RESUMEN
Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7 (KCNQ, M-type) voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca2+/calmodulin (CaM) and zinc-mediated potentiation. We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.
Asunto(s)
Calcio , Calmodulina , Espacio Intracelular , Canales de Potasio KCNQ , Zinc , Señalización del Calcio , Calmodulina/metabolismo , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/química , Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Mutación , Unión Proteica/genética , Zinc/farmacología , Zinc/metabolismo , Espacio Intracelular/metabolismo , Calcio/metabolismo , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/química , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/química , Canal de Potasio KCNQ3/genética , Canal de Potasio KCNQ3/metabolismoRESUMEN
Although the lateral habenula (LHb) is involved in the regulation of multiple brain functions and this region expresses abundant M-type potassium channel (M-channel) subunits Kv7.2 and Kv7.3, the role of M-channels in regulating working memory is unclear, particularly in Parkinson's disease (PD). Here we tested the effects of activation and blockade of LHb M-channels on working memory by the T-maze rewarded alternation test in rats with unilateral 6-hydroxydopamine lesions of the substantia nigra compacta (SNc). The SNc lesion induced working memory impairment, increased the firing rate of LHb neurons, decreased dopamine (DA) level in the ventral medial prefrontal cortex (vmPFC) and reduced the expression of Kv7.2 subunit in the LHb. Intra-LHb injection of M-channel activator retigabine induced enhancement of working memory in SNc sham-lesioned and SNc-lesioned rats; conversely, the injection of M-channel blocker XE-991 impaired working memory in the two groups of rats. However, doses producing significant effects in SNc-lesioned rats were higher than those in SNc sham-lesioned rats. Further, intra-LHb injection of retigabine decreased the firing rate of LHb neurons and increased release of DA and serotonin (5-HT) in the vmPFC, while XE-991 increased the firing rate and decreased DA and 5-HT release in the two groups of rats. Compared with SNc sham-lesioned rats, the duration of M-channel activation and blockade action on the firing rate of the neurons and release of DA and 5-HT was significantly shortened in SNc-lesioned rats, which was consistent with reduced expression of Kv7.2 subunit in the LHb after lesioning the SNc. Collectively, these findings suggest involvement of LHb Kv7.2 subunit-containing M-channels in working memory impairment in SNc-lesioned rats, and that enhanced or impaired working memory after activation or blockade of M-channels in the LHb is related to the changes in the firing activity of LHb neurons and DA and 5-HT release in the vmPFC.
Asunto(s)
Habénula/metabolismo , Canal de Potasio KCNQ2/biosíntesis , Memoria a Corto Plazo/fisiología , Trastornos Parkinsonianos/metabolismo , Animales , Habénula/efectos de los fármacos , Canal de Potasio KCNQ2/agonistas , Canal de Potasio KCNQ2/antagonistas & inhibidores , Masculino , Moduladores del Transporte de Membrana/farmacología , Memoria a Corto Plazo/efectos de los fármacos , Oxidopamina/toxicidad , Trastornos Parkinsonianos/inducido químicamente , Bloqueadores de los Canales de Potasio/farmacología , Subunidades de Proteína/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC50â¯=â¯0.16⯵M in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.
Asunto(s)
Antracenos/farmacología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ3/antagonistas & inhibidores , Trastornos Mentales/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Bloqueadores de los Canales de Potasio/farmacología , Antracenos/síntesis química , Antracenos/química , Relación Dosis-Respuesta a Droga , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Estructura Molecular , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Relación Estructura-ActividadRESUMEN
The Kv7 family of voltage-dependent non-inactivating potassium channels is composed of five members, of which four are expressed in the CNS. Kv7.2, 7.3 and 7.5 are responsible for the M-current, which plays a critical role in the regulation of neuronal excitability. Stimulation of M1 muscarinic acetylcholine receptor, M1 receptor, increases neuronal excitability by suppressing the M-current generated by the Kv7 channel family. The M-current modulation via M1 receptor is well-described in in vitro assays using cell lines and in native rodent tissue. However, this mechanism was not yet reported in human induced pluripotent stem cells (hiPSC) derived neurons. In the present study, we investigated the effects of both agonists and antagonists of Kv7.2/7.3 channel and M1 receptor in hiPSC derived neurons and in primary rat cortical neuronal cells. The role of M1 receptors in the modulation of neuronal excitability could be demonstrated in both rat primary and hiPSC neurons. The M1 receptors agonist, xanomeline, increased neuronal excitability in both rat cortical and the hiPSC neuronal cells. Furthermore, M1 receptor agonist-induced neuronal excitability in vitro was reduced by an agonist of Kv7.2/7.3 in both neuronal cells. These results show that hiPSC derived neurons recreate the modulation of the M-current by the muscarinic receptor in hiPSC neurons similarly to rat native neurons. Thus, hiPSC neurons could be a useful human-based cell assay for characterization of drugs that affect neuronal excitability and/or induce seizure activity by modulation of M1 receptors or inhibition of Kv7 channels.
Asunto(s)
Fenómenos Electrofisiológicos , Células Madre Pluripotentes Inducidas/citología , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Neuronas/citología , Receptor Muscarínico M1/metabolismo , Animales , Fenómenos Electrofisiológicos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Canal de Potasio KCNQ2/agonistas , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/agonistas , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/genética , Antagonistas Muscarínicos/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidoresRESUMEN
Cholinergic interneurons (ChIs) of the striatum pause their firing in response to salient stimuli and conditioned stimuli after learning. Several different mechanisms for pause generation have been proposed, but a unifying basis has not previously emerged. Here, using in vivo and ex vivo recordings in rat and mouse brain and a computational model, we show that ChI pauses are driven by withdrawal of excitatory inputs to striatum and result from a delayed rectifier potassium current (IKr) in concert with local neuromodulation. The IKr is sensitive to Kv7.2/7.3 blocker XE-991 and enables ChIs to report changes in input, to pause on excitatory input recession, and to scale pauses with input strength, in keeping with pause acquisition during learning. We also show that although dopamine can hyperpolarize ChIs directly, its augmentation of pauses is best explained by strengthening excitatory inputs. These findings provide a basis to understand pause generation in striatal ChIs. VIDEO ABSTRACT.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Interneuronas/metabolismo , Aprendizaje , Animales , Antracenos/farmacología , Neuronas Colinérgicas/efectos de los fármacos , Simulación por Computador , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Interneuronas/efectos de los fármacos , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ3/antagonistas & inhibidores , Ratones , Modelos Neurológicos , Bloqueadores de los Canales de Potasio/farmacología , RatasRESUMEN
Multidrug resistance along with side-effects of available anti-epileptic drugs and unavailability of potent and effective agents in submicromolar quantities presents the biggest therapeutic challenges in anti-epileptic drug discovery. The molecular modeling techniques allow us to identify agents with novel structures to match the continuous urge for its discovery. KCNQ2 channel represents one of the validated targets for its therapy. The present study involves identification of newer anti-epileptic agents by means of a computer-aided drug design adaptive protocol involving both structure-based virtual screening of Asinex library using homology model of KCNQ2 and 3D-QSAR based virtual screening with docking analysis, followed by dG bind and ligand efficiency calculations with ADMET studies, of which 20 hits qualified all the criterions. The best ligands of both screenings with least potential for toxicity predicted computationally were then taken for molecular dynamic simulations. All the crucial amino acid interactions were observed in hits of both screenings such as Glu130, Arg207, Arg210 and Phe137. Robustness of docking protocol was analyzed through Receiver operating characteristic (ROC) curve values 0.88 (Area under curve AUC = 0.87) in Standard Precision and 0.84 (AUC = 0.82) in Extra Precision modes. Novelty analysis indicates that these compounds have not been reported previously as anti-epileptic agents.
Asunto(s)
Anticonvulsivantes/química , Epilepsia/tratamiento farmacológico , Canal de Potasio KCNQ2/química , Modelos Moleculares , Anticonvulsivantes/uso terapéutico , Epilepsia/patología , Humanos , Canal de Potasio KCNQ2/antagonistas & inhibidores , Ligandos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad Cuantitativa , Interfaz Usuario-ComputadorRESUMEN
Biological mechanism attributing mutations in KCNQ2/Q3 results in benign familial neonatal epilepsy (BFNE), a rare form of epilepsy and thus neglected. It offers a potential target for antiepileptic drug discovery. In the present work, a pharmacophore-based 3D-QSAR model was generated for a series of N-pyridyl and pyrimidine benzamides possessing KCNQ2/Q3 opening activity. The pharmacophore model generated contains one hydrogen bond donor (D), one hydrophobic (H), and two aromatic rings (R). They are the crucial molecular write-up detailing predicted binding efficacy of high affinity and low affinity ligands for KCNQ2/Q3 opening activity. Furthermore, it has been validated by using a biological correlation between pharmacophore hypothesis-based 3D-QSAR variables and functional fingerprints of openers responsible for the receptor binding and also by docking of these benzamides into the validated homology model. Excellent statistical computational tools of QSAR model such as good correlation coefficient (R2 > 0.80), higher F value (F > 39), and excellent predictive power (Q2 > 0.7) with low standard deviation (SD <0.3) strongly suggest that the developed model could be used for prediction of antiepileptic activity of newer analogs. A preliminary pharmacokinetic profile of these derivatives was also performed on the basis of QikProp predictions.
Asunto(s)
Benzamidas/química , Descubrimiento de Drogas , Epilepsia Benigna Neonatal/tratamiento farmacológico , Canal de Potasio KCNQ2/química , Canal de Potasio KCNQ3/química , Anticonvulsivantes/química , Anticonvulsivantes/uso terapéutico , Benzamidas/uso terapéutico , Sitios de Unión , Simulación por Computador , Epilepsia Benigna Neonatal/genética , Epilepsia Benigna Neonatal/patología , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/genética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Pirimidinas/química , Relación Estructura-Actividad CuantitativaRESUMEN
Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels.
Asunto(s)
Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ3/antagonistas & inhibidores , Oxotremorina/análogos & derivados , Bloqueadores de los Canales de Potasio/farmacología , Animales , Humanos , Oxotremorina/farmacología , Piridinas/farmacología , Receptor Muscarínico M1/metabolismo , Tiadiazoles/farmacología , XenopusRESUMEN
PURPOSE: KCNQ2/3 channels play an important role in controlling neuronal excitability. Agents that decrease KCNQ2/3 current amplitudes are proconvulsant, whereas KCNQ2/3 current enhancers are anticonvulsant. Levobupivacaine is able to block the KCNQ2/3 channels and enhance neuronal excitation, whereas retigabine is able to reopen the channels and thus reduce overexcitation of neurons. In this study, we aimed to determine if retigabine is able to abolish local-anesthetic-induced seizures. METHODS: Twenty New Zealand rabbits were randomly divided into two groups of ten. Levobupivacaine (0.5 %) was infused into conscious rabbits via the marginal ear vein at 8 ml/kg/h until the rabbits seized, and 5 mg/kg of retigabine were injected intravenously to terminate the seizure. The corresponding volume of saline was used as a control. The behavior of and the electroencephalogram (EEG) for each rabbit were continually monitored. Before levobupivacaine infusion, the rabbits were placed in a prostrate position calmly on the experimental platform, and the EEG pattern exhibited ß waves. Intravenous levobupivacaine induced a typical EEG seizure characterized by multiple spike and slow wave complexes. The EEG changes were accompanied by behavioral convulsions which were characterized by clonic activity and opisthotonus. RESULTS: Retigabine effectively terminated the electrographic and behavioral seizures. After receiving 5 mg/kg of retigabine, the animals became drowsy, and the EEG changed to δ waves. CONCLUSIONS: We propose that KCNQ2/3 channels play an important role in levobupivacaine-induced central nervous system toxicity, and a KCNQ2/3 channel activator may be used to treat levobupivacaine-induced convulsions.
Asunto(s)
Anticonvulsivantes/farmacología , Bupivacaína/análogos & derivados , Carbamatos/farmacología , Fenilendiaminas/farmacología , Convulsiones/prevención & control , Animales , Bupivacaína/toxicidad , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Levobupivacaína , Neuronas/efectos de los fármacos , Estudios Prospectivos , Conejos , Distribución AleatoriaRESUMEN
Spatial memory retrieval and hippocampal long-term potentiation (LTP) are impaired by stress. KCNQ/Kv7 channels are closely associated with memory and the KCNQ/Kv7 channel activator flupirtine represents neuroprotective effects. This study aims to test whether KCNQ/Kv7 channel activation prevents acute stress-induced impairments of spatial memory retrieval and hippocampal LTP. Rats were placed on an elevated platform in the middle of a bright room for 30 min to evoke acute stress. The expression of KCNQ/Kv7 subunits was analyzed at 1, 3 and 12 h after stress by Western blotting. Spatial memory was examined by the Morris water maze (MWM) and the field excitatory postsynaptic potential (fEPSP) in the hippocampal CA1 area was recorded in vivo. Acute stress transiently decreased the expression of KCNQ2 and KCNQ3 in the hippocampus. Acute stress impaired the spatial memory retrieval and hippocampal LTP, the KCNQ/Kv7 channel activator flupirtine prevented the impairments, and the protective effects of flupirtine were blocked by XE-991 (10,10-bis(4-Pyridinylmethyl)-9(10H)-anthracenone), a selective KCNQ channel blocker. Furthermore, acute stress decreased the phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9 in the hippocampus, and flupirtine inhibited the reduction. These results suggest that the KCNQ/Kv7 channels may be a potential target for protecting both hippocampal synaptic plasticity and spatial memory retrieval from acute stress influences.
Asunto(s)
Aminopiridinas/farmacología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Trastornos de la Memoria/prevención & control , Nootrópicos/farmacología , Estrés Psicológico/tratamiento farmacológico , Animales , Antracenos/farmacología , Carbamatos/farmacología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/fisiopatología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/metabolismo , Potenciación a Largo Plazo/fisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/fisiopatología , Fenilendiaminas/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Ratas Sprague-Dawley , Memoria Espacial/efectos de los fármacos , Memoria Espacial/fisiología , Estrés Psicológico/fisiopatologíaRESUMEN
The voltage-gated potassium channels of the KV7 family (KV7.1-5) play important roles in controlling neuronal excitability and are therefore attractive targets for treatment of CNS disorders linked to hyperexcitability. One of the main challenges in developing KV7 channel active drugs has been to identify compounds capable of discriminating between the neuronally expressed subtypes (KV7.2-5), aiding the identification of the subunit composition of KV7 currents in various tissues, and possessing better therapeutic potential for particular indications. By taking advantage of the structure-activity relationship of acrylamide KV7 channel openers and the effects of these compounds on mutant KV7 channels, we have designed and synthesized a novel KV7 channel modulator with a unique profile. The compound, named SMB-1, is an inhibitor of KV7.2 and an activator of KV7.4. SMB-1 inhibits KV7.2 by reducing the current amplitude and increasing the time constant for the slow component of the activation kinetics. The activation of KV7.4 is seen as an increase in the current amplitude and a slowing of the deactivation kinetics. Experiments studying mutant channels with a compromised binding site for the KV7.2-5 opener retigabine indicate that SMB-1 binds within the same pocket as retigabine for both inhibition of KV7.2 and activation of KV7.4. SMB-1 may serve as a valuable tool for KV7 channel research and may be used as a template for further design of better subtype selective KV7 channel modulators. A compound with this profile could hold novel therapeutic potential such as the treatment of both positive and cognitive symptoms in schizophrenia.
Asunto(s)
Canales de Potasio KCNQ , Canal de Potasio KCNQ2 , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/farmacología , Mutación Missense , Sustitución de Aminoácidos , Animales , Humanos , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Canal de Potasio KCNQ2/agonistas , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/metabolismo , Xenopus laevisRESUMEN
KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels.
Asunto(s)
Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/metabolismo , Tamoxifeno/farmacología , Células HEK293 , Humanos , Concentración 50 Inhibidora , Canal de Potasio KCNQ2/genética , Mutación Missense/genética , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
AIM: Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies. METHODS: A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System. RESULTS: A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC50 value=0.10 ± 0.05 µmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 µmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 µmol/L) did not affect the kinetics of channel activation and deactivation. CONCLUSION: The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.
Asunto(s)
Anticonvulsivantes/farmacología , Carbamatos/farmacología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Fenilendiaminas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Animales , Antracenos/farmacología , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Células CHO , Carbamatos/síntesis química , Carbamatos/química , Cricetinae , Cricetulus , Diseño de Fármacos , Concentración 50 Inhibidora , Técnicas de Placa-Clamp , Fenilendiaminas/síntesis química , Fenilendiaminas/química , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Relación Estructura-ActividadRESUMEN
Skin keratinocytes fulfil important signalling and protective functions. Immunocytochemical experiments revealed the unexpected presence of immunoreactivity for the M-type potassium channel subunit Kv7.2 in the keratinocyte layer of intact rat paw skin and in keratinocytes isolated from the skin of 1-day-old rats and cultured in vitro for 3-10 days. Application of the M-channel enhancer retigabine (3-10 µM) to isolated cultured rat keratinocytes: (a) increased outward membrane currents recorded under voltage clamp, (b) produced ~3 mV hyperpolarization at rest, (c) enhanced ~3-fold the release of ATP induced by the TRPV3 agonist carvacrol (1 mM) and (d) increased the amplitude of the carvacrol-induced intracellular Ca(2+) transient measured with Fura-2. The effect of retigabine on ATP release was prevented by the M-channel blocking agent XE991. We conclude that rat skin keratinocytes possess M-channels that, when activated, can modify their physiological properties, with potential significance for their sensory and other biological functions.
Asunto(s)
Canal de Potasio KCNQ2/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Potenciales de Acción , Adenosina Trifosfato/metabolismo , Animales , Antracenos/farmacología , Calcio/metabolismo , Carbamatos/farmacología , Células Cultivadas , Cimenos , Canal de Potasio KCNQ2/antagonistas & inhibidores , Queratinocitos/fisiología , Monoterpenos/farmacología , Fenilendiaminas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Piel/citología , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/metabolismoRESUMEN
The parasubiculum (PaS) is a component of the hippocampal formation that sends its major output to layer II of the entorhinal cortex. The PaS receives strong cholinergic innervation from the basal forebrain that is likely to modulate neuronal excitability and contribute to theta-frequency network activity. The present study used whole cell current- and voltage-clamp recordings to determine the effects of cholinergic receptor activation on layer II PaS neurons. Bath application of carbachol (CCh; 10-50 µM) resulted in a dose-dependent depolarization of morphologically-identified layer II stellate and pyramidal cells that was not prevented by blockade of excitatory and inhibitory synaptic inputs. Bath application of the M1 receptor antagonist pirenzepine (1 µM), but not the M2-preferring antagonist methoctramine (1 µM), blocked the depolarization, suggesting that it is dependent on M1 receptors. Voltage-clamp experiments using ramped voltage commands showed that CCh resulted in the gradual development of an inward current that was partially blocked by concurrent application of the selective Kv7.2/3 channel antagonist XE-991, which inhibits the muscarine-dependent K(+) current I M. The remaining inward current also reversed near EK and was inhibited by the K(+) channel blocker Ba(2+), suggesting that M1 receptor activation attenuates both I M as well as an additional K(+) current. The additional K(+) current showed rectification at depolarized voltages, similar to K(+) conductances mediated by Kir 2.3 channels. The cholinergic depolarization of layer II PaS neurons therefore appears to occur through M1-mediated effects on I M as well as an additional K(+) conductance.
Asunto(s)
Potenciales de la Membrana/fisiología , Neuronas/metabolismo , Células Piramidales/metabolismo , Receptor Muscarínico M1/metabolismo , Animales , Antracenos/farmacología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Diaminas/farmacología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Antagonistas Muscarínicos/farmacología , Neuronas/citología , Parasimpatolíticos/farmacología , Pirenzepina/farmacología , Células Piramidales/citología , Ratas , Ratas Long-Evans , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidoresRESUMEN
Bone cancer pain has a strong impact on the quality of life of patients, but is difficult to treat. Better understanding of the pathogenic mechanisms underlying bone cancer pain will likely lead to the development of more effective treatments. In the present study, we investigated whether inhibition of KCNQ/M channels contributed to the hyperexcitability of primary sensory neurons and to the pathogenesis of bone cancer pain. By using a rat model of bone cancer pain based on intratibial injection of MRMT-1 tumour cells, we documented a prominent decrease in expression of KCNQ2 and KCNQ3 proteins and a reduction of M-current density in small-sized dorsal root ganglia (DRG) neurons, which were associated with enhanced excitability of these DRG neurons and the hyperalgesic behaviours in bone cancer rats. Coincidently, we found that inhibition of KCNQ/M channels with XE-991 caused a robust increase in the excitability of small-sized DRG neurons and produced an obvious mechanical allodynia in normal rats. On the contrary, activation of the KCNQ/M channels with retigabine not only inhibited the hyperexcitability of these small DRG neurons, but also alleviated mechanical allodynia and thermal hyperalgesia in bone cancer rats, and all of these effects of retigabine could be blocked by KCNQ/M-channel antagonist XE-991. These results suggest that repression of KCNQ/M channels leads to the hyperexcitability of primary sensory neurons, which in turn causes bone cancer pain. Thus, suppression of KCNQ/M channels in primary DRG neurons plays a crucial role in the development of bone cancer pain.
Asunto(s)
Neoplasias Óseas/fisiopatología , Carcinoma/fisiopatología , Ganglios Espinales/fisiopatología , Hiperalgesia/etiología , Canal de Potasio KCNQ2/fisiología , Canal de Potasio KCNQ3/fisiología , Nocicepción/fisiología , Dolor/etiología , Células Receptoras Sensoriales/fisiología , Animales , Antracenos/farmacología , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Carbamatos/farmacología , Carbamatos/uso terapéutico , Carcinoma/patología , Carcinoma/secundario , Regulación hacia Abajo , Femenino , Calor/efectos adversos , Hiperalgesia/fisiopatología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/biosíntesis , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/biosíntesis , Canal de Potasio KCNQ3/genética , Neoplasias Mamarias Experimentales/patología , Trasplante de Neoplasias , Técnicas de Placa-Clamp , Fenilendiaminas/farmacología , Fenilendiaminas/uso terapéutico , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismo , Estrés Mecánico , Transmisión Sináptica , Tibia/patologíaAsunto(s)
Epilepsia , Canal de Potasio KCNQ2 , Canal de Potasio KCNQ3 , Potasio/metabolismo , Convulsiones , Animales , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/terapia , Humanos , Recién Nacido , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/antagonistas & inhibidores , Canal de Potasio KCNQ3/genética , Terapia Molecular Dirigida , Mutación , Ratas , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/terapiaRESUMEN
Benign familial neonatal epilepsy is a neuronal channelopathy most commonly caused by mutations in KCNQ2, which encodes the K(v)7.2 subunit of the slow K(+) channel. K(v)7.2 is expressed in both central and peripheral nervous systems. Seizures occur in the neonatal period, often in clusters within the first few days of life, and usually remit by 12 months of age. The mechanism of involvement of K(v)7.2 mutations in the process of seizure generation has not been established in vivo. In peripheral axons, K(v)7.2 contributes to the nodal slow K(+) current. The present study aimed to determine whether axonal excitability studies could detect changes in peripheral nerve function related to dysfunction or loss of slow potassium channel activity. Nerve excitability studies were performed on eight adults with KCNQ2 mutations and a history of benign familial neonatal epilepsy, now in remission. Studies detected distinctive changes in peripheral nerve, indicating a reduction in slow K(+) current. Specifically, accommodation to long-lasting depolarizing currents was reduced in mutation carriers by 24% compared with normal controls, and the threshold undershoot after 100 ms depolarizing currents was reduced by 22%. Additional changes in excitability included a reduction in the relative refractory period, an increase in superexcitability and a tendency towards reduced sub-excitability. Modelling of the nerve excitability changes suggested that peripheral nerve hyperexcitability may have been ameliorated by upregulation of other potassium channels. We conclude that subclinical dysfunction of K(v)7.2 in peripheral axons can be reliably detected non-invasively in adulthood. Related alterations in neuronal excitability may contribute to epilepsy associated with KCNQ2 mutations.
Asunto(s)
Canalopatías/genética , Canalopatías/metabolismo , Epilepsia Benigna Neonatal/genética , Epilepsia Benigna Neonatal/metabolismo , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ2/genética , Adulto , Axones/patología , Axones/fisiología , Canalopatías/fisiopatología , Estudios de Cohortes , Epilepsia Benigna Neonatal/fisiopatología , Femenino , Humanos , Canal de Potasio KCNQ2/metabolismo , Masculino , Persona de Mediana Edad , Mutación/genética , Prevención SecundariaRESUMEN
A potent and selective inhibitor of KCNQ2, (S)-5 (ML252, IC(50) = 69 nM), was discovered after a high-throughput screen of the MLPCN library was performed. SAR studies revealed a small structural change (ethyl group to hydrogen) caused a functional shift from antagonist to agonist activity (37, EC(50) = 170 nM), suggesting an interaction at a critical site for controlling gating of KCNQ2 channels.
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Encéfalo/metabolismo , Canal de Potasio KCNQ2/antagonistas & inhibidores , Fenilbutiratos/síntesis química , Bloqueadores de los Canales de Potasio/síntesis química , Pirrolidinas/síntesis química , Animales , Bases de Datos Factuales , Ensayos Analíticos de Alto Rendimiento , Humanos , Canal de Potasio KCNQ2/química , Microsomas Hepáticos/metabolismo , Permeabilidad , Fenilbutiratos/química , Fenilbutiratos/farmacocinética , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacocinética , Pirrolidinas/química , Pirrolidinas/farmacocinética , Ratas , Bibliotecas de Moléculas Pequeñas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Dysfunction of brain-gut interaction is thought to underlie visceral hypersensitivity which causes unexplained abdominal pain syndromes. However, the mechanism by which alteration of brain function in the brain-gut axis influences the perception of visceral pain remains largely elusive. In this study we investigated whether altered brain activity can generate visceral hyperalgesia. RESULTS: Using a forebrain specific αCaMKII promoter, we established a line of transgenic (Tg) mice expressing a dominant-negative pore mutant of the Kv7.2/KCNQ2 channel which suppresses native KCNQ/M-current and enhances forebrain neuronal excitability. Brain slice recording of hippocampal pyramidal neurons from these Tg mice confirmed the presence of hyperexcitable properties with increased firing. Behavioral evaluation of Tg mice exhibited increased sensitivity to visceral pain induced by intraperitoneal (i.p.) injection of either acetic acid or magnesium sulfate, and intracolon capsaicin stimulation, but not cutaneous sensation for thermal or inflammatory pain. Immunohistological staining showed increased c-Fos expression in the somatosensory SII cortex and insular cortex of Tg mice that were injected intraperitoneally with acetic acid. To mimic the effect of cortical hyperexcitability on visceral hyperalgesia, we injected KCNQ/M channel blocker XE991 into the lateral ventricle of wild type (WT) mice. Intracerebroventricular injection of XE991 resulted in increased writhes of WT mice induced by acetic acid, and this effect was reversed by co-injection of the channel opener retigabine. CONCLUSIONS: Our findings provide evidence that forebrain hyperexcitability confers visceral hyperalgesia, and suppression of central hyperexcitability by activation of KCNQ/M-channel function may provide a therapeutic potential for treatment of abdominal pain syndromes.