作者: Meng, Yuhuan ; Peng, Ziyue ; Mao, Di ; Yu, Shihui ; Zhang, Yi ; Song, Huilin ; Qiao, Shupei ; Xia, Yang ; Zhang, Jiahui ; Xie, Li ; Cao, Tianhua ; Jing, Wenwen ; Sui, Shengbin ; Jia, Ying ; Meng, Lingdi ; Zhang, Xue ; Song, Yue

BackgroundIncreasing evidence indicates a robust correlation between epilepsy and variants of the Kv7.2 (KCNQ2) channel, which is critically involved in directing M-currents and regulating neuronal excitability within the nervous system. With the advancement of next-generation sequencing, the identification ofKCNQ2variants has surged. Nonetheless, their functional impacts are still being determined, introducing uncertainty into the diagnostic process for affected families and potentially hindering their ability to participate in targeted precision medicine trials. This study aims to elucidate the pathogenicity of these novel variants and explore potential therapeutic interventions.MethodsWhole-cell patch-clamp recordings, western blotting, and immunofluorescent staining were performed to elucidate the functional consequences of the identified variants. Moreover, coimmunoprecipitation techniques were conducted to explore protein interactions, thus facilitating a deeper understanding of the underlying pathogenetic mechanisms contributing to the disease. Ultimately, the effects of pharmacological interventions were evaluated in vitro using the patch-clamp technique.ResultsHerein, we identified 12 novelKCNQ2variants, further expanding the mutational spectrum ofKCNQ2. Our investigation revealed that one gain-of-function variant (p.L102V (c.304C>G)) and three loss-of-function variants (p.H328Q (c.984C>G), p.A336V (c.1007C>T) and p.D563Efs*22 (c.1688_1689insACTT)) had different impacts on the binding of calmodulin and phosphati-dylinositol-4,5-bisphosphate, potentially altering their localisation and protein stability. Furthermore, the application of ML213, unlike Retigabine and ICA-069673, led to a significant increase in the current of p.H328Q.ConclusionThis study expanded the mutational spectrum ofKCNQ2and analysed the genetic and functional consequences, as well as the pharmacological rescue, of fourde novo KCNQ2variants. These findings offer valuable insights into the precise medicine ofKCNQ2-related epilepsy.

2023-06-19·Function

Site and Mechanism of ML252 Inhibition of Kv7 Voltage-Gated Potassium Channels

Article

作者: Kurata, Harley T ; Allison, W Ted ; Urrutia, Arturo ; Kanyo, Richard ; Lamothe, Shawn M ; Goodchild, Samuel J ; Dean, Richard

AbstractKv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators.ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.

2023-01-01·British Journal of Pharmacology

Marked oestrous cycle‐dependent regulation of rat arterial K_V7.4 channels driven by GPER1

Article

作者: Stott, Jennifer B. ; Homer, Natalie Z. M. ; Isakson, Brant E. ; Baldwin, Samuel N. ; Albert, Anthony P. ; Greenwood, Iain A. ; Barrese, Vincenzo ; Andrew, Ruth ; Forrester, Elizabeth A.

Background and PurposeKcnq‐encoded KV7 channels (termed KV7.1–5) regulate vascular smooth muscle cell (VSMC) contractility at rest and as targets of receptor‐mediated responses. However, the current data are mostly derived from males. Considering the known effects of sex, the oestrous cycle and sex hormones on vascular reactivity, here we have characterised the molecular and functional properties of KV7 channels from renal and mesenteric arteries from female Wistar rats separated into di‐oestrus and met‐oestrus (F‐D/M) and pro‐oestrus and oestrus (F‐P/E).Experimental ApproachRT‐qPCR, immunocytochemistry, proximity ligation assay and wire myography were performed in renal and mesenteric arteries. Circulating sex hormone concentrations were determined by liquid chromatography–tandem mass spectrometry. Whole‐cell electrophysiology was undertaken on cells expressing KV7.4 channels in association with G‐protein‐coupled oestrogen receptor 1 (GPER1).Key ResultsThe KV7.2–5 activators S‐1 and ML213 and the pan‐KV7 inhibitor linopirdine were more effective in arteries from F‐D/M compared with F‐P/E animals. In VSMCs isolated from F‐P/E rats, exploratory evidence indicates reduced membrane abundance of KV7.4 but not KV7.1, KV7.5 and Kcne4 when compared with cells from F‐D/M. Plasma oestradiol was higher in F‐P/E compared with F‐D/M, and progesterone showed the converse pattern. Oestradiol/GPER1 agonist G‐1 diminished KV7.4 encoded currents and ML213 relaxations and reduced the membrane abundance of KV7.4 and interaction between KV7.4 and heat shock protein 90 (HSP90), in arteries from F‐D/M but not F‐P/E.Conclusions and ImplicationsGPER1 signalling decreased KV7.4 membrane abundance in conjunction with diminished interaction with HSP90, giving rise to a ‘pro‐contractile state’.

100 项与 ML213 相关的药物交易

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