltage-gated potassium channels within the KV7 family (comprising KV7.1-7.5), sometimes also co-assembled with auxiliary subunits in the KCNE family (comprising KCNE1-5). An ongoing challenge to KV7 drug development is creating subtype-selective compounds to limit adverse effects. Prior work has shown that the antiepileptic cannabidiol (CBD), a pan-KV7 modulator, inhibits cardiac- and epithelia-associated KV7.1 and KV7.1/KCNE1 channels, while activating neuronal KV7 subtypes (KV7.2-7.5). However, little is known about the binding sites through which CBD mediates inhibitory effects on KV7.1 and KV7.1/KCNE1, limiting insight towards the development of selective KV7 modulators. To address this knowledge gap, we used a combination of the Chai-1 artificial intelligence model (to generate CBD binding site predictions in human KV7.1 and KV7.1/KCNE1 channels), site-directed mutagenesis and electrophysiology of these channels expressed in Xenopus laevis oocytes (to corroborate CBD binding site predictions), and molecular dynamics simulations (to study the biophysical mechanisms underlying CBD binding). We found that CBD binds to two unique sites within KV7.1 and KV7.1/KCNE1. In KV7.1 alone, CBD was bound to an intrasubunit S5-S6 pore domain binding site; referred to as the S5-S6 site. In KV7.1/KCNE1, the addition of the KCNE1 subunit created a novel binding site for CBD, sandwiched between two KV7.1 subunits and one KCNE1 subunit; referred to here as the S6-S5'-E1 site. Molecular dynamics simulations showed that CBD binding to the S6-S5'-E1 KV7.1/KCNE1 site closes off the KV7.1 S5-S6 site. A sequence comparison between KV7 channels revealed key amino acid differences at both the S5-S6 and S6-S5'-E1 sites relative to neuronal KV7s. These support the notion that CBD binds differently in KV7.1 and KV7.1/KCNE1 channels in accordance with its unique inhibitory pharmacological effects on these channels compared to the activating effect in neuronal KV7s. Thus, we provide support for KV7.1 and KV7.1/KCNE1 being inhibited by CBD via distinct binding sites, which can guide future research focused on the rational development of drugs that avoid inhibitory effects on KV7.1 and KV7.1/KCNE1 channels or utilize these sites to modulate channel activity.