RESUMO
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
AIMS: Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM) could be a helpful tool to study the physiology and diseases of the human atrium. To fulfil this expectation, the electrophysiology of hiPSC-aCM should closely resemble the situation in the human atrium. Data on the contribution of the slowly activating delayed rectifier currents (IKs) to repolarization are lacking for both human atrium and hiPSC-aCM. METHODS AND RESULTS: Human atrial tissues were obtained from patients with sinus rhythm (SR) or atrial fibrillation (AF). Currents were measured in human atrial cardiomyocytes (aCM) and compared with hiPSC-aCM and used to model IKs contribution to action potential (AP) shape. Action potential was recorded by sharp microelectrodes. HMR-1556 (1â µM) was used to identify IKs and to estimate IKs contribution to repolarization. Less than 50% of hiPSC-aCM and aCM possessed IKs. Frequency of occurrence, current densities, activation/deactivation kinetics, and voltage dependency of IKs did not differ significantly between hiPSC-aCM and aCM, neither in SR nor AF. ß-Adrenoceptor stimulation with isoprenaline did not increase IKs neither in aCM nor in hiPSC-aCM. In tissue from SR, block of IKs with HMR-1556 did not lengthen the action potential duration, even when repolarization reserve was reduced by block of the ultra-rapid repolarizing current with 4-aminopyridine or the rapidly activating delayed rectifier potassium outward current with E-4031. CONCLUSION: I Ks exists in hiPSC-aCM with biophysics not different from aCM. As in adult human atrium (SR and AF), IKs does not appear to relevantly contribute to repolarization in hiPSC-aCM.
Assuntos
Potenciais de Ação , Fibrilação Atrial , Canais de Potássio de Retificação Tardia , Átrios do Coração , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Átrios do Coração/fisiopatologia , Canais de Potássio de Retificação Tardia/metabolismo , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/metabolismo , Feminino , Células Cultivadas , Masculino , Pessoa de Meia-Idade , Cinética , Idoso , Diferenciação Celular , Modelos Cardiovasculares , Bloqueadores dos Canais de Potássio/farmacologiaRESUMO
Gemcitabine is an antineoplastic drug commonly used in the treatment of several types of cancers including pancreatic cancer and non-small cell lung cancer. Although gemcitabine-induced cardiotoxicity is widely recognized, the exact mechanism of cardiac dysfunction causing arrhythmias remains unclear. The objective of this study was to electrophysiologically evaluate the proarrhythmic cardiotoxicity of gemcitabine focusing on the human rapid delayed rectifier potassium channel, hERG channel. In heterologous hERG expressing HEK293 cells (hERG-HEK cells), hERG channel current (IhERG) was reduced by gemcitabine when applied for 24 h but not immediately after the application. Gemcitabine modified the activation gating properties of the hERG channel toward the hyperpolarization direction, while inactivation, deactivation or reactivation gating properties were unaffected by gemcitabine. When gemcitabine was applied to hERG-HEK cells in combined with tunicamycin, an inhibitor of N-acetylglucosamine phosphotransferase, gemcitabine was unable to reduce IhERG or shift the activation properties toward the hyperpolarization direction. While a mannosidase I inhibitor kifunensine alone reduced IhERG and the reduction was even larger in combined with gemcitabine, kifunensine was without effect on IhERG when hERG-HEK cells were pretreated with gemcitabine for 24 h. In addition, gemcitabine down-regulated fluorescence intensity for hERG potassium channel protein in rat neonatal cardiomyocyte, although hERG mRNA was unchanged. Our results suggest the possible mechanism of arrhythmias caused by gemcitabine revealing a down-regulation of IhERG through the post-translational glycosylation disruption possibly at the early phase of hERG channel glycosylation in the endoplasmic reticulum that alters the electrical excitability of cells.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Ratos , Gencitabina , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Regulação para Baixo , Cardiotoxicidade/etiologia , Células HEK293 , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/genética , Canais de Potássio de Retificação Tardia/genética , Canais de Potássio de Retificação Tardia/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismoRESUMO
BACKGROUND: Voltage-gated potassium channel Kv2 is the primarily delayed rectifier in insect nerves and muscles involved in several crucial biological processes, including action potential regulation, photoreceptor performance, and larval locomotor. It is a potential molecular target for developing a novel pesticide for mosquitos. However, there are few studies on the Kv2 channel in agricultural pests. RESULTS: The only α-subunit gene of the Kv2 channel in Plutella xylostella (L.), PxShab, was cloned, and its expression profile was analyzed. The relative expression level of PxShab was highest in the pupal stage of both sexes and male adults but lowest in female adults. Meanwhile, PxShab had the highest expression in the head in both larvae and adults. Then, PxShab was stably expressed in the HEK-293 T cell line. Whole cell patch clamp recordings showed an outward current whose current-voltage relationship conformed to a typical delayed-rectifier potassium channel. 20 µM quinidine could effectively inhibit the potassium current, while the channel was insensitive to 4-AP even at 10 mM. Several potential compounds and botanical pesticides were assessed, and carvedilol (IC50 = 0.53 µM) and veratrine (IC50 = 2.22 µM) had a good inhibitory effect on the channel. CONCLUSION: This study revealed the pharmacological properties of PxShab and screened out several high potency inhibitors, which laid the foundation for further functional research of PxShab and provides new insight into designing novel insecticides. © 2022 Society of Chemical Industry.
Assuntos
Mariposas , Animais , Masculino , Feminino , Humanos , Canais de Potássio de Retificação Tardia , Mariposas/genética , Células HEK293 , Canais de Potássio Shab , Larva/genéticaRESUMO
Lacosamide (Vimpat®, LCS) is widely known as a functionalized amino acid with promising anti-convulsant properties; however, adverse events during its use have gradually appeared. Despite its inhibitory effect on voltage-gated Na+ current (INa), the modifications on varying types of ionic currents caused by this drug remain largely unexplored. In pituitary tumor (GH3) cells, we found that the presence of LCS concentration-dependently decreased the amplitude of A-type K+ current (IK(A)) elicited in response to membrane depolarization. The IK(A) amplitude in these cells was sensitive to attenuation by the application of 4-aminopyridine, 4-aminopyridine-3-methanol, or capsaicin but not by that of tetraethylammonium chloride. The effective IC50 value required for its reduction in peak or sustained IK(A) was calculated to be 102 or 42 µM, respectively, while the value of the dissociation constant (KD) estimated from the slow component in IK(A) inactivation at varying LCS concentrations was 52 µM. By use of two-step voltage protocol, the presence of this drug resulted in a rightward shift in the steady-state inactivation curve of IK(A) as well as in a slowing in the recovery time course of the current block; however, no change in the gating charge of the inactivation curve was detected in its presence. Moreover, the LCS addition led to an attenuation in the degree of voltage-dependent hysteresis for IK(A) elicitation by long-duration triangular ramp voltage commands. Likewise, the IK(A) identified in mouse mHippoE-14 neurons was also sensitive to block by LCS, coincident with an elevation in the current inactivation rate. Collectively, apart from its canonical action on INa inhibition, LCS was effective at altering the amplitude, gating, and hysteresis of IK(A) in excitable cells. The modulatory actions on IK(A), caused by LCS, could interfere with the functional activities of electrically excitable cells (e.g., pituitary tumor cells or hippocampal neurons).
Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Ativação do Canal Iônico , Lacosamida/farmacologia , Neoplasias Hipofisárias/tratamento farmacológico , Potássio/metabolismo , Adenoma/tratamento farmacológico , Adenoma/metabolismo , Adenoma/patologia , Animais , Anticonvulsivantes/farmacologia , Transporte de Íons , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Ratos , Células Tumorais CultivadasRESUMO
BACKGROUND: Slow delayed rectifier potassium current (IKs) is an important component of repolarization reserve during sympathetic nerve excitement. However, little is known about age-related functional changes of IKs and its involvement in age-dependent arrhythmogenesis. OBJECTIVE: The purpose of this study was to investigate age-related alteration of the IKs response to ß-adrenergic receptor (ßAR) activation. METHODS: Dunkin-Hartley guinea pigs were used. Whole-cell patch-clamp recording was used to record K+ currents. Optical mapping of membrane potential was performed in ex vivo heart. RESULTS: There was no difference in IKs density in ventricular cardiomyocytes between young and old guinea pigs. However, in contrast to IKs potentiation in young hearts, isoproterenol (ISO) evoked an acute inhibition on IKs in a concentration-dependent manner in old guinea pig hearts. The ß2AR antagonist, but not ß1AR antagonist, reversed the inhibitory response. Preincubation of cardiomyocytes with the inhibitory G protein (Gi) inhibitor pertussis toxin (PTX) also reversed the inhibitory response. In HEK293 cells cotransfected with cloned IKs channel and ß2AR, ISO enhanced the current but reduced it when cells were cotransfected with Gi2, and PTX restored the ISO-induced excitatory response. Moreover, in aging cardiomyocytes, Gßγ inhibitor gallein, PLC inhibitor U73122, or protein kinase C inhibitor Bis-1 prevented the reduction of IKs by ISO. Furthermore, cardiac-specific Gi2 overexpression in young guinea pigs predisposed the heart to ventricular tachyarrhythmias. PTX pretreatment protected the hearts from ventricular arrhythmias. CONCLUSION: ßAR activation acutely induces an inhibitory IKs response in aging guinea pig hearts through ß2AR-Gi signaling, which contributes to increased susceptibility to arrhythmogenesis in aging hearts.
Assuntos
Arritmias Cardíacas/metabolismo , Senescência Celular/fisiologia , Canais de Potássio de Retificação Tardia/metabolismo , Potenciais da Membrana , Miócitos Cardíacos , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Antiarrítmicos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Cobaias , Células HEK293 , Humanos , Isoproterenol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Toxina Pertussis/farmacologiaRESUMO
Hypoxia typically accompanies acute inflammatory responses in patients and animal models. However, a limited number of studies have examined the effect of hypoxia in combination with inflammation (Hypo-Inf) on neural function. We previously reported that neuronal excitability in hippocampal CA1 neurons decreased during hypoxia and greatly rebounded upon reoxygenation. We attributed this altered excitability mainly to the dynamic regulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and input resistance. However, the molecular mechanisms underlying input resistance changes by Hypo-Inf and reperfusion remained unclear. In the present study, we found that a change in the density of the delayed rectifier potassium current (IDR) can explain the input resistance variability. Furthermore, voltage-dependent inactivation of A-type potassium (IA) channels shifted in the depolarizing direction during Hypo-Inf and reverted to normal upon reperfusion without a significant alteration in the maximum current density. Our results indicate that changes in the input resistance, and consequently excitability, caused by Hypo-Inf and reperfusion are at least partially regulated by the availability and voltage dependence of KV channels. Moreover, these results suggest that selective KV channel modulators can be used as potential neuroprotective drugs to minimize hypoxia- and reperfusion-induced neuronal damage.
Assuntos
Região CA1 Hipocampal/fisiopatologia , Hipóxia Celular/fisiologia , Canais de Potássio de Retificação Tardia/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Meios de Cultura/farmacologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Técnicas In Vitro , Inflamação , Cinética , Potenciais da Membrana/fisiologia , Fármacos Neuroprotetores/farmacologia , Técnicas de Patch-Clamp , Ratos , Reperfusão , Tetrodotoxina/farmacologiaRESUMO
Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both ß1- and ß2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by ß2-adrenoceptor stimulation than ß1-adrenoceptor stimulation. Both ß-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the ß-adrenoceptor-potentiated firing rate. The stimulatory effects of ß- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under ß-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.
Assuntos
Canais de Potássio de Retificação Tardia/metabolismo , Miócitos Cardíacos/metabolismo , Veias Pulmonares/metabolismo , Receptores Adrenérgicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Agonistas alfa-Adrenérgicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Cobaias , Átrios do Coração/metabolismo , Isoproterenol/farmacologia , Canal de Potássio KCNQ1/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Norepinefrina/farmacologia , Técnicas de Patch-Clamp , Proteína Quinase C/metabolismo , Veias Pulmonares/citologia , Transdução de SinaisRESUMO
Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell's K+ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in concentration-, time-, and state-dependent manners. The IC50 for MDZ-mediated reduction of IK(DR) density was 5.87 µM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on IK(DR) density was estimated with a dissociation constant of 5.14 µM. In addition, the inactivation curve of IK(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of IK(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both IK(DR) and IKCa-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of IK(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.
Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Midazolam/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Citocinas/metabolismo , Canais de Potássio de Retificação Tardia/metabolismo , Relação Dose-Resposta a Droga , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Cinética , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Microscopia Confocal , Midazolam/administração & dosagem , Técnicas de Patch-Clamp , Fito-Hemaglutininas/farmacologia , Linfócitos T/imunologiaRESUMO
Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. SBMA is characterized by selective dysfunction and degeneration of motor neurons in the brainstem and spinal cord through still unclear mechanisms in which ion channel modulation might play a central role as for other neurodegenerative diseases. The beta2-adrenergic agonist clenbuterol was observed to ameliorate the SBMA phenotype in mice and patient-derived myotubes. However, the underlying molecular mechanism has yet to be clarified. Here, we unveil that ionic current alterations induced by the expression of polyQ-expanded AR in motor neuron-derived MN-1 cells are attenuated by the administration of clenbuterol. Our combined electrophysiological and pharmacological approach allowed us to reveal that clenbuterol modifies delayed outward potassium currents. Overall, we demonstrated that the protection provided by clenbuterol restores the normal function through the modulation of KV2-type outward potassium currents, possibly contributing to the protective effect on motor neuron toxicity in SBMA.
Assuntos
Atrofia Bulboespinal Ligada ao X/etiologia , Canais de Potássio de Retificação Tardia/metabolismo , Animais , Proteínas de Artrópodes , Atrofia Bulboespinal Ligada ao X/metabolismo , Linhagem Celular , Clembuterol , Humanos , Camundongos , Técnicas de Patch-Clamp , Venenos de AranhaRESUMO
Robust, spontaneous pacemaker activity originating in the sinoatrial node (SAN) of the heart is essential for cardiovascular function. Anatomical, electrophysiological, and molecular methods as well as mathematical modeling approaches have quite thoroughly characterized the transmembrane fluxes of Na+, K+ and Ca2+ that produce SAN action potentials (AP) and 'pacemaker depolarizations' in a number of different in vitro adult mammalian heart preparations. Possible ionic mechanisms that are responsible for SAN primary pacemaker activity are described in terms of: (i) a Ca2+-regulated mechanism based on a requirement for phasic release of Ca2+ from intracellular stores and activation of an inward current-mediated by Na+/Ca2+ exchange; (ii) time- and voltage-dependent activation of Na+ or Ca2+ currents, as well as a cyclic nucleotide-activated current, If; and/or (iii) a combination of (i) and (ii). Electrophysiological studies of single spontaneously active SAN myocytes in both adult mouse and rabbit hearts consistently reveal significant expression of a rapidly activating time- and voltage-dependent K+ current, often denoted IKr, that is selectively expressed in the leading or primary pacemaker region of the adult mouse SAN. The main goal of the present study was to examine by combined experimental and simulation approaches the functional or physiological roles of this K+ current in the pacemaker activity. Our patch clamp data of mouse SAN myocytes on the effects of a pharmacological blocker, E4031, revealed that a rapidly activating K+ current is essential for action potential (AP) repolarization, and its deactivation during the pacemaker potential contributes a small but significant component to the pacemaker depolarization. Mathematical simulations using a murine SAN AP model confirm that well known biophysical properties of a delayed rectifier K+ current can contribute to its role in generating spontaneous myogenic activity.
Assuntos
Canais de Potássio de Retificação Tardia/metabolismo , Miócitos Cardíacos/fisiologia , Potássio/metabolismo , Potenciais de Ação , Animais , Cátions Monovalentes/metabolismo , Células Cultivadas , Coração/fisiologia , Transporte de Íons , Camundongos , Modelos Cardiovasculares , Marca-Passo Artificial , Coelhos , Trocador de Sódio e Cálcio/metabolismoRESUMO
Renshaw cells (V1R) are excitable as soon as they reach their final location next to the spinal motoneurons and are functionally heterogeneous. Using multiple experimental approaches, in combination with biophysical modeling and dynamical systems theory, we analyzed, for the first time, the mechanisms underlying the electrophysiological properties of V1R during early embryonic development of the mouse spinal cord locomotor networks (E11.5-E16.5). We found that these interneurons are subdivided into several functional clusters from E11.5 and then display an unexpected transitory involution process during which they lose their ability to sustain tonic firing. We demonstrated that the essential factor controlling the diversity of the discharge pattern of embryonic V1R is the ratio of a persistent sodium conductance to a delayed rectifier potassium conductance. Taken together, our results reveal how a simple mechanism, based on the synergy of two voltage-dependent conductances that are ubiquitous in neurons, can produce functional diversity in embryonic V1R and control their early developmental trajectory.
Assuntos
Potenciais de Ação , Canais de Potássio de Retificação Tardia/metabolismo , Potássio/metabolismo , Células de Renshaw/metabolismo , Canais de Sódio/metabolismo , Sódio/metabolismo , Medula Espinal/metabolismo , Animais , Feminino , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos Transgênicos , Modelos Neurológicos , Morfogênese , Fenótipo , Medula Espinal/embriologia , Teoria de Sistemas , Fatores de TempoRESUMO
Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca2+ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH3 cells and HL-1 atrial cardiomyocytes). GH3-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na+ current (INa) with effective IC50 of 14.7 or 2.8 µM, respectively. The inactivation time course of INa activated by short depolarization became fastened in the presence of CBN with estimated KD value of 3.15 µM. The peak INa diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of INa. CBN (10 µM) shifted the midpoint of inactivation curve of INa to the leftward direction. The CBN-mediated inhibition of peak INa exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent INa enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K+ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak INa and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in INa. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies INa in electrically excitable cells.
Assuntos
Cumarínicos/farmacologia , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Angelica/química , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular Tumoral , Cumarínicos/química , Cumarínicos/isolamento & purificação , Canais de Potássio de Retificação Tardia/metabolismo , Transporte de Íons/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Hipófise/citologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , RatosRESUMO
Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Gânglios Sensitivos/efeitos dos fármacos , Gentamicinas/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/toxicidade , Nervo Vestibular/efeitos dos fármacos , Potenciais de Ação , Animais , Células Cultivadas , Canais de Potássio de Retificação Tardia/metabolismo , Feminino , Gânglios Sensitivos/enzimologia , Masculino , Camundongos Endogâmicos ICR , Neurônios/enzimologia , Fosforilação , Transdução de Sinais , Nervo Vestibular/enzimologiaRESUMO
Activation of transient receptor potential vanilloid 4 (TRPV4) can increase hippocampal neuronal excitability. TRPV4 has been reported to be involved in the pathogenesis of epilepsy. Voltage-gated potassium channels (VGPCs) play an important role in regulating neuronal excitability and abnormal VGPCs expression or function is related to epilepsy. Here, we examined the effect of TRPV4 activation on the delayed rectifier potassium current (IK ) in hippocampal pyramidal neurons and on the Kv subunits expression in male mice. We also explored the role of TRPV4 in changes in Kv subunits expression in male mice following pilocarpine-induced status epilepticus (PISE). Application of TRPV4 agonists, GSK1016790A and 5,6-EET, markedly reduced IK in hippocampal pyramidal neurons and shifted the voltage-dependent inactivation curve to the hyperpolarizing direction. GSK1016790A- and 5,6-EET-induced inhibition of IK was blocked by TRPV4 specific antagonists, HC-067047 and RN1734. GSK1016790A-induced inhibition of IK was markedly attenuated by calcium/calmodulin-dependent kinase II (CaMKII) antagonist. Application of GSK1016790A for up to 1 hr did not change the hippocampal protein levels of Kv1.1, Kv1.2, or Kv2.1. Intracerebroventricular injection of GSK1016790A for 3 d reduced the hippocampal protein levels of Kv1.2 and Kv2.1, leaving that of Kv1.1 unchanged. Kv1.2 and Kv2.1 protein levels as well as IK reduced markedly in hippocampi on day 3 post PISE, which was significantly reversed by HC-067047. We conclude that activation of TRPV4 inhibits IK in hippocampal pyramidal neurons, possibly by activating CaMKII. TRPV4-induced decrease in Kv1.2 and Kv2.1 expression and IK may be involved in the pathological changes following PISE.
Assuntos
Canais de Potássio de Retificação Tardia/metabolismo , Células Piramidais/fisiologia , Estado Epiléptico/fisiopatologia , Canais de Cátion TRPV/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Canais de Potássio de Retificação Tardia/farmacologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfolinas/farmacologia , Pilocarpina , Células Piramidais/metabolismo , Pirróis/farmacologia , Estado Epiléptico/induzido quimicamente , Sulfonamidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Ribociclib (RIB, LE011, Kisqali®), an orally administered inhibitor of cyclin-dependent kinase-4/6 (CDK-4/6) complex, is clinically effective for the treatment of several malignancies, including advanced breast cancer. However, information regarding the effects of RIB on membrane ion currents is limited. In this study, the addition of RIB to pituitary tumor (GH3) cells decreased the peak amplitude of erg-mediated K+ current (IK(erg)), which was accompanied by a slowed deactivation rate of the current. The IC50 value for RIB-perturbed inhibition of deactivating IK(erg) in these cells was 2.7 µM. In continued presence of µM RIB, neither the subsequent addition of 17ß-estradiol (30 µM), phorbol 12-myristate 13-acetate (10 µM), or transforming growth factor-ß (1 µM) counteracted the inhibition of deactivating IK(erg). Its presence affected the decrease in the degree of voltage-dependent hysteresis for IK(erg) elicitation by long-duration triangular ramp voltage commands. The presence of RIB differentially inhibited the peak or sustained component of delayed rectifier K+ current (IK(DR)) with an effective IC50 of 28.7 or 11.4 µM, respectively, while it concentration-dependently decreased the amplitude of M-type K+ current with IC50 of 13.3 µM. Upon 10-s long membrane depolarization, RIB elicited a decrease in the IK(DR) amplitude, which was concomitant with an accelerated inactivation time course. However, the inability of RIB (10 µM) to modify the magnitude of the hyperpolarization-activated cation current was disclosed. The mean current-voltage relationship of IK(erg) present in HL-1 atrial cardiomyocytes was inhibited in the presence of RIB (10 µM). Collectively, the hyperpolarization-activated cation current was observed. RIB-mediated perturbations in ionic currents presented herein are upstream of its suppressive action on cytosolic CDK-4/6 activities and partly participates in its modulatory effects on the functional activities of pituitary tumor cells (e.g., GH3 cells) or cardiac myocytes (e.g., HL-1 cells).
Assuntos
Aminopiridinas/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Canal de Potássio ERG1/antagonistas & inibidores , Neoplasias Hipofisárias/tratamento farmacológico , Purinas/farmacologia , Potenciais de Ação , Animais , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Células Tumorais CultivadasRESUMO
Channelopathies involving acquired or genetic modifications of the delayed rectifier K+ channel Kv1.1 include phenotypes characterized by enhanced neuronal excitability. Affected Kv1.1 channels exhibit combinations of altered expression, voltage sensitivity, and rates of activation and deactivation. Computational modeling and analysis can reveal the potential of particular channelopathies to alter neuronal excitability. A dynamical systems approach was taken to study the excitability and underlying dynamical structure of the Hodgkin-Huxley (HH) model of neural excitation as properties of the delayed rectifier K+ channel were altered. Bifurcation patterns of the HH model were determined as the amplitude of steady injection current was varied simultaneously with single parameters describing the delayed rectifier rates of activation and deactivation, maximal conductance, and voltage sensitivity. Relatively modest changes in the properties of the delayed rectifier K+ channel analogous to what is described for its channelopathies alter the bifurcation structure of the HH model and profoundly modify excitability of the HH model. Channelopathies associated with Kv1.1 can reduce the threshold for onset of neural activity. These studies also demonstrate how pathological delayed rectifier K+ channels could lead to the observation of the generalized Hopf bifurcation and, perhaps, other variants of the Hopf bifurcation. The observed bifurcation patterns collectively demonstrate that properties of the nominal delayed rectifier in the HH model appear optimized to permit activation of the HH model over the broadest possible range of input currents.
Assuntos
Canalopatias/fisiopatologia , Canais de Potássio de Retificação Tardia/genética , Potenciais da Membrana/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Animais , Canalopatias/genética , Simulação por ComputadorRESUMO
Ca2+ spikes initiated in the distal trunk of layer 5 pyramidal cells (PCs) underlie nonlinear dynamic changes in the gain of cellular response, critical for top-down control of cortical processing. Detailed models with many compartments and dozens of ionic channels can account for this Ca2+ spike-dependent gain and associated critical frequency. However, current models do not account for all known Ca2+-dependent features. Previous attempts to include more features have required increasing complexity, limiting their interpretability and utility for studying large population dynamics. We overcome these limitations in a minimal two-compartment biophysical model. In our model, a basal-dendrites/somatic compartment included fast-inactivating Na+ and delayed-rectifier K+ conductances, while an apical-dendrites/trunk compartment included persistent Na+, hyperpolarization-activated cation (I h ), slow-inactivating K+, muscarinic K+, and Ca2+ L-type. The model replicated the Ca2+ spike morphology and its critical frequency plus three other defining features of layer 5 PC synaptic integration: linear frequency-current relationships, back-propagation-activated Ca2+ spike firing, and a shift in the critical frequency by blocking I h Simulating 1000 synchronized layer 5 PCs, we reproduced the current source density patterns evoked by Ca2+ spikes and describe resulting medial-frontal EEG on a male macaque monkey. We reproduced changes in the current source density when I h was blocked. Thus, a two-compartment model with five crucial ionic currents in the apical dendrites reproduces all features of these neurons. We discuss the utility of this minimal model to study the microcircuitry of agranular areas of the frontal lobe involved in cognitive control and responsible for event-related potentials, such as the error-related negativity.SIGNIFICANCE STATEMENT A minimal model of layer 5 pyramidal cells replicates all known features crucial for distal synaptic integration in these neurons. By redistributing voltage-gated and returning transmembrane currents in the model, we establish a theoretical framework for the investigation of cortical microcircuit contribution to intracranial local field potentials and EEG. This tractable model will enable biophysical evaluation of multiscale electrophysiological signatures and computational investigation of cortical processing.
Assuntos
Biofísica , Modelos Neurológicos , Neocórtex/fisiologia , Rede Nervosa/fisiologia , Células Piramidais/fisiologia , Algoritmos , Animais , Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Simulação por Computador , Canais de Potássio de Retificação Tardia/fisiologia , Dendritos/fisiologia , Eletroencefalografia , Potenciais Evocados/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Macaca radiata , Masculino , Neocórtex/citologia , Rede Nervosa/citologia , Canais de Sódio/fisiologiaRESUMO
FTY720 (fingolimod), a modulator of sphingosine-1-phosphate receptors, is known to produce the immunomodulatory actions and to be beneficial for treating the relapsing multiple sclerosis. However, whether it exerts any effects on membrane ion currents in immune cells remains largely unknown. Herein, the effects of FTY720 on ionic currents in Jurkat T-lymphocytes were investigated. Cell exposure to FTY720 suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner with an IC50 value of 1.51 µM. Increasing the FTY720 concentration not only decreased the IK(DR) amplitude but also accelerated the inactivation time course of the current. By using the minimal reaction scheme, the effect of FTY720 on IK(DR) inactivation was estimated with a dissociation constant of 3.14 µM. FTY720 also shifted the inactivation curve of IK(DR) to a hyperpolarized potential with no change in the slope factor, and recovery from IK(DR) became slow during the exposure to this compound. Cumulative inactivation for IK(DR) in response to repetitive depolarizations was enhanced in the presence of FTY720. In SEW2871-treated cells, FTY720-induced inhibition of IK(DR) was attenuated. This compound also exerted a stimulatory action on the activity of intermediate-conductance Ca2+-activated K+ channels in Jurkat T-lymphocytes. However, in NSC-34 neuronal cells, FTY720 did not modify the inactivation kinetics of KV3.1-encoded IK(DR), although it suppressed IK(DR) amplitude in these cells. Collectively, the perturbations by FTY720 on different types of K+ channels may contribute to the functional activities of immune cells, if similar findings appear in vivo.
Assuntos
Canais de Potássio de Retificação Tardia/metabolismo , Cloridrato de Fingolimode/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Linfócitos T/metabolismo , Animais , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Células Jurkat , Cinética , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenantrenos/farmacologia , Linfócitos T/efeitos dos fármacosRESUMO
A rare mutation affecting the Forkhead-box protein P2 (FOXP2) transcription factor causes a severe monogenic speech and language disorder. Mice carrying an identical point mutation to that observed in affected patients (Foxp2+/R552H mice) display motor deficits and impaired synaptic plasticity in the striatum. However, the consequences of the mutation on neuronal function, in particular in the cerebral cortex, remain little studied. Foxp2 is expressed in a subset of Layer VI cortical neurons. Here, we used Ntsr1-EGFP mice to identify Foxp2+ neurons in the mouse auditory cortex ex vivo. We studied the functional impact of the R552H mutation on the morphologic and functional properties of Layer VI cortical neurons from Ntsr1-EGFP; Foxp2+/R552H male and female mice. The complexity of apical, but not basal dendrites was significantly lower in Foxp2+/R552H cortico-thalamic neurons than in control Foxp2+/+ neurons. Excitatory synaptic inputs, but not inhibitory synaptic inputs, were decreased in Foxp2+/R552H mice. In response, homeostatic mechanisms would be expected to increase neuronal gain, i.e., the conversion of a synaptic input into a firing output. However, the intrinsic excitability of Foxp2+ cortical neurons was lower in Foxp2+/R552H neurons. A-type and delayed-rectifier (DR) potassium currents, two putative transcriptional targets of Foxp2, were not affected by the mutation. In contrast, GABAB/GIRK signaling, another presumed target of Foxp2, was increased in mutant neurons. Blocking GIRK channels strongly attenuated the difference in intrinsic excitability between wild-type (WT) and Foxp2+/R552H neurons. Our results reveal a novel role for Foxp2 in the control of neuronal input/output homeostasis.SIGNIFICANCE STATEMENT Mutations of the Forkhead-box protein 2 (FOXP2) gene in humans are the first known monogenic cause of a speech and language disorder. The Foxp2 mutation may directly affect neuronal development and function in neocortex, where Foxp2 is expressed. Brain imaging studies in patients with a heterozygous mutation in FOXP2 showed abnormalities in cortical language-related regions relative to the unaffected members of the same family. However, the role of Foxp2 in neocortical neurons is poorly understood. Using mice with a Foxp2 mutation equivalent to that found in patients, we studied functional modifications in auditory cortex neurons ex vivo We found that mutant neurons exhibit alterations of synaptic input and GABAB/GIRK signaling, reflecting a loss of neuronal homeostasis.