ABSTRACT
Background Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. Methods Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. Results About 10 μM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 μM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 μM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 μM. Moreover, 10 μM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. Conclusions Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.(AU)
Subject(s)
Proline/analysis , Conotoxins/analysis , Potassium , Sodium , CalciumABSTRACT
OBJECTIVES: Until recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs. METHODS: IHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25degrees C) and physiological temperatures (35degrees C-37degrees C). RESULTS: IHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36degrees C than at room temperature. Half-time for activation was shorter at 36degrees C than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q10 for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36degrees C than at room temperature between the membrane potentials of -20 and +60 mV. Q10 for the inactivation time constant was 3.19. CONCLUSION: The results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.
Subject(s)
Cochlea , Dependency, Psychological , Hair , Ion Channels , Kinetics , Membrane Potentials , PotassiumABSTRACT
Voltage gated potassium channels present in T lymphocytes play an important role during lymphocyte activation. Though an increase in potassium currents has been reported in activated lymphocytes, changes in potassium currents in culture without activation by antigen or mitogen has not been reported. The peak potassium current densities on day 1 and day 5 of culture have been compared in this study. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. Lymphocytes were separated from PBMCs by negative selection using anti-CD14 coated magnetic beads and cultured under appropriate conditions without antigenic or mitogenic stimulation. Lymphocytes were patched on day 1 or day 5 of culture. Voltage gated potassium currents were recorded by whole cell patch clamp technique using a depolarizing protocol. The mean of peak current densities recorded at +60 mV on day 1 of culture was 228.12± 89.39 pA/pF (n=7) and on day 5 of culture was 468.96 ± 192.07 pA/pF (n=7). The difference between the current densities on day 1 and day 5 was found to be significant. Change in electrophysiological characteristics can lead to functional changes in the lymphocytes and this should be considered when culturing lymphocytes in vitro for research and clinical use.
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Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in humans, however its mechanisms are poorly understood and its therapy is often sub-optimal. This article reviews recent experimental, numerical and clinical data on dynamics of wave propagation during AF and its mechanistic link to ionic and structural properties of the atria. At the onset, the article presents numerical and optical mapping data suggesting that a presence of periodic source with increasingly high dominant frequency (DF) of excitation underlies observations of dispersion of local activation rate during AF. Further optical mapping studies in isolated normal sheep hearts in the presence of acetylcholine (ACh) reveals that rotors localized to the left atrium (LA) drive the arrhythmia and are faster than those in the right atrium (RA). Patch-clamp data from isolated cardiomycytes shows that the ACh-modulated potassium inward rectifier current is higher in the LA than in the RA which may explain the higher DFs and sensitivity of LA rotors to ACh compared with RA rotors. Following, the role of fibrosis in governing the propagation dynamics with a decrease in excitation frequency is presented in AF in failing sheep hearts and complex activation in cell cultures. Translation into the clinical setting is then discussed: DF distribution in patients with paroxysmal AF follows the LA-to-RA gradients found in the acute cholinergic AF of sheep hearts with highest DFs localized primarily to the posterior LA wall and pulmonary veins (PV) region; however in patients with persistent AF, the highest DFs localize mainly outside of the PVs region with possible implication on the outcome of ablation procedures. Next, intravenous injection of adenosine to patients in AF is demonstrated to result in further acceleration of high DF sites and suggests that reentrant activity, rather than triggered or automatic activity, maintains the arrhythmia. Finally, analysis of excitation during AF developed in patients post-cardiac surgery suggests a DF distribution similar to that of patients with paroxysmal AF with dependency on fibrosis as found in sheep failing hearts and cell cultures. In sum, the article presents data demonstrating the use of DF of excitation in linking wave propagation mechanisms to ionic and structural properties in both experimental and human AF.
La fibrilación auricular es la arritmia cardiaca más frecuente en los seres humanos; sus mecanismos aún no se entienden totalmente y la terapia utilizada para su tratamiento es, a menudo insuficiente. En este artículo se revisan los resultados numéricos y clínicos de experiencias recientes sobre la dinámica de la propagación de ondas durante la fibrilación auricular, y su relación mecánica con las propiedades iónicas y estructurales de la aurícula. Al inicio, el artículo presenta datos sobre el mapeo numérico y óptico, que sugieren que la presencia de una fuente periódica con un incremento de frecuencia dominante de excitación, sustenta las observaciones de la dispersión de la tasa de activación local durante la fibrilación auricular. Estudios subsecuentes de mapeo óptico en corazones normales de borregos con adición de acetilcolina, revelan que los rotores localizados en la auricular izquierda impulsan la arritmia y son más rápidos que los de la aurícula derecha. Evidencias de "patch-clamp" en cardiomiocitos aislados muestran que la corriente rectificadora de entrada de potasio modulada por la acetilcolina es más alta en la aurícula izquierda que en la aurícula derecha, lo cual podría explicar las frecuencias dominantes más altas y la mayor sensibilidad a la acetilcolina de los rotores de la aurícula izquierda, comparados con los de la aurícula derecha. A seguir, se presenta el papel de la fibrosis en la dinámica de propagación, con una disminución de la frecuencia de excitación durante la fibrilación auricular en corazones enfermos de borregos, así como la activación completa en cultivos de células. Posteriormente se discute la aplicación al escenario clínico: la distribución de la frecuencia dominante en pacientes con fibrilación auricular paroxística sigue el gradiente: aurícula izquierda hacia la aurícula derecha hallados en la fibrilación auricular colinérgica aguda en corazones de borregos con las frecuencias dominantes más altas, principalmente localizadas en la pared posterior de la aurícula izquierda y en la región de las venas pulmonares; sin embargo en pacientes con fibrilación auricular persistente, la frecuencia dominante más alta se localiza principalmente fuera de la región de las venas pulmonares, con una posible implicación en los resultados de los procedimientos de ablación. A seguir, se demuestra que la inyección intravenosa de adenosina a pacientes con fibrilación auricular, resulta en mayor aceleración de los sitios de frecuencias dominantes altas, lo que sugiere que la actividad de re-entrada más bien mantiene la arritmia y no la actividad automática. Finalmente, el análisis de la excitación durante la fibrilación auricular desarrollada en pacientes post-cirugía cardiaca, sugiere una distribución de frecuencia dominante similar a la de los pacientes con fibrilación auricular paroxística con dependencia de fibrosis, tal como se evidenció en los corazones enfermos de borregos y en los cultivos celulares. En suma, el artículo presenta datos que demuestran el uso de la frecuencia dominante para correlacionar los mecanismos de propagación de onda con las propiedades iónicas y estructurales, tanto en la fibrilación auricular experimental como en la de los humanos.
Subject(s)
Animals , Humans , Atrial Fibrillation/physiopathology , Cardiac Surgical Procedures , Cells, Cultured , Electrophysiologic Techniques, Cardiac , Electrophysiological Phenomena , Fibrosis , Myocardium/pathology , Postoperative Complications/pathology , SheepABSTRACT
This study analyzes outward currents in freshly isolated goat chondrocytes patched in the whole cell mode. Capacitance tracings were recorded from the cells by the time domain method. The average capacitance was 6.33 pF ±2.15pF (Mean±SD, n=60). The range was 2.7 pF to 11.2 pF. A family of outward currents was seen when the cell was depolarized from –70 mV to +70 mV in 10 mV increments. The current density at +60 mV varied from 125 pA/pF to 2410 pA/pF. The currents were inhibited by 10 mM tetraethylammonium chloride (TEA) and the current-voltage profile suggests that these are voltage gated K+ channels. The currents were also recordable in a chloride-free external solution, thereby proving that these currents are not chloride currents. There was no evidence of voltage-gated sodium channels in these cells.
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Lithium carbonate could be used to treat or prevent brain damage following traumatic injury and neurodegenerative diseases.It has been shown that its protective effect is related to protein kinase C (PKC) and extracellular signal-related kinase (ERK).It was demonstrated that PDBu,a PKC activator,inhibited amplitudes of delayed rectifier potassium current (It,) and produced a hyperpolarizing shift in the activation-voltage curve.The responses to PDBu were inhibited by lithium carbonate (50μmol/L).Further studies showed that when pretreated with MEK/ERK inhibitor U0126 (20 μmol/L),although PDBu significantly reduced IK,lithium did not reverse the effect of PDBu.Thus,the results suggested that PKC signaling cascades,along with MAPK (mitogen-activated protein kinase) pathway,were required in the phosphorylation of potassium channel,which was presented by regulation of potassium channel characteristic.AC-cAMP and their eross-talk with GC-cGMP pathway could also modulate the effect of lithium on PKC activation,which could be one of underlying mechanisms likely related to neuroprotective effect of lithium.
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Objective To investigate the effects of methylmercury on outward potassium currents in isolated outer hair cells of guinea pig cochlea.Methods The whole-cell patch clamp technique was used.Calcium sensitive outword potassium currents were recorded when the concentrations of mercurial were 1/1000 LD_(50),1/100 LD_(50) and 1/10 LD_(50).Results After methylmercury poisoning,amplitudes of the outword potassium currents I_(K(Ca)) were(1.48?0.28),(1.36?0.16) and(1.22?0.13)nA.Compared with control group,the amplitudes in three methylmercury poisoning groups were decreased by 8%,16% and 25%(P
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Coeruleo-vestibular pathway which connects locus coeruleus and vestibular nuclei is noradrenergic. This study was designed to elucidate the effects of phenylephrine on the spontaneous activity of acutely isolated medial vestibular nuclear neurons of rat by whole-cell patch-clamp technique. Sprague-Dawley rats, aged 14 to 16 days, were used. After enzymatic digestion, dissociated medial vestibular neurons were transferred to a recording chamber mounted on an inverted microscope, and spontaneous action potentials were recorded by standard patch-clamp techniques. In current-clamp mode, the frequency of spontaneous action potential of medial vestibular nuclear neurons was decreased by phenylephrine (n=15). Phenylephrine increased the amplitude of afterhyperpolarization without changes in the resting membrane potential and spike width. In voltage-clamp mode, the whole potassium currents of the medial vestibular nuclear neurons were increased by phenylephrine (n=12). These experimental results suggest that alpha-receptor mediates the inhibitory effects on the neuronal activity of the medial vestibular nuclear neuron.
Subject(s)
Animals , Rats , Action Potentials , Digestion , Locus Coeruleus , Membrane Potentials , Neurons , Patch-Clamp Techniques , Phenylephrine , Potassium , Rats, Sprague-Dawley , Vestibular NucleiABSTRACT
Objective:To study the e ects of anthopleurin-Q(AP-Q) on potassium currents of ventricular myocytes in rats and guinea pig.Methods:The ventricular cells of guinea pigs and S.D.rats were obtained by enzymatic method.Whole cell patch clamp technique was used to record potassium currents,including transient outward potassium currents(Ito),delayed recti er potassium current(IK) and inward recti er potassium current(IK1).Results:AP-Q 3-100 nmol/L blocked Ito in a concentration-dependent manner with an EC50 value of 10.5 nmol/L.With +50 mV depolarized pulse and 10nmol/LAP-Q,the Ito increased from(13.3?3.4)pA/pFto(19.46?4.3)pA/pF.AP-Q 0.1-100 nmol/Lincreased IK and tail current(IK tail) in a concentration-dependent manner with EC50 value of 4.7 nmol/Land 5.0 nmol/L,respectively.AP-Q 1 pmol/L-100 nmol/Lincrease IK1 in a concentration-dependent manner with EC50 value of 0.2 nmol/L.Conclusion:Augmented e ect of AP-Q on Ito,IK and IK1 may partly explain its e ects of shortening APD and increasing absolute value of RP.
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AIM To investigate the effect of ke tamine on ATP-sensitive K + currents (I K ATP ) in single airway smooth muscle (ASM) cells of asthmatic rat. METHODS Single ASM cells of asthmatic rat were isolated with enzymatic dissociation technique. Effect of ketamine on I K ATP in single ASM cells was studied using the whole-cell configu ration of patch clamp technique. RESULTS Ketamine opened the ATP -sensitive K + channel (K ATP channel) in a dose-dependent manner. When the concentrations of ketamine were 1?10 -7 ,1?10 -6 ,1?10 -5 and 1?10 -4 mol?L -1 , the amplitude values of I K ATP were increased to 63 86?19 33 pA/pF(n=8,P
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BACKGROUND: Endothelin (ET), potent vasoconstricting peptide, are produced by endothelial cells after brain insults such as ischemia or infection and may play a role in the amplification or regulation of the microglia. In pathology of central nervous system, microglia might become activated leading to altered cellular function in response to brain injury. METHODS: Whole-cell patch clamp technique was used to study the effects of endothelin on changes of potassium currents in cultured rat microglia. Outward and inward rectifying potassium currents were recorded after application of depolarization and hyperpolarization pulses from -60 mV holding potential, respectively. RESULT: ET (100 nM) decreased the amplitude of outward rectifying potassium current and ET-3 was the most potent isoform. Both amplitude and slope conductance of inward rectifying potassium current were increased by ET. After application of ET, the reversal potential of inward potassium current was shifted to left relative to control. CONCLUSION: These results suggest that ET changes the potassium currents and membrane potential of microglia and this depolarizing effect of ET is related with the activation of microglia under pathological conditions of brain
Subject(s)
Animals , Rats , Brain , Brain Injuries , Central Nervous System , Endothelial Cells , Endothelins , Ischemia , Membrane Potentials , Microglia , Pathology , PotassiumABSTRACT
Objective To investigate the effect of carboxymethyl chitosan on ATP-sensitive K+ currents(IKATP) in single ventricular myocyte of rat.Methods Single ventricular myocyte of rat was isolated with enzymatic dissociation technique.Whole-cell patch clamp technique was used.The stimulating protocol was:holding potential-40 mV,command potential-100 mV to +50 mV,step potential + 10 mV,duration 200 ms,stimulating interval 6 s.Results Carboxymethyl chitosan inhibited the ATP-sensitive K+ channel(KATP channel) in a dose-dependent manner.When holding potential was-60 mV and stimulating potential was + 50 mV,carboxymethyl chitosan(0.1%,0.2%,0.3%) decreased the amplitude values of IKATP to 66.1%?9.9%,50.3%?13.3%and 39.8%?9.5%(n=7)of control,respectively.The changes of IKATP under other command potentials were consistent with this.Conclusion Carboxymethyl chitosan inhibits KATP channel in single ventricular myocyte of rat in a dose-dependent manner,and may be one of the mechanisms of antiarrhythmia.
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AIM To investigate the effect of N n butyl haloperidol iodide (F 2) on potassium currents in enzymatically isolated vascular smooth muscle cells (VSMC) from thoracic aortas and the effect of F 2 on aortic rings of rat. METHODS The whole cell patch clamp technique and the contraction of rats thoracic aortic rings were used in experiments. RESULTS The outward currents were observed when holding potential was -40 mV and the cell was depolarized from -30 mV to +100 mV (in 10 mV increase) for 400 ms. At the point of the test potential of +70 mV, solutions of F 2 (0 1,1, 5 ?mol?L -1 ) were added into bath (external) solution, which led to the increase of the outward currents from (229?28)pA,(226?57)pA and(228?42) pA to (354?29) pA ( n =6, P
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AIM To investigate the effect of N methyl berbamine(NMB)on ATP sensitive potassium currents( I KATP )in single ventricular myocytes of guinea pig. METHODS Patch clamp technique with whole cell configuration. Holding potential was -40 mV, commanding potential was -100～+50 mV and duration was 600 ms. Pipette solution contained 0 3 mmol?L -1 ATP. RESULTS NMB inhibited I KATP in a concentration dependent manner. When holding potential was -40 mV and command potential was 0 mV, I KATP were reduced from (0 46?0 09) nA, (0 43?0 15) nA, and(0 47?0 10) nA to (0 37?0 07) nA( n=4,P