RESUMEN
Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.
Asunto(s)
Trastornos Motores/prevención & control , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Proteínas Serina-Treonina Quinasas/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidores , Ataxias Espinocerebelosas/complicaciones , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos Motores/etiología , Trastornos Motores/metabolismo , Trastornos Motores/patología , Proteínas Serina-Treonina Quinasas/genética , Canales de Potasio Shaw/genética , Canales de Potasio Shaw/metabolismoRESUMEN
The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.
Asunto(s)
Simulación del Acoplamiento Molecular , Bloqueadores de los Canales de Potasio/química , Venenos de Escorpión/química , Canales de Potasio Shaw , Animales , Humanos , Escorpiones/química , Canales de Potasio Shaw/antagonistas & inhibidores , Canales de Potasio Shaw/química , Xenopus laevisRESUMEN
Cell migration is a complex and important process in cancer progression. Vimentin has pivotal roles in cancer cell migration, and various signaling pathways including the AKT pathway are involved in cancer cell migration via vimentin regulation. Recent studies have revealed that voltage-gated potassium (Kv) channels have important functions in cancer cell migration; however, the exact mechanism is still unclear. In the present study, we focused on Kv3 channels with vimentin in cancer migration using human cervical cancer cells (HeLa) and canine mammary tumor cells (CHMp). Cancer cell migration was significantly inhibited, and vimentin expression was significantly decreased by Kv3 blocker, BDS-II. The Kv3 blocker also inactivated the AKT pathway in HeLa cells. In addition, reduced expressions of vimentin and Kv3.4 were observed in HeLa cells when treated with AKT blocker, MK2206. These results suggest that Kv3 channels play important roles in cancer cell migration by regulating vimentin and having closely related with the AKT pathway in human cervical cancer cells.
Asunto(s)
Movimiento Celular , Neoplasias/metabolismo , Neoplasias/patología , Canales de Potasio Shaw/metabolismo , Vimentina/metabolismo , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Perros , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidores , Vimentina/biosíntesisRESUMEN
3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach â¼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.
Asunto(s)
Amifampridina/farmacología , Fármacos Neuromusculares/farmacología , Unión Neuromuscular/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Canales de Potasio Shaw/metabolismo , Acetilcolina/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica , Masculino , Ratones , Microelectrodos , Unión Neuromuscular/metabolismo , Terminales Presinápticos/metabolismo , Rana pipiens , Canales de Potasio Shaw/antagonistas & inhibidores , Canales de Potasio Shaw/genética , Técnicas de Cultivo de TejidosRESUMEN
Oxidative stress is one of the most important risk factors for cataractogenesis. Previous studies have indicated that BDS-II, a Kv3 channel blocker, plays pivotal roles in oxidative stress-related diseases. This study demonstrates that BDS-II exerts a protective effect on cataractogenesis. Specifically, BDS-II was observed to inhibit lens opacity induced by H2O2. BDS-II was also determined to inhibit cataract progression in a sodium selenite-induced in vivo cataract model by inhibiting reduction of the total GSH. In addition, BDS-II was demonstrated to protect human lens epithelial cells against H2O2-induced cell death. Our results suggest that BDS-II is a potential pharmacological candidate in cataract therapy.
Asunto(s)
Catarata/prevención & control , Estrés Oxidativo/efectos de los fármacos , Bloqueadores de los Canales de Potasio/uso terapéutico , Canales de Potasio Shaw/antagonistas & inhibidores , Animales , Muerte Celular , Línea Celular , Progresión de la Enfermedad , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Cristalino/citología , Masculino , Bloqueadores de los Canales de Potasio/farmacología , Ratas Sprague-Dawley , Canales de Potasio Shaw/metabolismoRESUMEN
BACKGROUND: Hypoxic pulmonary vasoconstriction (HPV) is a reaction of the pulmonary vasculature upon hypoxia, diverting blood flow into ventilated areas to preserve oxygenation. It is impaired in endotoxemia or ARDS. Voltage gated potassium channels have been shown to play a key role in the regulation of HPV. The aim of the study was to identify a voltage gated potassium channel involved in dysregulated HPV during endotoxemia. METHODS: Lungs of male C57BL/6 mice with and without endotoxemia (n = 6 ea. group) were analyzed for Kv3.4 gene and protein expression. HPV was examined in isolated perfused lungs of mice with and without endotoxemia and with and without selective Kv3.4 blocker BDS-I (n = 7 ea. group). Pulmonary artery pressure (PAP) and pressure-flow curves were measured during normoxic (FiO2 0.21) and hypoxic (FiO2 0.01) ventilation. HPV was quantified as the increase in perfusion pressure in response to hypoxia in percent of baseline perfusion pressure (ΔPAP) in the presence and absence of BDS-I. RESULTS: Kv3.4 gene (3.2 ± 0.5-fold, p < 0.05) and protein (1.5 ± 0.1-fold p < 0.05) expression levels were increased in endotoxemic mouse lungs. Endotoxemia reduced HPV (∆PAP control: 121.2 ± 8.7% vs. LPS 19.5 ± 8.0%, means ± SEM) while inhibition of Kv3.4 with 50 nM BDS-I augmented HPV in endotoxemic but not in control lungs (∆PAP control BDS-I: 116.6 ± 16.0% vs. LPS BDS-I 84.4 ± 18.2%, means ± SEM). CONCLUSIONS: Kv3.4 gene and protein expressions are increased in endotoxemic mouse lungs. Selective inhibition of Kv3.4 augments HPV in lungs of endotoxemic mice, but not in lungs of control mice.
Asunto(s)
Endotoxemia/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , Canales de Potasio Shaw/biosíntesis , Vasoconstricción/fisiología , Animales , Endotoxemia/patología , Hipoxia/patología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Perfusión , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Circulación Pulmonar/efectos de los fármacos , Canales de Potasio Shaw/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacosRESUMEN
KV3.1 blockers can serve as modulators of the rate of action potential firing in neurons with high rates of firing such as those of the auditory system. We studied the effects of several bioisosteres of N-alkylbenzenesulfonamides, and molecules derived from sulfanilic acid on KV3.1 channels, heterologously expressed in L-929 cells, using the whole-cell patch-clamp technique. Only the N-alkyl-benzenesulfonamides acted as open-channel blockers on KV3.1, while molecules analogous to PABA (p-aminobenzoic acid) and derived from sulfanilic acids did not block the channel. The IC50 of six N-alkyl-benzenesulfonamides ranged from 9 to 55 µM; and the Hill coefficient suggests the binding of two molecules to block KV3.1. Also, the effects of all molecules on KV3.1 were fully reversible. We look for similar features amongst the molecules that effectively blocked the channel and used them to model a blocker prototype. We found that bulkier groups and amino-lactams decreased the effectiveness of the blockage, while the presence of NO2 increased the effectiveness of the blockage. Thus, we propose N-alkylbenzenesulfonamides as a new class of KV3.1 channel blockers.
Asunto(s)
Activación del Canal Iónico , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Shaw/antagonistas & inhibidores , Sulfonamidas/química , Sulfonamidas/farmacología , Ácido 4-Aminobenzoico/metabolismo , Animales , Línea Celular , Lactamas/metabolismo , Ratones , Neuronas/metabolismo , Dióxido de Nitrógeno/metabolismo , Bloqueadores de los Canales de Potasio/síntesis química , Ácidos Sulfanílicos/metabolismo , Sulfonamidas/síntesis química , BencenosulfonamidasRESUMEN
Presynaptic voltage-gated K+ (Kv) channels in dorsal root ganglion (DRG) neurons are thought to regulate nociceptive synaptic transmission in the spinal dorsal horn. However, the Kv channel subtypes responsible for this critical role have not been identified. The Kv3.4 channel is particularly important because it is robustly expressed in DRG nociceptors, where it regulates action potential (AP) duration. Furthermore, Kv3.4 dysfunction is implicated in the pathophysiology of neuropathic pain in multiple pain models. We hypothesized that, through their ability to modulate AP repolarization, Kv3.4 channels in DRG nociceptors help to regulate nociceptive synaptic transmission. To test this hypothesis, we investigated Kv3.4 immunoreactivity (IR) in the rat cervical superficial dorsal horn (sDH) in both sexes and implemented an intact spinal cord preparation to investigate glutamatergic synaptic currents from second order neurons in the sDH under conditions that selectively inhibit the Kv3.4 current. We found presynaptic Kv3.4 IR in peptidergic and nonpeptidergic nociceptive fibers of the sDH. The Kv3.4 channel is hypersensitive to 4-aminopyridine and tetraethylammonium (TEA). Accordingly, 50 µm 4-aminopyridine and 500 µm TEA significantly prolong the AP, slow the maximum rate of repolarization in small-diameter DRG neurons, and potentiate monosynaptic excitatory postsynaptic currents (EPSCs) in dorsal horn laminae I and II through a presynaptic mechanism. In contrast, highly specific inhibitors of BK, Kv7, and Kv1 channels are less effective modulators of the AP and have little to no effect on EPSCs. The results strongly suggest that presynaptic Kv3.4 channels are major regulators of nociceptive synaptic transmission in the spinal cord.SIGNIFICANCE STATEMENT Intractable neuropathic pain can result from disease or traumatic injury and many studies have been conducted to determine the underlying pathophysiological changes. Voltage-gated ion channels, including the K+ channel Kv3.4, are dysregulated in multiple pain models. Kv3.4 channels are ubiquitously expressed in the dorsal root ganglion (DRG), where they are major regulators of DRG excitability. However, little is known about the ionic mechanisms that regulate nociceptive synaptic transmission at the level of the first synapse in the spinal cord, which is critical to pain transmission in both intact and pathological states. Here, we show that Kv3.4 channels have a significant impact on glutamatergic synaptic transmission in the dorsal horn, further illuminating its potential as a molecular pain therapeutic target.
Asunto(s)
Potenciales Postsinápticos Excitadores , Nocicepción , Células del Asta Posterior/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Células Cultivadas , Femenino , Ácido Glutámico/metabolismo , Masculino , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shaw/antagonistas & inhibidoresRESUMEN
We have recently developed a simple and effective bioengineering approach to large-scale production of alpha-KTx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. This approach was successfully approved for different peptides containing three disulfide bonds. To extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction with tobacco etch virus (TEV) protease need to be found. For this, the interplay between efficiency of TEV hydrolysis and sensitivity of the target peptides to disulfide reducing agents was studied, and optimized protocols of TEV cleavage reaction were worked out. Maurotoxin and hetlaxin were produced in a folded form avoiding in vitro renaturation step with yields of 14 and 12 mg/liter of culture, respectively.
Asunto(s)
Endopeptidasas/química , Bloqueadores de los Canales de Potasio/química , Venenos de Escorpión/química , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Canales de Potasio Shaw/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Clonación Molecular , Disulfuros , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Hidrólisis , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/metabolismo , Oxidación-Reducción , Plásmidos/química , Plásmidos/metabolismo , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Bloqueadores de los Canales de Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/metabolismo , Venenos de Escorpión/farmacología , Escorpiones/química , Canales de Potasio de la Superfamilia Shaker/metabolismo , Canales de Potasio Shaw/metabolismoRESUMEN
The intrinsic electrical characteristics of different types of neurons are shaped by the K+ channels they express. From among the more than 70 different K+ channel genes expressed in neurons, Kv3 family voltage-dependent K+ channels are uniquely associated with the ability of certain neurons to fire action potentials and to release neurotransmitter at high rates of up to 1,000 Hz. In general, the four Kv3 channels Kv3.1-Kv3.4 share the property of activating and deactivating rapidly at potentials more positive than other channels. Each Kv3 channel gene can generate multiple protein isoforms, which contribute to the high-frequency firing of neurons such as auditory brain stem neurons, fast-spiking GABAergic interneurons, and Purkinje cells of the cerebellum, and to regulation of neurotransmitter release at the terminals of many neurons. The different Kv3 channels have unique expression patterns and biophysical properties and are regulated in different ways by protein kinases. In this review, we cover the function, localization, and modulation of Kv3 channels and describe how levels and properties of the channels are altered by changes in ongoing neuronal activity. We also cover how the protein-protein interaction of these channels with other proteins affects neuronal functions, and how mutations or abnormal regulation of Kv3 channels are associated with neurological disorders such as ataxias, epilepsies, schizophrenia, and Alzheimer's disease.
Asunto(s)
Neuronas/metabolismo , Neurotransmisores/metabolismo , Canales de Potasio Shaw/metabolismo , Secuencia de Aminoácidos , Animales , Orientación del Axón , Humanos , Datos de Secuencia Molecular , Enfermedades del Sistema Nervioso/metabolismo , Fosfotransferasas/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidores , Transducción de SeñalRESUMEN
Action potential (AP) shape is a key determinant of cellular electrophysiological behavior. We found that in small-diameter, capsaicin-sensitive dorsal root ganglia neurons corresponding to nociceptors (from rats of either sex), stimulation at frequencies as low as 1 Hz produced progressive broadening of the APs. Stimulation at 10 Hz for 3 s resulted in an increase in AP width by an average of 76 ± 7% at 22°C and by 38 ± 3% at 35°C. AP clamp experiments showed that spike broadening results from frequency-dependent reduction of potassium current during spike repolarization. The major current responsible for frequency-dependent reduction of overall spike-repolarizing potassium current was identified as Kv3 current by its sensitivity to low concentrations of 4-aminopyridine (IC50 <100 µm) and block by the peptide inhibitor blood depressing substance I (BDS-I). There was a small component of Kv1-mediated current during AP repolarization, but this current did not show frequency-dependent reduction. In a small fraction of cells, there was a component of calcium-dependent potassium current that showed frequency-dependent reduction, but the contribution to overall potassium current reduction was almost always much smaller than that of Kv3-mediated current. These results show that Kv3 channels make a major contribution to spike repolarization in small-diameter DRG neurons and undergo frequency-dependent reduction, leading to spike broadening at moderate firing frequencies. Spike broadening from frequency-dependent reduction in Kv3 current could mitigate the frequency-dependent decreases in conduction velocity typical of C-fiber axons.SIGNIFICANCE STATEMENT Small-diameter dorsal root ganglia (DRG) neurons mediating nociception and other sensory modalities express many types of potassium channels, but how they combine to control firing patterns and conduction is not well understood. We found that action potentials of small-diameter rat DRG neurons showed spike broadening at frequencies as low as 1 Hz and that spike broadening resulted predominantly from frequency-dependent inactivation of Kv3 channels. Spike width helps to control transmitter release, conduction velocity, and firing patterns and understanding the role of particular potassium channels can help to guide new pharmacological strategies for targeting pain-sensing neurons selectively.
Asunto(s)
Potenciales de Acción/fisiología , Capsaicina/farmacología , Ganglios Espinales/fisiología , Neuronas/fisiología , Canales de Potasio Shaw/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Femenino , Ganglios Espinales/efectos de los fármacos , Masculino , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Long-Evans , Canales de Potasio Shaw/antagonistas & inhibidoresRESUMEN
The Kv3.4 channel is characterized by fast inactivation and sensitivity to oxidation. However, the physiological role of Kv3.4 as an oxidation-sensitive channel has yet to be investigated. Here, we demonstrate that Kv3.4 plays a pivotal role in oxidative stress-related neural cell damage as an oxidation-sensitive channel and that HIF-1α down-regulates Kv3.4 function, providing neuroprotection. MPP+ and CoCl2 are reactive oxygen species (ROS)-generating reagents that induce oxidative stress. However, only CoCl2 decreases the expression and function of Kv3.4. HIF-1α, which accumulates in response to CoCl2 treatment, is a key factor in Kv3.4 regulation. In particular, mitochondrial Kv3.4 was more sensitive to CoCl2. Blocking Kv3.4 function using BDS-II, a Kv3.4-specific inhibitor, protected SH-SY5Y cells against MPP+-induced neural cell death. Kv3.4 inhibition blocked MPP+-induced cytochrome c release from the mitochondrial intermembrane space to the cytosol and mitochondrial membrane potential depolarization, which are characteristic features of apoptosis. Our results highlight Kv3.4 as a possible new therapeutic paradigm for oxidative stress-related diseases, including Parkinson's disease.
Asunto(s)
Apoptosis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neuronas/metabolismo , Estrés Oxidativo , Canales de Potasio Shaw/metabolismo , 1-Metil-4-fenilpiridinio/toxicidad , Línea Celular Tumoral , Cobalto/toxicidad , Humanos , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Shaw/antagonistas & inhibidoresRESUMEN
In the last years it has been increasingly clear that KV-channel activity modulates neurotransmitter release. The subcellular localization and composition of potassium channels are crucial to understanding its influence on neurotransmitter release. To investigate the role of KV in corticostriatal synapses modulation, we combined extracellular recording of population-spike and pharmacological blockage with specific and nonspecific blockers to identify several families of KV channels. We induced paired-pulse facilitation (PPF) and studied the changes in paired-pulse ratio (PPR) before and after the addition of specific KV blockers to determine whether particular KV subtypes were located pre- or postsynaptically. Initially, the presence of KV channels was tested by exposing brain slices to tetraethylammonium or 4-aminopyridine; in both cases we observed a decrease in PPR that was dose dependent. Further experiments with tityustoxin, margatoxin, hongotoxin, agitoxin, dendrotoxin, and BDS-I toxins all rendered a reduction in PPR. In contrast heteropodatoxin and phrixotoxin had no effect. Our results reveal that corticostriatal presynaptic KV channels have a complex stoichiometry, including heterologous combinations KV1.1, KV1.2, KV1.3, and KV1.6 isoforms, as well as KV3.4, but not KV4 channels. The variety of KV channels offers a wide spectrum of possibilities to regulate neurotransmitter release, providing fine-tuning mechanisms to modulate synaptic strength.
Asunto(s)
Corteza Cerebral/fisiología , Cuerpo Estriado/fisiología , Terminales Presinápticos/fisiología , Canales de Potasio de la Superfamilia Shaker/fisiología , Canales de Potasio Shaw/fisiología , Sinapsis/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Corteza Cerebral/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Masculino , Bloqueadores de los Canales de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/fisiología , Ratas , Ratas Wistar , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Canales de Potasio Shaw/antagonistas & inhibidores , Sinapsis/efectos de los fármacosRESUMEN
Marine ladder-shaped polyether toxins are implicated in neurological symptoms of fish-borne food poisonings. The toxin gambierol, produced by the marine dinoflagellate Gambierdiscus toxicus, belongs to the group of ladder-shaped polyether toxins and inhibits Kv3.1 channels with nanomolar affinity through a mechanism of gating modification. Binding determinants for gambierol localize at the lipid-exposed interface of the pore forming S5 and S6 segments, suggesting that gambierol binds outside of the permeation pathway. To explore a possible involvement of the voltage-sensing domain (VSD), we made different chimeric channels between Kv3.1 and Kv2.1, exchanging distinct parts of the gating machinery. Our results showed that neither the electro-mechanical coupling nor the S1-S3a region of the VSD affect gambierol sensitivity. In contrast, the S3b-S4 part of the VSD (paddle motif) decreased gambierol sensitivity in Kv3.1 more than 100-fold. Structure determination by homology modeling indicated that the position of the S3b-S4 paddle and its primary structure defines the shape and∖or the accessibility of the binding site for gambierol, explaining the observed differences in gambierol affinity between the channel chimeras. Furthermore, these findings explain the observed difference in gambierol affinity for the closed and open channel configurations of Kv3.1, opening new possibilities for exploring the VSDs as selectivity determinants in drug design.
Asunto(s)
Ciguatoxinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Shab/antagonistas & inhibidores , Canales de Potasio Shab/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidores , Canales de Potasio Shaw/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Relación Dosis-Respuesta a Droga , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Modelos Moleculares , Proteínas Mutantes Quiméricas , Técnicas de Placa-Clamp , Conformación Proteica , Canales de Potasio Shab/genética , Canales de Potasio Shaw/genéticaRESUMEN
The intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels play a pivotal role in the proliferation and collagen secretion of cardiac fibroblasts. However, their contribution in cardiac fibrosis remains unknown. This study was designed to investigate whether KCa3.1 channels mediate the development of cardiac fibrosis. Pressure-overloaded rats were induced by abdominal aortic constriction and treated without or with KCa3.1 blocker (TRAM-34) or angiotensin type 1 receptor blocker (losartan) for 2 weeks. Besides the increase of blood pressure, angiotensin (Ang) II level in the plasma and myocardium, left ventricle mass and hydroxyproline concentration, myocardial hypertrophy, as well as significant collagen deposition in the perivascular regions and interstitium of the myocardium were observed in pressure-overloaded rats. The expression of leukocyte differentiation antigens (CD45 and CD3), macrophage surface marker (F4/80), tumor necrosis factor alpha, and monocyte chemotactic protein-1 (MCP-1) also significantly increased. All these alterations were prevented by losartan and TRAM-34. TRAM-34 also reduced the increase of renin and angiotensinogen in the plasma and myocardium of pressure-overloaded rats. Ang II promoted the migration of monocytes through endothelial cells and the secretion of MCP-1 from human umbilical vein endothelial cells in vitro, which was inhibited by TRAM-34. In conclusion, the present study demonstrates that TRAM-34 alleviates cardiac fibrosis induced by pressure overload, which is related to its inhibitory action on KCa3.1 channels and Ang II level. Our findings indicate that the inhibition of KCa3.1 channels may represent a novel approach of preventing the progression of cardiac fibrosis, and also add to the already developing literature of promising targets for TRAM-34.
Asunto(s)
Miocardio/metabolismo , Miocardio/patología , Canales de Potasio Shaw/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Angiotensinógeno/sangre , Angiotensinógeno/metabolismo , Animales , Aorta Abdominal/efectos de los fármacos , Presión Sanguínea , Cardiomegalia/patología , Citocinas/metabolismo , Fibrosis , Hidroxiprolina/metabolismo , Losartán/farmacología , Masculino , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Renina/sangre , Renina/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidoresRESUMEN
AahG50, the toxic fraction of Androctonus australis hector venom, was studied on human Kv3.1 channels activation, stably expressed in Xenopus oocytes using the two-electrode voltage clamp technique. AahG50 reduced Kv3.1 currents in a reversible concentration-dependent manner, with an IC50 value and a Hill coefficient of 40.4 ± 0.2 µg/ml and 1.3 ± 0.05, respectively. AahG50 inhibited IKv3.1 without modifying the current activation kinetics. The AahG50-induced inhibition of Kv3.1 channels was voltage-dependent, with a gradual increase at lower concentrations and over the voltage range of channels opening. However, at higher concentrations, the inhibition exhibited voltage dependence only in the first range of channels opening from -20 to +10 mV, but demonstrates a low degree of voltage-dependence when channels are fully activated. In the literature, toxins have previously been isolated from AahG50, KAaH1 and KAaH2 and were reported not to have any effect on IKv3.1. The present article's findings suggest that AahG50 may contain a peptidic component active on Kv3.1 channels, which inhibits IKv3.1 in a selective manner.
Asunto(s)
Oocitos/metabolismo , Fragmentos de Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/farmacología , Canales de Potasio Shaw/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Fragmentos de Péptidos/química , Bloqueadores de los Canales de Potasio/química , Venenos de Escorpión/química , Transfección , Xenopus laevisRESUMEN
Diclofenac (DIC), a nonsteroidal anti-inflammatory drug, is known to exert anti-nociceptive and anti-convulsant actions; however, its effects on ion currents, in neurons remain debatable. We aimed to investigate (1) potential effects of diclofenac on membrane potential and potassium currents in differentiated NSC-34 neuronal cells and dorsal root ganglion (DRG) neurons with whole-cell patch-clamp technology, and (2) firing of action potentials (APs), using a simulation model from hippocampal CA1 pyramidal neurons based on diclofenac's effects on potassium currents. In the NSC-34 cells, diclofenac exerted an inhibitory effect on delayed-rectifier K⺠current (I(KDR)) with an IC50 value of 73 µM. Diclofenac not merely inhibited the I(KDR) amplitude in response to membrane depolarization, but also accelerated the process of current inactivation. The inhibition by diclofenac of IK(DR) was not reversed by subsequent application of either naloxone. Importantly, diclofenac (300 µM) increased the amplitude of M-type K⺠current (I)(KM)), while flupirtine (10 µM) or meclofenamic acid (10 µM) enhanced it effectively. Consistently, diclofenac (100 µM) increased the amplitude of I(KM) and diminished the I(KDR) amplitude, with a shortening of inactivation time constant in DRG neurons. Furthermore, by using the simulation modeling, we demonstrated the potential electrophysiological mechanisms underlying changes in AP firing caused by diclofenac. During the exposure to diclofenac, the actions on both I(KM) and I(KDR) could be potential mechanism through which it influences the excitability of fast-spiking neurons. Caution needs to be made in attributing the effects of diclofenac primarily to those produced by the activation of I(KM).
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Diclofenaco/farmacología , Ganglios Espinales/efectos de los fármacos , Canales de Potasio KCNQ/antagonistas & inhibidores , Neuronas Motoras/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Shaw/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/antagonistas & inhibidores , Anticonvulsivantes/antagonistas & inhibidores , Anticonvulsivantes/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Diclofenaco/antagonistas & inhibidores , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Ratones , Modelos Biológicos , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Bloqueadores de los Canales de Potasio/antagonistas & inhibidores , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Ratas , Canales de Potasio Shaw/agonistas , Canales de Potasio Shaw/genética , Canales de Potasio Shaw/metabolismoRESUMEN
Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.
Asunto(s)
Anestésicos Generales/farmacología , Simulación de Dinámica Molecular , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio Shaw/antagonistas & inhibidores , Canales de Potasio Shaw/metabolismo , 1-Butanol/farmacología , Secuencia de Aminoácidos , Biología Computacional , Relación Dosis-Respuesta a Droga , Fenómenos Electrofisiológicos/efectos de los fármacos , Halotano/farmacología , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis , Mutación , Estructura Secundaria de Proteína , Canales de Potasio Shaw/química , Canales de Potasio Shaw/genéticaRESUMEN
Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation-a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (ß) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-ß coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.
Asunto(s)
Conductividad Eléctrica , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidores , Canales de Potasio Shaw/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Endocitosis , Regulación de la Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Canales de Potasio Shaw/químicaRESUMEN
The goal of the present study was to examine the effects of lobeline, an agonist at nicotinic receptors, on cloned Kv channels, Kv1.5, Kv3.1, Kv4.3, and human ether-a-gogo-related gene (HERG), which are stably expressed in Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. Whole-cell patch-clamp experiments revealed that lobeline accelerated the decay rate of Kv1.5 inactivation, decreasing the current amplitude at the end of the pulse in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) value of 15.1 µM. Using a time constant for the time course of drug-channel interaction, the apparent association (k(+1)), and dissociation rate (k(-1)) constants were 2.4 µΜ(-1) s(-1) and 40.9 s(-1), respectively. The calculated K(D) was 17.0 µΜ. Lobeline slowed the decay rate of the tail current, resulting in a tail crossover phenomenon. The inhibition of Kv1.5 by lobeline steeply increased at potentials between -10 and +10 mV, which corresponds to the voltage range of channel activation. At more depolarized potentials a weaker voltage dependence was observed (δ=0.26). The voltage dependence of the steady-state activation curve was not affected by lobeline, but lobeline shifted the steady-state inactivation curve of Kv1.5 in the hyperpolarizing direction. Lobeline produced use-dependent inhibition of Kv1.5 at frequencies of 1 and 2 Hz, and slowed the recovery from inactivation. Lobeline also inhibited Kv3.1, Kv4.3, and HERG in a concentration-dependent manner, with IC(50) values of 21.7, 28.2, and 0.34 µM, respectively. These results indicate that lobeline produces a concentration-, time-, voltage-, and use-dependent inhibition of Kv1.5, which can be interpreted as an open-channel block mechanism.