RESUMO
Depression is a common and complex psychiatric illness with multiple clinical symptoms, even leading to the disability and suicide. Owing to the partial understanding of the pathogenesis of depressive-like disorders, available pharmacotherapeutic strategies are developed mainly based on the "monoamine hypothesis", resulting in a limited effectiveness and a number of adverse effects in the clinical practice. The concept of multiple pathogenic factors be helpful for clarifying the etiology of depression and developing the antidepressants. It is well documented that K+ channels serve crucial roles in modulating the neuronal excitability and neurotransmitter release in the brain, and abnormality of these channels participated in the pathogenic process of diverse central nervous system (CNS) pathologies, such as seizure and Alzheimer's disease (AD). The clinical and preclinical evidence also delineates that the involvement of several types of K+ channels in depressive-like behaviors appear to be evident, suggesting these channels being one of the multiple factors in the etiology of this debilitating disorder. Emerging data manifest that diverse antidepressants impact distinct K+ channels, such as Kv, Kir and K2P, meaning the functioning of these drug via a "multi-target" manner. On the other hand, the scenario of antidepressants impinging K+ channels could render an alternative interpretation for the pharmacological effectiveness and numerous side effects in clinical trials. Furthermore, these channels serve to be considered as a "druggable target" to develop novel therapeutic compound to antagonize this psychiatry.
Assuntos
Antidepressivos , Canais de Potássio , Humanos , Antidepressivos/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio/efeitos dos fármacos , Animais , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/metabolismo , Depressão/tratamento farmacológico , Depressão/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismoRESUMO
BACKGROUND: Neuronal ceroid lipofuscinosis (NCL) is a heterogeneous group of 13 rare, progressive neurodegenerative diseases of the brain and retina. CLN14 is a very rare subtype of NCL caused by pathogenic variants in the KCTD7 gene. Only four cases of this subtype have been reported in the literature. CASE PRESENTATION: A nine-month-old, previously healthy male who was firstborn to first-cousin parents presented with progressive psychomotor regression, dysmorphic facial features, myoclonus, and vision loss. Neurological examination showed generalized hypotonia and brisk reflexes. He continued to deteriorate until age 18 months, when he developed his first generalized tonic-clonic seizure. An ophthalmological examination showed a hypopigmented fundus and slight temporal disc pallor. Brain MRI showed mild generalized brain atrophy and white matter disease. EEG revealed a severely abnormal trace marked by generalized, high amplitude, sharply contoured, polymorphic delta slowing intermixed with theta slowing and some alpha activity, with disorganized and scattered spikes and sharp waves. The patient continued to have uncontrolled seizures and further psychomotor regression until he died of status epilepticus and pneumonia at the age of 44 months. WES identified a novel homozygous variant c.413T > C, p.(Leu138Pro) in the KCTD7 gene, causing an amino acid transition from leucine to proline at position 138. Both parents were carriers of the same variant. CONCLUSIONS: We present the fifth known case of CLN14 in the literature and report the clinical course and a novel underlying likely causative variant in the KCTD7 gene. The improving accessibility and affordability of genetic testing will likely uncover more NCL cases and further expand the disease's genotypic and phenotypic spectrum.
Assuntos
Lipofuscinoses Ceroides Neuronais , Humanos , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/diagnóstico , Masculino , Lactente , Canais de PotássioRESUMO
Studies have demonstrated the therapeutic effects of Lindera plants. This study was undertaken to reveal the antihypertensive properties of Lindera erythrocarpa leaf ethanolic extract (LEL). Aorta segments of Sprague-Dawley rats were used to study the vasodilatory effect of LEL, and the mechanisms involved were evaluated by treating specific inhibitors or activators that affect the contractility of blood vessels. Our results revealed that LEL promotes a vasorelaxant effect through the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway, blocking the Ca2+ channels, opening the K+ channels, and inhibiting the vasoconstrictive action of angiotensin II. In addition, the effects of LEL on blood pressure were investigated in spontaneously hypertensive rats by the tail-cuff method. LEL (300 or 1000 mg/kg) was orally administered to the rats, and 1000 mg/kg of LEL significantly lowered the blood pressure. Systolic blood pressure decreased by -20.06 ± 4.87%, and diastolic blood pressure also lowered by -30.58 ± 5.92% at 4 h in the 1000 mg/kg LEL group. Overall, our results suggest that LEL may be useful to treat hypertensive diseases, considering its vasorelaxing and hypotensive effects.
Assuntos
Anti-Hipertensivos , Pressão Sanguínea , GMP Cíclico , Hipertensão , Lindera , Óxido Nítrico , Extratos Vegetais , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Animais , Anti-Hipertensivos/farmacologia , Extratos Vegetais/farmacologia , Óxido Nítrico/metabolismo , Pressão Sanguínea/efeitos dos fármacos , GMP Cíclico/metabolismo , Masculino , Hipertensão/tratamento farmacológico , Ratos , Lindera/química , Canais de Potássio/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Folhas de Planta/química , Vasodilatação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
The relative lack of marine venom could be attributed to the difficulty in dealing with venomous marine animals. Moreover, the venom of marine animals consists of various bioactive molecules, many of which are proteins with unique properties. In this study, we investigated the potential toxic proteins of jellyfish collected for ligand screening to understand the mechanism of the toxic effects of jellyfish. Since taxonomic identification is problematic due to the lack of proper keys, we conducted morphological and molecular mitochondrial DNA sequencing from COI and ITS regions. The venom extract from nematocysts found along the bell margins was used for protein characterization using SDS-gel electrophoresis and nano-liquid chromatography-tandem mass spectrometry. Ligand screening for the most potent toxin and antibacterial and cytotoxicity assays were carried out. The phylogenetic tree showed distinct clustering from other Catostylus sp. The proteomic analysis revealed venom with many bioactive proteins. Only 13 venom proteins were identified with molecular weights ranging from 4318 to 184,923 Da, exhibiting the venom's complexity. The overall toxin protein composition of Catostylus sp. venom was dominated by potassium channel toxin alpha-KTx. Molecular docking of toxin alpha-KTx 1.13 revealed high specificity towards the human voltage-gated potassium channel Kv3 with a high fitness score and a minimum energy barrier of -17.9 kcal/mol. Disc diffusion and cytotoxicity assays revealed potent antibacterial activity against Klebsiella pneumoniae with no cytotoxicity. Further studies on detailed characterization and therapeutic potentials are warranted.
Assuntos
Antibacterianos , Venenos de Cnidários , Simulação de Acoplamento Molecular , Peptídeos , Animais , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Venenos de Cnidários/farmacologia , Venenos de Cnidários/química , Peptídeos/farmacologia , Peptídeos/química , Cifozoários , Ligantes , Filogenia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Proteômica/métodosRESUMO
Previous studies have suggested a role for selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac®) in the treatment of dizziness and inner ear vestibular dysfunction. The potential mechanism of action within the vestibular system remains unclear; however, fluoxetine has been reported to block certain types of K+ channel in other systems. Here, we investigated the direct actions of fluoxetine on membrane currents in presynaptic hair cells and postsynaptic calyx afferents of the gerbil peripheral vestibular system using whole cell patch clamp recordings in crista slices. We explored differences in K+ currents in peripheral zone (PZ) and central zone (CZ) calyces of the crista and their response to fluoxetine application. Outward K+ currents in PZ calyces showed greater inactivation at depolarized membrane potentials compared to CZ calyces. The application of 100 µM fluoxetine notably reduced K+ currents in calyx terminals within both zones of the crista, and the remaining currents exhibited distinct traits. In PZ cells, fluoxetine inhibited a non-inactivating K+ current and revealed a rapidly activating and inactivating K+ current, which was sensitive to blocking by 4-aminopyridine. This was in contrast to CZ calyces, where low-voltage-activated and non-inactivating K+ currents persisted following application of 100 µM fluoxetine. Additionally, marked inhibition of transient inward Na+ currents by fluoxetine was observed in calyces from both crista zones. Different concentrations of fluoxetine were tested, and the EC50 values were found to be 40 µM and 32 µM for K+ and Na+ currents, respectively. In contrast, 100 µM fluoxetine had no impact on voltage-dependent K+ currents in mechanosensory type I and type II vestibular hair cells. In summary, micromolar concentrations of fluoxetine are expected to strongly reduce both Na+ and K+ conductance in afferent neurons of the peripheral vestibular system in vivo. This would lead to inhibition of action potential firing in vestibular sensory neurons and has therapeutic implications for disorders of balance.
Assuntos
Fluoxetina , Gerbillinae , Fluoxetina/farmacologia , Animais , Potenciais da Membrana/efeitos dos fármacos , Vestíbulo do Labirinto/efeitos dos fármacos , Vestíbulo do Labirinto/metabolismo , Técnicas de Patch-Clamp , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Canais de Potássio/metabolismo , Masculino , Células Ciliadas Vestibulares/efeitos dos fármacos , Células Ciliadas Vestibulares/metabolismoRESUMO
The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.
Assuntos
Proteínas de Transporte , Oócitos , Canais de Potássio , Ubiquitinação , Animais , Canais de Potássio/metabolismo , Canais de Potássio/genética , Oócitos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Xenopus laevis , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ligação Proteica , Potássio/metabolismo , Proteínas de XenopusRESUMO
Biological ion channels usually conduct the high-flux transport of 107 ~ 108 ions·s-1; however, the underlying mechanism is still lacking. Here, by applying the KcsA potassium channel as a typical example, and performing multitimescale molecular dynamics simulations, we demonstrate that there is coherence of the K+ ions confined in biological channels, which determines transport. The coherent oscillation state of confined K+ ions with a nanosecond-level lifetime in the channel dominates each transport event, serving as the physical basis for the high flux of ~108 ionsâs-1. The coherent transfer of confined K+ ions only takes several picoseconds and has no perturbation effect on the ion coherence, acting as the directional key of transport. Such ion coherence is allowed by quantum mechanics. An increase in the coherence can significantly enhance the ion conductance. These findings provide a potential explanation from the perspective of coherence for the high-flux ion transport with ultralow energy consumption of biological channels.
Assuntos
Transporte de Íons , Simulação de Dinâmica Molecular , Canais de Potássio , Potássio , Teoria Quântica , Canais de Potássio/metabolismo , Canais de Potássio/química , Potássio/metabolismo , Potássio/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Íons/metabolismoRESUMO
BACKGROUND: Activation of the NLRP3 inflammasome is critical in the inflammatory response to gout. Potassium ion (K+) efflux mediated by the TWIK2 channel is an important upstream mechanism for NLRP3 inflammasome activation. Therefore, the TWIK2 channel may be a promising therapeutic target for MSU crystal-induced inflammation. In the present study, we investigated the effect of ML335, a known K2P channel modulator, on MSU crystal-induced inflammatory responses and its underlying molecular mechanisms. METHODS: By molecular docking, we calculated the binding energies and inhibition constants of five K2P channel modulators (Hydroxychloroquine, Fluoxetine, DCPIB, ML365 and ML335) with TWIK2. Intracellular potassium ion concentration and mitochondrial function were assessed by flow cytometry. The interaction between MARCH5 and SIRT3 was demonstrated by immunoprecipitation and Western blotting assay. MSU suspensions were injected into mouse paw and peritoneal cavity to induce acute gout model. RESULTS: ML335 has the highest binding energy and the lowest inhibition constant with TWIK2 in the five calculated K2P channel modulators. In comparison, among these five compounds, ML335 efficiently inhibited the release of IL-1ß from MSU crystal-treated BMDMs. ML335 decreased MSU crystal-induced K+ efflux mainly dependent on TWIK2 channel. More importantly, ML335 can effectively inhibit the expression of the mitochondrial E3 ubiquitin ligase MARCH5 induced by MSU crystals, and MARCH5 can interact with the SIRT3 protein. ML335 blocked MSU crystal-induced ubiquitination of SIRT3 protein by MARCH5. In addition, ML335 improved mitochondrial dynamics homeostasis and mitochondrial function by inhibiting MARCH5 protein expression. ML335 attenuated the inflammatory response induced by MSU crystals in vivo and in vitro. CONCLUSION: Inhibition of TWIK2-mediated K+ efflux by ML335 alleviated mitochondrial injury via suppressing March5 expression, suggesting that ML335 may be an effective candidate for the future treatment of gout.
Assuntos
Inflamação , Mitocôndrias , Potássio , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Inflamação/patologia , Potássio/metabolismo , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Masculino , Gota/metabolismo , Gota/patologia , Gota/tratamento farmacológico , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Canais de Potássio/metabolismo , Sirtuína 3/metabolismo , Interleucina-1beta/metabolismo , Inflamassomos/metabolismo , HumanosRESUMO
Plants often face high salinity as a significant environmental challenge with roots being the first responders to this stress. Maintaining K+/Na+ ratio within plant cells is crucial for survival, as the intracellular K+ level decreases and the intracellular Na+ level increases under saline conditions. However, knowledge about the molecular regulatory mechanisms of K+ loss in response to salt stress through outward-rectifying K+ channels in plants is largely unknown. In this study, we found that the Arabidopsis double mutant gorkskor, in which the GORK and SKOR genes are disrupted, showed an improved primary root growth under salt stress compared to wild-type (WT) and the gork and skor single-mutant plants. No significant differences in the sensitivity to mannitol stress between the WT and gorkskor mutant were observed. Accumulation of ROS induced by salt stress was reduced in the gorkskor roots. The gorkskor mutant seedlings had significantly higher K+ content, lower Na+ content, and a greater resultant K+/Na+ ratio than the WT under salt stress. Moreover, salt-stress-induced elevation of cytosolic free Ca2+ concentration was reduced in the gorkskor roots. Taken together, these results suggest that Arabidopsis Shaker-type outward-rectifying K+ channels GORK and SKOR may redundantly function in regulation of primary root growth under salt stress and are involved in not only the late-stage response (e.g. K+ leakage) but also the early response including ROS production and [Ca2+]cyt elevation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Raízes de Plantas , Canais de Potássio , Estresse Salino , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Canais de Potássio/metabolismo , Canais de Potássio/genética , Potássio/metabolismo , Sódio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mutação , Superfamília Shaker de Canais de PotássioRESUMO
BACKGROUND: Cardiovascular disease remains one of the leading causes of death globally. Myocardial ischemia and infarction, in particular, frequently cause disturbances in cardiac electrical activity that can trigger ventricular arrhythmias. We aimed to investigate whether catestatin, an endogenous catecholamine-inhibiting peptide, ameliorates myocardial ischemia-induced ventricular arrhythmias in rats and the underlying ionic mechanisms. METHODS AND RESULTS: Adult male Sprague-Dawley rats were randomly divided into control and catestatin groups. Ventricular arrhythmias were induced by ligation of the left anterior descending coronary artery and electrical stimulation. Action potential, transient outward potassium current, delayed rectifier potassium current, inward rectifying potassium current, and L-type calcium current (ICa-L) of rat ventricular myocytes were recorded using a patch-clamp technique. Catestatin notably reduced ventricular arrhythmia caused by myocardial ischemia/reperfusion and electrical stimulation of rats. In ventricular myocytes, catestatin markedly shortened the action potential duration of ventricular myocytes, which was counteracted by potassium channel antagonists TEACl and 4-AP, and ICa-L current channel agonist Bay K8644. In addition, catestatin significantly increased transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current density in a concentration-dependent manner. Catestatin accelerated the activation and decelerated the inactivation of the transient outward potassium current channel. Furthermore, catestatin decreased ICa-L current density in a concentration-dependent manner. Catestatin also accelerated the inactivation of the ICa-L channel and slowed down the recovery of ICa-L from inactivation. CONCLUSIONS: Catestatin enhances the activity of transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current, while suppressing the ICa-L in ventricular myocytes, leading to shortened action potential duration and ultimately reducing the ventricular arrhythmia in rats.
Assuntos
Potenciais de Ação , Cromogranina A , Miócitos Cardíacos , Fragmentos de Peptídeos , Ratos Sprague-Dawley , Animais , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cromogranina A/farmacologia , Cromogranina A/metabolismo , Potenciais de Ação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Arritmias Cardíacas/metabolismo , Antiarrítmicos/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Modelos Animais de Doenças , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Técnicas de Patch-Clamp , Canais de Potássio de Retificação Tardia/metabolismo , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Canais de Potássio/metabolismo , Canais de Potássio/efeitos dos fármacosRESUMO
TMEM175 is a lysosomal potassium and proton channel that is associated with the development of Parkinson's disease. Advances in understanding the physiological roles of TMEM175 have been hampered by the absence of selective inhibitors, and studies involving genetic perturbations have yielded conflicting results. Here, we report the discovery and characterization of the first reported TMEM175-selective inhibitors, 2-phenylpyridin-4-ylamine (2-PPA), and AP-6. Cryo-EM structures of human TMEM175 bound by 2-PPA and AP-6 reveal that they act as pore blockers, binding at distinct sites in the pore and occluding the ion permeation pathway. Acute inhibition of TMEM175 by 2-PPA or AP-6 increases the level of lysosomal macromolecule catabolism, thereby accelerating macropinocytosis and other digestive processes. These inhibitors may serve as valuable tools to study the roles of TMEM175 in regulating lysosomal function and provide useful templates for future therapeutic development in Parkinson's disease.
Assuntos
Lisossomos , Doença de Parkinson , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Lisossomos/metabolismo , Descoberta de Drogas , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Canais Iônicos/química , Piridinas/química , Piridinas/farmacologia , Modelos Moleculares , Microscopia Crioeletrônica , Canais de PotássioRESUMO
Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis, efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3, a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium-mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications.
Assuntos
Antifúngicos , Bacillus subtilis , Peptídeos Cíclicos , Saccharomyces cerevisiae , Antifúngicos/farmacologia , Antifúngicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Bacillus subtilis/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Lipopeptídeos/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio/genética , Ergosterol , Aspergillus/efeitos dos fármacos , Aspergillus/metabolismo , Potássio/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.
Assuntos
Epilepsia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Mutação , Canais de Potássio , Propofol , Humanos , Sítios de Ligação , Microscopia Crioeletrônica , Eletrofisiologia , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/metabolismo , Células HEK293 , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/ultraestrutura , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Movimento/efeitos dos fármacos , Fenilalanina/genética , Fenilalanina/metabolismo , Polimorfismo Genético , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio/ultraestrutura , Propofol/farmacologia , Propofol/químicaRESUMO
The significant effects of lipid binding on the functionality of potassium channel KcsA have been validated by brilliant studies. However, the specific interactions between lipids and KcsA, such as binding parameters for each binding event, have not been fully elucidated. In this study, we employed native mass spectrometry to investigate the binding of lipids to KcsA and their effects on the channel. The tetrameric structure of KcsA remains intact even in the absence of lipid binding. However, the subunit architecture of the E71A mutant, which is constantly open at low pH, relies on tightly associated copurified lipids. Furthermore, we observed that lipids exhibit weak binding to KcsA at high pH when the channel is at a closed/inactivation state in the absence of permeant cation K+. This feeble interaction potentially facilitates the association of K+ ions, leading to the transition of the channel to a resting closed/open state. Interestingly, both anionic and zwitterionic lipids strongly bind to KcsA at low pH when the channel is in an open/inactivation state. We also investigated the binding patterns of KcsA with natural lipids derived from E. coli and Streptomyces lividans. Interestingly, lipids from E. coli exhibited much stronger binding affinity compared to the lipids from S. lividans. Among the natural lipids from S. lividans, free fatty acids and triacylglycerols demonstrated the tightest binding to KcsA, whereas no detectable binding events were observed with natural phosphatidic acid lipids. These findings suggest that the lipid association pattern in S. lividans, the natural host for KcsA, warrants further investigation. In conclusion, our study sheds light on the role of lipids in stabilizing KcsA and highlights the importance of specific lipid-protein interactions in modulating its conformational states.
Assuntos
Proteínas de Bactérias , Canais de Potássio , Ligação Proteica , Streptomyces lividans , Canais de Potássio/metabolismo , Canais de Potássio/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Streptomyces lividans/metabolismo , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Escherichia coli/metabolismoRESUMO
Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gß and cullin3, along with elevated dissociation of Gß from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gß and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.
Assuntos
Adenilil Ciclases , Proteínas Culina , Dependência de Morfina , Animais , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética , Proteínas Culina/metabolismo , Camundongos , Dependência de Morfina/metabolismo , Células HEK293 , Humanos , Canais de Potássio/metabolismo , Canais de Potássio/genética , Camundongos Endogâmicos C57BL , Masculino , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Morfina/farmacologia , Camundongos Knockout , Transdução de Sinais , AMP Cíclico/metabolismoRESUMO
The objective of this investigation was to examine the impact of multiple exposures to general anesthesia (GA) with sevoflurane on the offspring of pregnant mice, as well as to elucidate the underlying mechanism. Neurodevelopmental assessments, including various reflexes and behavioral tests, were conducted on the offspring in the GA group to evaluate neuronal cell development. Furthermore, neonatal mouse neuronal cells were isolated and transfected with a high-expression CREB vector (pcDNA3.1-CREB), followed by treatment with sevoflurane (0.72 mol/L), ZD7288 (50 µmol/L), and KN-62 (10 µmol/L), or a combination of these compounds. The expression of relevant genes was then analyzed using qRT-PCR and western blot techniques. In comparison to the sham group, neonatal mice in the GA group exhibited significantly prolonged latencies in surface righting reflex, geotaxis test, and air righting reflex. Furthermore, there was a notable deceleration in the development of body weight and tail in the GA group. These mice also displayed impairments in social ability, reduced reciprocal social interaction behaviors, diminished learning capacity, and heightened levels of anxious behaviors. Additionally, synaptic trigger malfunction was observed, along with decreased production of c-Fos and neurotrophic factors. Sevoflurane was found to notably decrease cellular c-Fos and neurotrophic factor production, as well as the expression of HCN2 and CaMKII/CREB-related proteins. The inhibitory effects of sevoflurane on HCN2 or CaMKII channels were similar to those observed with ZD7288 or KN-62 inhibition. However, overexpression of CREB mitigated the impact of sevoflurane on neuronal cells. Repetitive exposure to sevoflurane general anesthesia while pregnant suppresses the CaMKII/CREB pathway, leading to the development of autism-like characteristics in offspring mice through the reduction of HCN2 expression.
Assuntos
Anestésicos Inalatórios , Transtorno Autístico , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Regulação para Baixo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Efeitos Tardios da Exposição Pré-Natal , Sevoflurano , Animais , Sevoflurano/farmacologia , Sevoflurano/toxicidade , Camundongos , Gravidez , Feminino , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Anestésicos Inalatórios/farmacologia , Anestésicos Inalatórios/toxicidade , Anestésicos Inalatórios/efeitos adversos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Canais de Potássio/metabolismo , Canais de Potássio/genética , Células Cultivadas , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold. RESULTS: Our screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses. CONCLUSIONS: Our functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.
Assuntos
Canais de Potássio , Animais , Canais de Potássio/metabolismo , Canais de Potássio/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Filogenia , Camundongos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Evolução Molecular , Humanos , Venenos de Artrópodes/químicaRESUMO
BACKGROUND & AIMS: The treatment of cardiovascular diseases (CVD) could greatly benefit from using nitric oxide (NO) donors. This study aimed to investigate the mechanisms of action of NONO2P that contribute to the observed responses in the mesenteric artery. The hypothesis was that NONO2P would have similar pharmacological actions to sodium nitroprusside (SNP) and NO. METHODS: Male Wistar rats were euthanized to isolate the superior mesenteric artery for isometric tension recordings. NO levels were measured using the DAF-FM/DA dye, and cyclic guanosine monophosphate (cGMP) levels were determined using a cGMP-ELISA Kit. RESULTS: NONO2P presented a similar maximum efficacy to SNP. The free radical of NO (NOâ¢) scavengers (PTIO; 100 µM and hydroxocobalamin; 30 µM) and nitroxyl anion (NO-) scavenger (L-cysteine; 3 mM) decreased relaxations promoted by NONO2P. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor (ODQ; 10 µM) nearly abolished the vasorelaxation. The cGMP-dependent protein kinase (PKG) inhibition (KT5823; 1 µM) attenuated the NONO2P relaxant effect. The vasorelaxant response was significantly attenuated by blocking inward rectifying K+ channels (Kir), voltage-operated K+ channels (KV), and large conductance Ca2+-activated K+ channels (BKCa). NONO2P-induced relaxation was attenuated by cyclopiazonic acid (10 µM), indicating that sarcoplasmic reticulum Ca2+-ATPase (SERCA) activation is involved in this relaxation. Moreover, NONO2P increased NO levels in endothelial cells and cGMP production. CONCLUSIONS: NONO2P induces vasorelaxation with the same magnitude as SNP, releasing NO⢠and NO-. Its vasorelaxant effect involves sGC, PKG, K+ channels opening, and SERCA activation, suggesting its potential as a therapeutic option for CVD.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico , GMP Cíclico , Doadores de Óxido Nítrico , Óxido Nítrico , Canais de Potássio , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais , Guanilil Ciclase Solúvel , Vasodilatação , Animais , Masculino , Vasodilatação/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Guanilil Ciclase Solúvel/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Ratos , Canais de Potássio/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Guanilato Ciclase/metabolismo , Ativação Enzimática/efeitos dos fármacosRESUMO
Parkinson's disease (PD), recognized as the second most prevalent neurodegenerative disease in the aging population, presents a significant challenge due to the current lack of effective treatment methods to mitigate its progression. Many pathogenesis of PD are related to lysosomal dysfunction. Moreover, extensive genetic studies have shown a significant correlation between the lysosomal membrane protein TMEM175 and the risk of developing PD. Building on this discovery, TMEM175 has been identified as a novel potassium ion channel. Intriguingly, further investigations have found that potassium ion channels gradually close and transform into hydrion "excretion" channels in the microenvironment of lysosomes. This finding was further substantiated by studies on TMEM175 knockout mice, which exhibited pronounced motor dysfunction in pole climbing and suspension tests, alongside a notable reduction in dopamine neurons within the substantia nigra compacta. Despite these advancements, the current research landscape is not without its controversies. In light of this, the present review endeavors to methodically examine and consolidate a vast array of recent literature on TMEM175. This comprehensive analysis spans from the foundational research on the structure and function of TMEM175 to expansive population genetics studies and mechanism research utilizing cellular and animal models.A thorough understanding of the structure and function of TMEM175, coupled with insights into the intricate mechanisms underpinning lysosomal dysfunction in PD dopaminergic neurons, is imperative. Such knowledge is crucial for pinpointing precise intervention targets, thereby paving the way for novel therapeutic strategies that could potentially alter the neurodegenerative trajectory of PD.