Activation Of Kv1 Research Articles

Flupirtine enhances NHE-3-mediated Na+ absorption in rat colon via an ENS-dependent mechanism.

Recent studies in our lab have shown that the KV7 channel activator, flupirtine, inhibits colonic epithelial Cl- secretion through effects on submucosal neurons of the enteric nervous system (ENS). We hypothesized that flupirtine would also stimulate Na+ absorption as a result of reduced secretory ENS input to the epithelium. To test this hypothesis, unidirectional 22Na+ fluxes were measured under voltage-clamped conditions. Pharmacological approaches using an Ussing-style recording chamber combined with immunofluorescence microscopy techniques were used to determine the effect of flupirtine on active Na+ transport in the rat colon. Flupirtine stimulated electroneutral Na+ absorption in partially seromuscular-stripped colonic tissues, while simultaneously inhibiting short-circuit current (ISC; i.e., Cl- secretion). Both of these effects were attenuated by pretreatment with the ENS inhibitor, tetrodotoxin. The Na+/H+ exchanger isoform 3 (NHE-3)-selective inhibitor, S3226, significantly inhibited flupirtine-stimulated Na+ absorption, whereas the NHE-2-selective inhibitor HOE-694 did not. NHE-3 localization near the apical membranes of surface epithelial cells was also more apparent in flupirtine-treated colon versus control. Flupirtine did not alter epithelial Na+ channel (ENaC)-mediated Na+ absorption in distal colonic tissues obtained from hyperaldosteronaemic rats and had no effect in the normal ileum but did stimulate Na+ absorption in the proximal colon. Finally, the parallel effects of flupirtine on ISC (Cl- secretion) and Na+ absorption were significantly correlated with each other. Together, these data indicate that flupirtine stimulates NHE-3-dependent Na+ absorption, likely as a result of reduced stimulatory input to the colonic epithelium by submucosal ENS neurons.NEW & NOTEWORTHY We present a novel mechanism regarding regulation of epithelial ion transport by enteric neurons. Activation of neuronal KV7 K+ channels markedly stimulates Na+ absorption and inhibits Cl- secretion across the colonic epithelium. This may be useful in developing new treatments for diarrheal disorders, such as irritable bowel syndrome with diarrhea (IBS-D).

Activation of KCNQ (KV7) K+ channels in enteric neurons inhibits epithelial Cl- secretion in mouse distal colon.

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).

Molecular expression and functional features of Kv7 channels in human prostate smooth muscle.

Relaxation of prostate smooth muscle tone is a key strategy for the medical treatment of lower urinary tract symptoms (LUTS) in men. However, potassium channel's physiological role inhuman prostatic smooth muscle (HPrSM) has yet to be determined. In this study, we examined the molecular characteristics and physiological roles of Kv7 channels in HPrSM. The expressions of KCNQ1-5 (Kv7 channel pore-forming α-subunits) and KCNE1-5 (β-regulatory subunits) isoforms in HPrSM were examined using real-time PCR. The relaxation effect of ML213 was investigated by cumulatively adding ML213 to the prostate strips. Kv7 currents were recorded using an amphotericin-B perforated patch-clamp technique. In HPrSM cells, KCNQ4, KCNQ5, and KCNE4 isoforms were predominantly expressed, while KCNQ1, KCNQ5, and KCNE3 isoforms were the most abundantly expressed in human prostatic tissues. Western blot analysis revealed the protein expression of the Kv7.1, 7.4, and 7.5 channel subtypes in human prostate tissues (n=3). ML213 (an activator of Kv7.2/7.4/7.5) induced the concentration-dependent relaxation of HPrSM strips (n=15, p=0.001), and this effect was attenuated by pretreatment with XE991 (a Kv7.1-7.5 blocker). In electrophysiology studies, ML213 significantly increased the amplitude of whole-cell Kv7 currents in HPrSM cells. ML213-induced outward currents were greater than retigabine (a Kv7.2-7.5 channel activator). The subsequent addition of XE991 completely inhibited the ML213-induced currents (n=9, p<0.01 vs. ML213). ML213 hyperpolarized the HPrSM cell membrane potential and was fully reversed by XE991. In situ PLA revealed the colocalization of heteromeric KV7.4/KV7.5 channels in HPrSM cells. Our findings suggest that Kv7.4 and 7.5 channels in prostatic smooth muscle play a critical role in the regulation of HPrSM tone and that Kv7 channel subtypes may be novel therapeutic targets for the treatment of LUTS associated with BPH.

The S2–S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation

Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2–S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM1234, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM3, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM1234 on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM1234 or CaM3 are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2–S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM3, indicating that these five mutations decrease the inhibitory effect of Ca2+/CaM. The double mutation C156A/R159A decreases Ca2+/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2–S3 loop in CaM regulation.

The Functional Availability of Arterial Kv7 Channels Is Suppressed Considerably by Large-Conductance Calcium-Activated Potassium Channels in 2- to 3-Month Old but Not in 10- to 15-Day Old Rats

BackgroundVoltage-gated potassium (Kv) channels, especially Kv7 channels, are major potassium channels identified in vascular smooth muscle cells with a great, albeit differential functional impact in various vessels. Vascular smooth muscle Kv7 channels always coexist with other K channels, in particular with BK channels. BK channels differ in the extent to which they influence vascular contractility. Whether this difference also causes the variability in the functional impact of Kv7 channels is unknown. Therefore, this study addressed the hypothesis that the functional impact of Kv7 channels depends on BK channels.Experimental ApproachExperiments were performed on young and adult rat gracilis and saphenous arteries using real-time PCR as well as pressure and wire myography.Key ResultsSeveral subfamily members of Kv7 (KCNQ) and BK channels were expressed in saphenous and gracilis arteries: the highest expression was observed for BKα, BKβ1 and KCNQ4. Arterial contractility was assessed with methoxamine-induced contractions and pressure-induced myogenic responses. In vessels of adult rats, inhibition of Kv7 channels or BK channels by XE991 or IBTX, respectively enhanced arterial contractility to a similar degree, whereas activation of Kv7 channels or BK channels by retigabine or NS19504, respectively reduced arterial contractility to a similar degree. Further, IBTX increased both the contractile effect of XE991 and the anticontractile effect of retigabine, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991. In vessels of young rats, inhibition of Kv7 channels by XE991 enhanced arterial contractility much stronger than inhibition of BK channels by IBTX, whereas activation of Kv7 by retigabine reduced arterial contractility to a greater extent than activation of BK channels by NS19504. Further, IBTX increased the anticontractile effect of retigabine but not the contractile effect of XE991, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991.ConclusionKv7 and BK channels are expressed in young and adult rat arteries and function as negative feedback modulators in the regulation of contractility of these arteries. Importantly, BK channels govern the extent of functional impact of Kv7 channels. This effect depends on the relationship between the functional activities of BK and Kv7 channels.

Familial neonatal seizures caused by the Kv7.3 selectivity filter mutation T313I.

ObjectiveA spectrum of seizure disorders is linked to mutations in Kv7.2 and Kv7.3 channels. Linking functional effects of identified mutations to their clinical presentation requires ongoing characterization of newly identified variants. In this study, we identified and functionally characterized a previously unreported mutation in the selectivity filter of Kv7.3.MethodsNext‐generation sequencing was used to identify the Kv7.3[T313I] mutation in a family affected by neonatal seizures. Electrophysiological approaches were used to characterize the functional effects of this mutation on ion channels expressed in Xenopus laevis oocytes.ResultsSubstitution of residue 313 from threonine to isoleucine (Kv7.3[T313I]) likely disrupts a critical intersubunit hydrogen bond. Characterization of the mutation in homomeric Kv7.3 channels demonstrated a total loss of channel function. Assembly in heteromeric channels (with Kv7.2) leads to modest suppression of total current when expressed in Xenopus laevis oocytes. Using a Kv7 activator with distinct effects on homomeric Kv7.2 vs heteromeric Kv7.2/Kv7.3 channels, we demonstrated that assembly of Kv7.2 and Kv7.3[T313I] generates functional channels.SignificanceBiophysical and clinical effects of the T313I mutation are consistent with Kv7.3 mutations previously identified in cases of pharmacoresponsive self‐limiting neonatal epilepsy. These findings expand our description of functionally characterized Kv7 channel variants and report new methods to distinguish molecular mechanisms of channel mutations.

Gabapentin treatment in a patient with KCNQ2 developmental epileptic encephalopathy

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations.

Functional and behavioral signatures of Kv7 activator drug subtypes.

Voltage-gated potassium channels of the KCNQ (Kv7) family are targeted by a variety of activator compounds with therapeutic potential for treatment of epilepsy. Exploration of this drug class has revealed a variety of effective compounds with diverse mechanisms. In this study, we aimed to clarify functional criteria for categorization of Kv7 activator compounds, and to compare the effects of prototypical drugs in a zebrafish larvae model. In vitro electrophysiological approaches with recombinant ion channels were used to highlight functional properties important for classification of drug mechanisms. We also benchmarked the effects of representative antiepileptic Kv7 activator drugs using behavioral seizure assays of zebrafish larvae and in vivo Ca2+ imaging with the ratiometric Ca2+ sensor CaMPARI. Drug effects on channel gating kinetics, and drug sensitivity profiles to diagnostic channel mutations, were used to highlight properties for categorization of Kv7 activator drugs into voltage sensor-targeted or pore-targeted subtypes. Quantifying seizures and ratiometric Ca2+ imaging in freely swimming zebrafish larvae demonstrated that while all Kv7 activators tested lead to suppression of neuronal excitability, pore-targeted activators (like ML213 and retigabine) strongly suppress seizure behavior, whereas ICA-069673 triggers a seizure-like hypermotile behavior. This study suggests criteria to categorize antiepileptic Kv7 activator drugs based on their underlying mechanism. We also establish the use of in vivo CaMPARI as a tool for screening effects of anticonvulsant drugs on neuronal excitability in zebrafish. In summary, despite a shared ability to suppress neuronal excitability, our findings illustrate how mechanistic differences between Kv7 activator subtypes influence their effects on heteromeric channels and lead to vastly different in vivo outcomes.

Heteromeric Assembly of Truncated Neuronal Kv7 Channels: Implications for Neurologic Disease and Pharmacotherapy.

Neuronal voltage-gated potassium channels (Kv) are critical regulators of electrical activity in the central nervous system. Mutations in the KCNQ (Kv7) ion channel family are linked to epilepsy and neurodevelopmental disorders. These channels underlie the neuronal "M-current" and cluster in the axon initial segment to regulate the firing of action potentials. There is general consensus that KCNQ channel assembly and heteromerization are controlled by C-terminal helices. We identified a pediatric patient with neurodevelopmental disability, including autism traits, inattention and hyperactivity, and ataxia, who carries a de novo frameshift mutation in KCNQ3 (KCNQ3-FS534), leading to truncation of ∼300 amino acids in the C terminus. We investigated possible molecular mechanisms of channel dysfunction, including haplo-insufficiency or a dominant-negative effect caused by the assembly of truncated KCNQ3 and functional KCNQ2 subunits. We also used a recently recognized property of the KCNQ2-specific activator ICA-069673 to identify assembly of heteromeric channels. ICA-069673 exhibits a functional signature that depends on the subunit composition of KCNQ2/3 channels, allowing us to determine whether truncated KCNQ3 subunits can assemble with KCNQ2. Our findings demonstrate that although the KCNQ3-FS534 mutant does not generate functional channels on its own, large C-terminal truncations of KCNQ3 (including the KCNQ3-FS534 mutation) assemble efficiently with KCNQ2 but fail to promote or stabilize KCNQ2/KCNQ3 heteromeric channel expression. Therefore, the frequent assumption that pathologies linked to KCNQ3 truncations arise from haplo-insufficiency should be reconsidered in some cases. Subtype-specific channel activators like ICA-069673 are a reliable tool to identify heteromeric assembly of KCNQ2 and KCNQ3. SIGNIFICANCE STATEMENT: Mutations that truncate the C terminus of neuronal Kv7/KCNQ channels are linked to a spectrum of seizure disorders. One role of the multifunctional KCNQ C terminus is to mediate subtype-specific assembly of heteromeric KCNQ channels. This study describes the use of a subtype-specific Kv7 activator to assess assembly of heteromeric KCNQ2/KCNQ3 (Kv7.2/Kv7.3) channels and demonstrates that large disease-linked and experimentally generated C-terminal truncated KCNQ3 mutants retain the ability to assemble with KCNQ2.

Chemical regulation of Kv7 channels: Diverse scaffolds, sites, and mechanisms of action.

Kv7 channels are powerfully regulated by a wide variety of physiological and pharmacological signals. Larsson et al. describe the direct modulation of Kv7 channels by endocannabinoids and explore how combinations of Kv7 activators with distinct subtype specificities might lead to effective and selective drug cocktails.

Probing the Molecular Basis of Opposing PUFA Effects on KV7 Channels

The human KV7 ion channel family consists of five members, KV7.1-7.5, distribution of which differs between tissues. As different KV7 subtypes play different roles in different physiological processes, developing pharmacological drugs that differentiate between KV7 subtypes is important in order to limit off-target effects. In pilot experiments, we have observed that free fatty acids, such as the omega-3 fatty acid docosahexaenoic acid, activated all KV7 channels save for KV7.4. In contrast, docosahexaenoic acid induced a slight inhibition of the KV7.4 channel. The aim of this study was to understand the molecular mechanism underlying the difference in observed effects of free fatty acid and their analogues on KV7.4. Using the two-electrode voltage clamp technique to measure potassium currents in Xenopus oocytes expressing hKV7.4 and hKV7.5, we compared the effect of free fatty acids and their analogues on these two KV7 channel subtypes. Our experiments found that neutralisation of the negative charge in the carboxylic head group of docosahexaenoic acid quenched both the inhibition and activation of KV7.4 and KV7.5, respectively, indicating an electrostatic element is involved in the mechanism of action. However, the importance of lipid tail properties differs between the two effects. To determine the contributions of ion channel structure to the observed difference in response, we created KV7.4 mutants in which native amino acids had been substituted for their KV7.5 counterparts. Our experiments found that mutation of residues in a putative fatty acid site either impaired or completely abolished fatty acid-mediated inhibition, but did not result in channel activation when docosahexaenoic acid was applied. Our preliminary studies have identified regions in KV7.4 that contribute to fatty acid-mediated inhibition. Further studies will be performed to fully understand the mechanistic basis for the opposing PUFA effects on KV7.4 and KV7.5.

Vasodilation of rat skeletal muscle arteries by the novel BK channel opener GoSlo is mediated by the simultaneous activation of BK and Kv 7 channels.

Background and PurposeBK channels play important roles in various physiological and pathophysiological processes and thus have been the target of several drug development programmes focused on creating new efficacious BK channel openers, such as the GoSlo‐SR compounds. However, the effect of GoSlo‐SR compounds on vascular smooth muscle has not been studied. Therefore, we tested the hypothesis that GoSlo‐SR compounds dilate arteries exclusively by activating BK channels.Experimental ApproachExperiments were performed on rat Gracilis muscle, saphenous, mesenteric and tail arteries using isobaric and isometric myography, sharp microelectrodes, digital droplet PCR and the patch‐clamp technique.Key ResultsGoSlo‐SR compounds dilated isobaric and relaxed and hyperpolarised isometric vessel preparations and their effects were abolished after (a) functionally eliminating K+ channels by pre‐constriction with 50 mM KCl or (b) blocking all K+ channels known to be expressed in vascular smooth muscle. However, these effects were not blocked when BK channels were inhibited. Surprisingly, the Kv7 channel inhibitor XE991 reduced their effects considerably, but neither Kv1 nor Kv2 channel blockers altered the inhibitory effects of GoSlo‐SR. However, the combined blockade of BK and Kv7 channels abolished the GoSlo‐SR‐induced relaxation. GoSlo‐SR compounds also activated Kv7.4 and Kv7.5 channels expressed in HEK 293 cells.Conclusion and ImplicationsThis study shows that GoSlo‐SR compounds are effective relaxants in vascular smooth muscle and mediate their effects by a combined activation of BK and Kv7.4/Kv7.5 channels. Activation of Kv1, Kv2 or Kv7.1 channels or other vasodilator pathways seems not to be involved.

Synthesis and Pharmacological Characterization of Conformationally Restricted Retigabine Analogues as Novel Neuronal Kv7 Channel Activators.

Kv7 K+ channels represent attractive pharmacological targets for the treatment of different neurological disorders, including epilepsy. In this paper, 42 conformationally restricted analogues of the prototypical Kv7 activator retigabine have been synthesized and tested by electrophysiological patch-clamp experiments as Kv7 agonists. When compared to retigabine (0.93 ± 0.43 μM), the EC50s for Kv7.2 current enhancements by compound 23a (0.08 ± 0.04 μM) were lower, whereas no change in potency was observed for 24a (0.63 ± 0.07 μM). In addition, compared to retigabine, 23a and 24a showed also higher potency in activating heteromeric Kv7.2/Kv7.3 and homomeric Kv7.4 channels. Molecular modeling studies provided new insights into the chemical features required for optimal interaction at the binding site. Stability studies evidenced improved chemical stability of 23a and 24a in comparison with retigabine. Overall, the present results highlight that the N5-alkylamidoindole moiety provides a suitable pharmacophoric scaffold for the design of chemically stable, highly potent and selective Kv7 agonists.

Activation of Kv7 Potassium Channels Inhibits Intracellular Ca2+ Increases Triggered By TRPV1-Mediated Pain-Inducing Stimuli in F11 Immortalized Sensory Neurons.

Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 µM) was higher than that of ICA-27243 (IC50~5 µM) and (S)-1 (IC50~7 µM). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.

Modulation of Kv7.1/KCNE1 Channel Activity by Navβ1

KV7.1 is a voltage-gated potassium channel encoded by the gene KCNQ1. This channel is found in several organs, including the heart and the brain. In the mammalian heart, KV7.1 assembles with KCNE1 forming a channel complex that is critically important for ventricular repolarization. The activity of the KV7.1/KCNE1 complex results in the delayed rectifier current known as IKs. The voltage-gated sodium channel β type 1 subunit (NaVβ1) is also expressed in the mammalian heart. Mutations in this protein have been implicated in disorders such as epilepsy and cardiopathies, including Brugada syndrome and atrial fibrillation. The NaVβ1 subunit modulates the activity, trafficking, and expression of NaV channels. It has been shown that NaVβ1 also modulates channels from the KV1 and KV4 families, and KV7.2 channels. Preliminary data from our laboratory show that NaVβ1 modulates the activity of KV7.1. When expressed in Xenopus oocytes, KV7.1 channel-mediated K+-currents display a mild slow inactivation. These channels recover from inactivation during deactivation driven at negative voltage, displaying a “hook” like kinetics. In the presence of NaVβ1, the inactivation is remarkably enhanced, producing a strong resurgent conductance during deactivation at negative potentials. Furthermore, the deactivation rate is decreased, while activation remains seemingly unaltered. Since KV7.1, KCNE1 and NaVβ1 are expressed in the heart, we decided to investigate whether NaVβ1 could modulate KV7.1/KCNE1 activity. We found that expressing KCNE1 subunit seems to override the NaVβ1 subunit effect on KV7.1 channels. However, decreasing the relative expression of KCNE1 resulted in K+-currents that displayed features of the activity of both type of complexes. We therefore conclude that NaVβ1 can modulate the KV7.1/KCNE1 complex activity. Our work suggests that, in the heart, the interaction of KCNE1 with KV7.1 has evolved to avoid the effect of NaVβ1 on the channel, so preventing its inactivation.

Electrophysiological and Pharmacological Characterization of a Novel and Potent Neuronal Kv7 Opener SCR2682 for Anti-Epilepsy

Voltage-gated Kv7/KCNQ/M-potassium channels play an essential role in control of membrane potential and neuronal excitability. Activation of neuronal Kv7/KCNQ/M-current represents an attractive therapeutic strategy for treatment of hyperexcitability-related neuropsychiatric disorders such as epilepsy, pain and schizophrenia. In this study, we synthesized and characterized a novel series of 2,6-dimethyl-4-(piperidin-yl)phenyl)-amide derivatives that exhibit selective and potent activation of neuronal Kv7/KCNQ/M channels. Whole-cell patch clamp recordings of HEK293 cells expressing Kv7.2/Kv7.3 channels show that a representative compound SCR2682 selectively activates the channel current in dose-dependent manner with an EC50 of 9.8 ± 0.4 nM, which is about 100-fold potent than antiepileptic drug retigabine approved by FDA for partial epilepsy. Compound SCR2682 shifts the voltage-dependent activation of Kv7.2/7.3 current towards more negative membrane potential about −37 mV (V1/2). SCR2682 also activates native M current of rat hippocampal or cortical neurons, causing a marked hyperpolarization and potent inhibition of evoked action potentials. In rat epileptic models, intraperitoneal and intragastric administrations of SCR2682 results in a dose-dependent inhibition of seizures. Taken together, our findings demonstrate that a novel SCR2682 selectively and potently activates neuronal Kv7 channels and reverses epileptic activity in rats. Thus, SCR2682 may warrant further evaluation for clinical development of antiepileptic therapy.

Neuropathic pain and Kv7 voltage-gated potassium channels: The potential role of Kv7 activators in the treatment of neuropathic pain.

Neuropathic pain conditions severely and chronically affect the quality of life in a large human population, but the pain conditions are not adequately managed due to poor understanding of their underlying mechanisms. There is a pressing need for further research into this field to help develop effective and nonaddictive medications to treat neuropathic pain. This article first describes general clinical classification of pain, types and symptoms of neuropathic pain, and current practices of clinical management for neuropathic pain. This is followed by a discussion of various cellular and molecular mechanisms responsible for the development and maintenance of neuropathic pain. In this review, we highlight the loss of function of Kv7 voltage-gated potassium as a mechanism of neuropathic pain and the potential use of Kv7 channel activator as subsequent treatment.

Investigating the Role of G Protein βγ in Kv7-Dependent Relaxations of the Rat Vasculature.

Objective- In renal arteries, inhibitors of G protein βγ subunits (Gβγ) reduce Kv7 activity and inhibit Kv7-dependent receptor-mediated vasorelaxations. However, the mechanisms underlying receptor-mediated relaxation are artery specific. Consequently, the aim of this study was to ascertain the role of Gβγ in Kv7-dependent vasorelaxations of the rat vasculature. Approach and Results- Isometric tension recording was performed in isolated rat renal, mesenteric, and cerebral arteries to study isoproterenol and calcitonin gene-related peptide relaxations. Kv7.4 was knocked down via morpholino transfection while inhibition of Gβγ was investigated with gallein and M119K. Proximity ligation assay was performed on isolated myocytes to study the association between Kv7.4 and G protein β subunits or signaling intermediaries. Isoproterenol or calcitonin gene-related peptide-induced relaxations were attenuated by Kv7.4 knockdown in all arteries studied. Inhibition of Gβγ with gallein or M119K had no effect on isoproterenol-mediated relaxations in mesenteric artery but had a marked effect on calcitonin gene-related peptide-induced responses in mesenteric artery and cerebral artery and isoproterenol responses in renal artery. Isoproterenol increased association with Kv7.4 and Rap1a in mesenteric artery which were not sensitive to gallein, whereas in renal artery, isoproterenol increased Kv7.4-AKAP (A-kinase anchoring protein) associations in a gallein-sensitive manner. Conclusions- The Gβγ-Kv7 relationship differs between vessels and is an essential requirement for AKAP, but not Rap-mediated regulation of the channel.

Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells.

β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.

Behavioral, cellular and molecular maladaptations covary with exposure to pyridostigmine bromide in a rat model of gulf war illness pain

Many veterans of Operation Desert Storm (ODS) struggle with the chronic pain of Gulf War Illness (GWI). Exposure to insecticides and pyridostigmine bromide (PB) have been implicated in the etiology of this multisymptom disease. We examined the influence of 3 (DEET (N,N-diethyl-meta-toluamide), permethrin, chlorpyrifos) or 4 GW agents (DEET, permethrin, chlorpyrifos, pyridostigmine bromide (PB)) on the post-exposure ambulatory and resting behaviors of rats. In three independent studies, rats that were exposed to all 4 agents consistently developed both immediate and delayed ambulatory deficits that persisted at least 16 weeks after exposures had ceased. Rats exposed to a 3 agent protocol (PB excluded) did not develop any ambulatory deficits. Cellular and molecular studies on nociceptors harvested from 16WP (weeks post-exposure) rats indicated that vascular nociceptor Nav1.9 mediated currents were chronically potentiated following the 4 agent protocol but not following the 3 agent protocol. Muscarinic linkages to muscle nociceptor TRPA1 were also potentiated in the 4 agent but not the 3 agent, PB excluded, protocol. Although Kv7 activity changes diverged from the behavioral data, a Kv7 opener, retigabine, transiently reversed ambulation deficits. We concluded that PB played a critical role in the development of pain-like signs in a GWI rat model and that shifts in Nav1.9 and TRPA1 activity were critical to the expression of these pain behaviors.