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.

Subtype-specific responses of hKv7.4 and hKv7.5 channels to polyunsaturated fatty acids reveal an unconventional modulatory site and mechanism.

The KV7.4 and KV7.5 subtypes of voltage-gated potassium channels play a role in important physiological processes such as sound amplification in the cochlea and adjusting vascular smooth muscle tone. Therefore, the mechanisms that regulate KV7.4 and KV7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human KV7.4 and KV7.5 channels expressed in Xenopus oocytes. We report that PUFAs facilitate activation of hKV7.5 by shifting the V50 of the conductance versus voltage (G(V)) curve toward more negative voltages. This response depends on the head group charge, as an uncharged PUFA analogue has no effect and a positively charged PUFA analogue induces positive V50 shifts. In contrast, PUFAs inhibit activation of hKV7.4 by shifting V50 toward more positive voltages. No effect on V50 of hKV7.4 is observed by an uncharged or a positively charged PUFA analogue. Thus, the hKV7.5 channel's response to PUFAs is analogous to the one previously observed in hKV7.1-7.3 channels, whereas the hKV7.4 channel response is opposite, revealing subtype-specific responses to PUFAs. We identify a unique inner PUFA interaction site in the voltage-sensing domain of hKV7.4 underlying the PUFA response, revealing an unconventional mechanism of modulation of hKV7.4 by PUFAs.

In order to carry out their roles in the body, cells need to send and receive electrical signals. They can do this by allowing ions to move in and out through dedicated pore-like structures studded through their membrane. These channels are specific to one type of ions, and their activity ­ whether they open or close ­ is carefully controlled. In humans, defective ion channels are associated with conditions such as irregular heartbeats, epileptic seizures or hearing loss. Research has identified molecules known as polyunsaturated fatty acids as being able to control the activity of certain members of the K_V7 family of potassium ion channels. The K_V7.1 and K_V7.2/7.3 channels are respectively present in the heart and the brain; K_V7.4 is important for hearing, while K_V7.5 plays a key role in regulating muscle tone in blood vessels. Polyunsaturated fatty acids can activate K_V7.1 and K_V7.2/7.3 but their impact on K_V7.4 and K_V7.5 remains unclear. Frampton et al. explored this question by studying human K_V7.4 and K_V7.5 channels expressed in frog egg cells. This showed that fatty acids activated K_V7.5 (as for K_V7.1 and K_V7.2/7.3), but that they reduced the activity of K_V7.4. Closely examining the structure of K_V7.4 revealed that the fatty acids were binding to a different region compared to the other K_V7 channels. When this site was made inaccessible, fatty acids increased the activity of K_V7.4, just as for the rest of the family. These results may help to understand the role of polyunsaturated fatty acids in the body. In addition, knowing how these molecules interact with channels in the same family will be useful for optimising a drug's structure to avoid side effects. However, further research will be needed to understand the broader impact in a more complex biological organism.

Polyunsaturated Fatty Acids as Modulators of KV7 Channels.

Voltage-gated potassium channels of the KV7 family are expressed in many tissues. The physiological importance of KV7 channels is evident from specific forms of disorders linked to dysfunctional KV7 channels, including variants of epilepsy, cardiac arrhythmia and hearing impairment. Thus, understanding how KV7 channels are regulated in the body is of great interest. This Mini Review focuses on the effects of polyunsaturated fatty acids (PUFAs) on KV7 channel activity and possible underlying mechanisms of action. By summarizing reported effects of PUFAs on KV7 channels and native KV7-mediated currents, we conclude that the generally observed effect is a PUFA-induced increase in current amplitude. The increase in current is commonly associated with a shift in the voltage-dependence of channel opening and in some cases with increased maximum conductance. Auxiliary KCNE subunits, which associate with KV7 channels in certain tissues, may influence PUFA effects, though findings are conflicting. Both direct and indirect activating PUFA effects have been described, direct effects having been most extensively studied on KV7.1. The negative charge of the PUFA head-group has been identified as critical for electrostatic interaction with conserved positively charged amino acids in transmembrane segments 4 and 6. Additionally, the localization of double bonds in the PUFA tail tunes the apparent affinity of PUFAs to KV7.1. Indirect effects include those mediated by PUFA metabolites. Indirect inhibitory effects involve KV7 channel degradation and re-distribution from lipid rafts. Understanding how PUFAs regulate KV7 channels may provide insight into physiological regulation of KV7 channels and bring forth new therapeutic strategies.