Subtype-specific modulation of human KV 7 channels by the anticonvulsant cannabidiol through a lipid-exposed pore-domain site.

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 KV 7.2/7.3 channel, which may be one important contributor to CBD anticonvulsant effect. Curiously, CBD inhibits the closely related cardiac KV 7.1/KCNE1 channel. Whether and how CBD affects other KV 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 KV 7 subtypes and that the effects are subtype dependent. CBD enhanced the activity of KV 7.2-7.5 subtypes, seen as a V50 shift towards more negative voltages or increased maximum conductance. In contrast, CBD inhibited the KV 7.1 and KV 7.1/KCNE1 channels, seen as a V50 shift towards more positive voltages and reduced conductance. In KV 7.2 and KV 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 KV 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 KV 7 subtypes.

Two Distinct Populations of α1α6-Containing GABAA-Receptors in Rat Cerebellum.

GABAA receptors are pentameric GABA-gated chloride channels. The existence of 19 different subunits (six α, three ß, three γ, δ, ε, θ, π, and three ρ) in mammalian systems gives rise to an enormous theoretical diversity of GABAA receptor subtypes with distinct subunit composition and unique pharmacological properties. These receptors are already now the drug targets of several clinically used compounds, such as benzodiazepines, anesthetics, and many more. There is a constant quest to identify novel molecules and possible future drug targets: Currently, α6-containing GABAA receptors are being discussed in the context of treating sensorimotor gating deficits in neuropsychiatric disorders, such as tic disorders and schizophrenia. Therefore, we aim to learn more about α6-containing GABAA receptors. They are mostly expressed in the cerebellar granule cell layer, where they form the following subtypes: α6ßxγ2, α1α6ßxγ2, α6ßxδ, and α1α6ßxδ. In former studies, α1α6ßxγ2-containing GABAA receptors were considered a single receptor population. In the current study, we investigate the possibility, that this population can consist of two subgroups with alternative arrangements depending if α1 neighbors γ2 (forming a "diazepam-sensitive" receptor), or if α6 neighbors γ2 (forming a "diazepam-insensitive" receptor) and aimed to prove the existence of both subtypes in native tissue. We performed immunoprecipitation experiments on rat cerebellar lysates using α1- or α6 subunit-specific antibodies followed by radioligand binding assays with either 3H-flunitrazepam or 3H-Ro 15-4513. Indeed, we were able to prove the existence of two distinct populations of α1α6-containing GABAA-receptors and could quantify the different receptor populations: α1ßxγ2 receptors constitute approximately 60% of all γ2-containing receptors in the rat cerebellum, α6ßxγ2 about 20%, and both isoforms of α1α6ßxγ2 9-15% each. The simple classification of GABAA-receptors into αx-containing subtypes seems not to reflect the complexity of nature; those receptors are more diverse than previously thought.