Cannabidiol inhibits both human KV7.1 and KV7.1/KCNE1 channels through distinct sites

Abstract Several essential physiological systems express voltage-gated potassium channels within the K V 7 family (comprising K V 7.1–7.5), sometimes also co-assembled with auxiliary subunits in the KCNE family (comprising KCNE1–5). An ongoing challenge to K V 7 drug development is creating subtype-selective compounds to limit adverse effects. Prior work has shown that the antiepileptic cannabidiol (CBD), a pan-K V 7 modulator, inhibits cardiac- and epithelia-associated K V 7.1 and K V 7.1/KCNE1 channels, while activating neuronal K V 7 subtypes (K V 7.2–7.5). However, little is known about the binding sites through which CBD mediates inhibitory effects on K V 7.1 and K V 7.1/KCNE1, limiting insight towards the development of selective K V 7 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 K V 7.1 and K V 7.1/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 K V 7.1 and K V 7.1/KCNE1. In K V 7.1 alone, CBD was bound to an intrasubunit S5–S6 pore domain binding site; referred to as the S5–S6 site. In K V 7.1/KCNE1, the addition of the KCNE1 subunit created a novel binding site for CBD, sandwiched between two K V 7.1 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 K V 7.1/KCNE1 site closes off the K V 7.1 S5–S6 site. A sequence comparison between K V 7 channels revealed key amino acid differences at both the S5–S6 and S6–S5’–E1 sites relative to neuronal K V 7s. These support the notion that CBD binds differently in K V 7.1 and K V 7.1/KCNE1 channels in accordance with its unique inhibitory pharmacological effects on these channels compared to the activating effect in neuronal K V 7s. Thus, we provide support for K V 7.1 and K V 7.1/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 K V 7.1 and K V 7.1/KCNE1 channels or utilize these sites to modulate channel activity.