Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.

The growing interest in Kv7.2/7.3 agonists originates from the involvement of these channels in several brain hyperexcitability disorders. In particular, Kv7.2/7.3 mutants have been clearly associated with epileptic encephalopathies (DEEs) as well as with a spectrum of focal epilepsy disorders, often associated with developmental plateauing or regression. Nevertheless, there is a lack of available therapeutic options, considering that retigabine, the only molecule used in clinic as a broad-spectrum Kv7 agonist, has been withdrawn from the market in late 2016. This is why several efforts have been made both by both academia and industry in the search for suitable chemotypes acting as Kv7.2/7.3 agonists. In this context, in silico methods have played a major role, since the precise structures of different Kv7 homotetramers have been only recently disclosed. In the present review, the computational methods used for the design of Kv.7.2/7.3 small molecule agonists and the underlying medicinal chemistry are discussed in the context of their biological and structure-function properties.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 μM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 μM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 μM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 μM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 μM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

Depression, a complex disorder with significant treatment challenges, necessitates innovative therapeutic approaches to address its multifaceted nature and enhance treatment outcomes. The modulation of KCNQ potassium (K+) channels, pivotal regulators of neuronal excitability and neurotransmitter release, is a promising innovative therapeutic target in psychiatry. Widely expressed across various tissues, including the nervous and cardiovascular systems, KCNQ channels play a crucial role in modulating membrane potential and regulating neuronal activity. Recent preclinical evidence suggests that KCNQ channels, particularly KCNQ3, contribute to the regulation of neuronal excitability within the reward circuitry, offering a potential target for alleviating depressive symptoms, notably anhedonia. Studies using animal models demonstrate that interventions targeting KCNQ channels can restore dopaminergic firing balance and mitigate depressive symptoms. Human studies investigating the effects of KCNQ channel activators, such as ezogabine, have shown promising results in alleviating depressive symptoms and anhedonia. The aforementioned observations underscore the therapeutic potential of KCNQ channel modulation in depression management and highlight the need and justification for phase 2 and phase 3 dose-finding studies as well as studies prespecifying symptomatic targets in depression including anhedonia.

Heightened excitability of vagal sensory neurons in inflammatory visceral diseases contributes to unproductive and difficult-to-treat neuronally based symptoms such as visceral pain and dysfunction. Identification of targets and modulators capable of regulating the excitability of vagal sensory neurons may lead to novel therapeutic options. KCNQ1-KCNQ5 genes encode KV7.1-7.5 potassium channel α-subunits. Homotetrameric or heterotetrameric KV7.2-7.5 channels can generate the so-called M-current (IM) known to decrease the excitability of neurons including visceral sensory neurons. This study aimed to address the hypothesis that KV7.2/7.3 channels are key regulators of vagal sensory neuron excitability by evaluating the effects of KCNQ2/3-selective activator, ICA-069673, on IM in mouse nodose neurons and determining its effects on excitability and action potential firings using patch clamp technique. The results showed that ICA-069673 enhanced IM density, accelerated the activation, and delayed the deactivation of M-channels in a concentration-dependent manner. ICA-069673 negatively shifted the voltage-dependent activation of IM and increased the maximal conductance. Consistent with its effects on IM, ICA-069673 induced a marked hyperpolarization of resting potential and reduced the input resistance. The hyperpolarizing effect was more pronounced in partially depolarized neurons. Moreover, ICA-069673 caused a 3-fold increase in the minimal amount of depolarizing current needed to evoke an action potential, and significantly limited the action potential firings in response to sustained suprathreshold stimulations. ICA-069673 had no effect on membrane currents when Kcnq2 and Kcnq3 were deleted. These results indicate that opening KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and enhance spike accommodation in vagal visceral sensory neurons. SIGNIFICANCE STATEMENT: This study supports the hypothesis that selectively activating KCNQ2/3-mediated M-channels is sufficient to suppress the excitability and action potential firings in vagal sensory neurons. These results provide evidence in support of further investigations into the treatment of various visceral disorders that involve nociceptor hyperexcitability with selective KCNQ2/3 M-channel openers.

Mutations within the Kv7.2 and Kv7.3 genes are well described causes for genetic childhood epilepsies. Knowledge on these channels in acquired focal epilepsy, especially in mesial temporal lobe epilepsy (mTLE), however, is scarce. Here, we used the rat pilocarpine model of drug-resistant mTLE to elucidate both expression and function by quantitative polymerase-chain reaction, immunohistochemistry, and electrophysiology, respectively. We found transcriptional downregulation of Kv7.2 and Kv7.3 as well as reduced Kv7.2 expression in epileptic CA1. Consequences were altered synaptic transmission, hyperexcitability which consisted of epileptiform afterpotentials, and increased susceptibility to acute GABAergic disinhibition. Importantly, blocking Kv7 channels with XE991 increased hyperexcitability in control tissue, but not in chronically epileptic tissue suggesting that the Kv7 deficit had precluded XE991 effects in this tissue. Conversely, XE991 resulted in comparable reduction of the paired-pulse ratio in both experimental groups implying preserved presynaptic Kv7.2 function of Schaffer collateral terminals. Consistent with Kv7.2/7.3 downregulation, the Kv7.3 channel opener β-hydroxybutyrate failed to mitigate hyperexcitability. Our findings demonstrate that compromised Kv7 function is not only relevant in genetic epilepsy, but also in acquired focal epilepsy. Moreover, they help explain reduced anti-seizure efficacy of Kv7 channel openers in drug-resistant epilepsy.

The human voltage-gated potassium channel KCNQ2/KCNQ3 carries the neuronal M-current, which helps to stabilize the membrane potential. KCNQ2 can be activated by analgesics and antiepileptic drugs but their activation mechanisms remain unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human KCNQ2-CaM in complex with three activators, namely the antiepileptic drug cannabidiol (CBD), the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and HN37 (pynegabine), an antiepileptic drug in the clinical trial, in an either closed or open conformation. The activator-bound structures, along with electrophysiology analyses, reveal the binding modes of two CBD, one PIP2, and two HN37 molecules in each KCNQ2 subunit, and elucidate their activation mechanisms on the KCNQ2 channel. These structures may guide the development of antiepileptic drugs and analgesics that target KCNQ2.

Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in ∼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.

Neuronal Kv7 voltage-gated potassium channels generate the M-current and regulate neuronal excitability. Here, we report that dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that attenuates Gq-coupled receptor-induced M-current suppression. DHEAS reduced muscarinic agonist-induced Kv7-current suppression of Kv7.1, Kv7.2, Kv7.4, or Kv7.5 homomeric currents and endogenous M-currents in rat sympathetic ganglion neurons. However, DHEAS per se did not alter the voltage dependence of these Kv7 homomeric channels or the m1 receptor-induced activation of phospholipase C or protein kinase C. DHEAS-treated Kv7.2 homomeric currents became resistant to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by voltage-activated phosphatase, Ci-VSP or eVSP. Our computational models predicted a novel binding site for DHEAS in the cytoplasmic domain of Kv7 subunits. A single-point mutation of the predicted key histidine into cysteine in the rat Kv7.2 subunit, rKv7.2(H558C), resulted in a loss of effects of DHEAS on muscarinic Kv7 current suppression. Furthermore, in vivo administration of DHEAS in mice of both sexes reduced late phase pain responses in the formalin paw test. However, it did not have effects on early phase responses in the formalin paw test or responses in the hot plate test. Coadministration of a selective Kv7 inhibitor, XE991, and DHEAS eliminated analgesic effects of DHEAS in late phase responses in the formalin paw test. Collectively, these results suggest that DHEAS attenuates M-current suppression by stabilizing PIP2-Kv7 subunit interaction and can mitigate inflammatory pain.SIGNIFICANCE STATEMENT M-current suppression induced by stimulation of Gq-coupled receptors is a form of Kv7 current modulation that can reversibly increase neuronal excitability. This study demonstrates that DHEAS, an endogenous steroid hormone, is a novel Kv7 channel modulator that can attenuate M-current suppression without affecting basal Kv7 channel kinetics. Administration of DHEAS in vivo alleviated inflammatory pain in rodents. These results suggest that the degree of M-current suppression can be dynamically regulated by small molecules. Therefore, this novel form of Kv7 channel regulation holds promising potential as a therapeutic target for sensitized nervous activities, such as inflammatory pain.