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Patch Clamp Technique (patch + clamp_technique)
Kinds of Patch Clamp Technique Selected AbstractsGating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+ACTA PHYSIOLOGICA, Issue 4 2006L. Lacinová Abstract Aim:, We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. Methods:, The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 ,m Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. Results:, The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ,100 mV for the Ca2+ current, but did not reach steady state even at ,150 mV for the Na+ current. Conclusions:, Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor. [source] Brain-derived neurotrophic factor induces long-lasting Ca2+ -activated K+ currents in rat visual cortex neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2002Yoshito Mizoguchi Abstract Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in various types of neurons. Ca2+ -activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute to hyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+ -activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At a holding potential of ,50 mV, BDNF application (20 ng/mL) for 1,2 min induced an outward current (IBDNF-OUT; 80.0 ± 29.0 pA) lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nm), an inhibitor of Trk receptor tyrosine kinase, and U73122 (3 ,m), a specific phospholipase C (PLC)-, inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both charybdotoxin- (600 nm) and apamin- (300 nm) sensitive, suggesting that this current was carried by Ca2+ -activated K+ channels. BAPTA-AM (150 ,m) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+ -activated K+ currents by sustained intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary for the induction of long-lasting Ca2+ -activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the maintenance of this current. [source] A slowly inactivating sodium current (INa2) in the plateau range in canine cardiac Purkinje single cellsEXPERIMENTAL PHYSIOLOGY, Issue 1 2007Mario Vassalle The action potential of Purkinje fibres is markedly shortened by tetrodotoxin, suggesting the possibility that a slowly inactivating sodium current might flow during the plateau. The aim of the present experiments was to investigate, in canine cardiac Purkinje single cells by means of a whole cell patch clamp technique, whether a sodium current slowly inactivates at less negative potentials and (if so) some of its distinctive characteristics. The results showed that a 500 ms depolarizing step from a holding potential of ,90 mV to ,50 mV induced the fast inward current INa (labelled here INa1). With steps to ,40 mV or less negative values, a slowly decaying component (tentatively labelled here INa2) appeared, which peaked at ,30 to ,20 mV and decayed slowly and incompletely during the 500 ms steps. The INa2 was present also during steps to ,10 mV, but then the transient outward current (Ito) appeared. When the holding potential (Vh) was decreased to ,60 to ,50 mV, INa2 disappeared even if a small INa1 might still be present. Tetrodotoxin (30 ,m), lignocaine (100 ,m) and cadmium (0.2 mm; but not manganese, 1 mm) blocked INa2. During fast depolarizing ramps, the rapid inactivation of INa1 was followed by a negative slope region. During repolarizing ramps, a region of positive slope was present, whereas INa1 was absent. At less negative values of Vh, the amplitude of the negative and positive slopes became gradually smaller. Gradually faster ramps increased the magnitude of the negative slope, and tetrodotoxin (30 ,m) reduced or abolished it. Thus, Purkinje cells have a slowly decaying inward current owing to Na+ entry (INa2) that is different in several ways from the fast INa1 and that appears important for the duration of the plateau. [source] Comparative Pharmacology of Guinea Pig Cardiac Myocyte and Cloned hERG (IKr) ChannelJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 11 2004CHRISTINA DAVIE Ph.D. Introduction: This study used whole-cell, patch clamp techniques on isolated guinea pig ventricular myocytes and HEK293 cells expressing cloned human ether-a-go-go-related gene (hERG) to examine the action of drugs causing QT interval prolongation and torsades de pointes (TdP) in man. Similarities and important differences in drug actions on cardiac myocytes and cloned hERG IKr channels were established. Qualitative actions of the drugs on cardiac myocytes corresponded with results obtained from Purkinje fibers and measurement of QT interval prolongation in animal and human telemetry studies. Methods and Results: Adult guinea pig ventricular myocytes were isolated by enzymatic digestion. Cells were continuously perfused with Tyrode's solution at 33,35°C. Recordings were made using the whole-cell, patch clamp technique. Action potentials (APs) were elicited under current clamp. Voltage clamp was used to study the effect of drugs on IKr (rapidly activating delayed rectifier potassium current), INa (sodium current), and ICa (L-type calcium current). Dofetilide increased the myocyte action potential duration (APD) in a concentration-dependent manner, with a pIC50 of 7.3. Dofetilide 1 ,M elicited early afterdepolarizations (EADs) but had little affect on ICa or INa. E-4031 increased APD in a concentration-dependent manner, with a pIC50 of 7.2. In contrast, 10 ,M loratadine, desloratadine, and cetirizine had little effect on APD or IKr. Interestingly, cisapride displayed a biphasic effect on myocyte APD and inhibited ICa at 1 ,M. Even at this high concentration, cisapride did not elicit EADs. A number of AstraZeneca compounds were tested on cardiac myocytes, revealing a mixture of drug actions that were not observed in hERG currents in HEK293 cells. One compound, particularly AR-C0X, was a potent blocker of myocyte AP (pIC50 of 8.4). AR-C0X also elicited EADs in cardiac myocytes. The potencies of the same set of drugs on the cloned hERG channel also were assessed. The pIC50 values for dofetilide, E-4031, terfenadine, loratadine, desloratadine, and cetirizine were 6.8, 7.1, 7.3, 5.1, 5.2, and <4, respectively. Elevation of temperature from 22 to 35°C significantly enhanced the current kinetics and amplitudes of hERG currents and resulted in approximately fivefold increase in E-4031 potency. Conclusion: Our study demonstrates the advantages of cardiac myocytes over heterologously expressed hERG channels in predicting QT interval prolongation and TdP in man. The potencies of some drugs in cardiac myocytes were similar to hERG, but only myocytes were able to detect important changes in APD characteristics and display EADs predictive of arrhythmia development. We observed similar qualitative drug profiles in cardiac myocytes, dog Purkinje fibers, and animal and human telemetry studies. Therefore, isolated native cardiac myocytes are a better predictor of drug-induced QT prolongation and TdP than heterologously expressed hERG channels. Isolated cardiac myocytes, when used with high-throughput patch clamp instruments, may have an important role in screening potential cardiotoxic compounds in the early phase of drug discovery. This would significantly reduce the attrition rate of drugs entering preclinical and/or clinical development. The current kinetics and amplitudes of the cloned hERG channel were profoundly affected by temperature, significantly altering the potency of one drug (E-4031). This finding cautions against routine drug testing at room temperature compared to physiologic temperature when using the cloned hERG channel. [source] Block of HERG-Carried K+ Currents by the New Repolarization Delaying Agent H 345/52JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 6 2003Gregory J. Amos M.D. Ph.D. Introduction: The aim of this study was to analyze the block of HERG-carried membrane currents caused by H 345/52, a new antiarrhythmic compound with low proarrhythmic activity, in transfected mouse fibroblasts. Methods and Results: Using the whole-cell configuration of the voltage patch clamp technique, it was demonstrated that H 345/52 concentration-dependently blocked HERG-carried currents with an IC50 of 230 nM. H 345/52 preferentially bound to the open channel with unusually rapid kinetics and was trapped by channel closure. Voltage-independent behavior of H 345/52 was observed during both square-pulse and action potential clamp protocols. In contrast, the Class III agents dofetilide (10 nM) and almokalant (250 nM) demonstrated significant membrane potential-dependent effects during square-pulse clamp protocols. When using action potential clamp protocols, voltage dependence was seen with dofetilide but not with almokalant. Mathematical simulations of human ventricular action potentials predicted that the different voltage-dependent behaviors would not produce marked variations in action potential duration prolongation patterns. Conclusion: We propose that block of IKr is the principal mechanism by which H 345/52 delays repolarization in human myocardium. The voltage independence of HERG/IKr block is unlikely to underlie the low proarrhythmic potential, and ancillary effects on other membrane currents must be considered. (J Cardiovasc Electrophysiol, Vol. 14, pp. 651-658, June 2003) [source] Changes in Left Ventricular Repolarization and Ion Channel Currents Following a Transient Rate Increase Superimposed on Bradycardia in Anesthetized DogsJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 6 2000MICHAEL RUBART M.D. Electrical Remodeling of the Heart due to Rate. Introduction: We previously demonstrated in dogs that a transient rate increase superimposed on bradycardia causes prolongation of ventricular refractoriness that persists for hours after resumption of bradycardia. In this study, we examined changes in membrane currents that are associated with this phenomenon. Methods and Results: The whole cell, patch clamp technique was used to record transmembrane voltages and currents, respectively, in single mid-myocardial left ventricular myocytes from dogs with 1 week of complete AV block; dogs either underwent 1 hour of left ventricular pacing at 120 beats/min or did not undergo pacing. Pacing significantly heightened mean phase 1 and peak plateau amplitudes by ,6 and ,3 mV, respectively (P < 0.02). and prolonged action potential duration at 90% repolarization from 235 ± 8 msec to 278 ± 8 msec (1 Hz; P = 0.02). Rapid pacing-induced changes in transmembrane ionic currents included (1) a more pronounced cumulative inactivation of the 4-aminopyridine-sensitive transient outward K+ current, I to over the range of physiologic frequencies, resulting from a ,30% decrease in the population of quickly reactivating channels; (2) increases in peak density of L-type Ca2+ currents, Ica.I.' by 15% to 35% between +10 and +60 mV; and (3) increases in peak density of the Ca2+ -activated chloride current, ICl.Ca' by 30% to 120% between +30 and +50 mV. Conclusion: Frequency-dependent reduction in Ito combined with enhanced ICa.I. causes an increase in net inward current that may he responsible for the observed changes in ventricular repolarization. This augmentation of net cation influx is partially antagonized by an increase in outward ICa.Cl. [source] Electrophysiological Identification of the Functional Presynaptic Nerve Terminals on an Isolated Single Vasopressin Neurone of the Rat Supraoptic NucleusJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2010T. Ohbuchi Release of arginine vasopressin (AVP) and oxytocin from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is under the control of glutamate-dependent excitation and GABA-dependent inhibition. The possible role of the synaptic terminals attached to SON neurones has been investigated using whole-cell patch-clamp recording in in vitro rat brain slice preparations. Recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of MNCs at the synaptic level in the SON. In the present study, excitatory glutamatergic and inhibitory GABAergic synaptic inputs were recorded from an isolated single SON neurone cultured for 12 h, using the whole-cell patch clamp technique. Neurones expressed an AVP-enhanced green fluorescent protein (eGFP) fusion gene in MNCs. In addition, native synaptic terminals attached to a dissociated AVP-eGFP neurone were visualised with synaptic vesicle markers. These results suggest that the function of presynaptic nerve terminals may be evaluated directly in a single AVP-eGFP neurone. These preparations would be helpful in future studies aiming to electrophysiologically distinguish between the functions of synaptic terminals and glial modifications in the SON neurones. [source] Alcohol-Induced Electrical Remodeling: Effects of Sustained Short-Term Ethanol Infusion on Ion Currents in Rabbit AtriumALCOHOLISM, Issue 10 2009Roman Laszlo Background:, In some patients, above-average alcohol consumption before occurrence of atrial fibrillation (AF) in terms of a "holiday heart syndrome" (HHS) can be determined. There is evidence that long before development of apparent alcohol-induced cardiomyopathy, above-average alcohol consumption generates an arrhythmogenic substrate which abets the onset of AF. Changes of atrial current densities in terms of an electrical remodeling after sustained short-term ethanol infusion in rabbits as a potential part of HHS pathophysiology were examined in this study. Methods:, Rabbits of the ethanol group (EG) received sustained short-term intravenous alcohol infusion for 120 hours (during infusion period, blood alcohol level did not fall below 158 mg/dl), whereas NaCl 0.9% was infused in the placebo group (PG). Using patch clamp technique in whole-cell mode, atrial current densities were measured and compared between both groups. Results:, Ethanol infusion did not alter current densities of Ito [58.7 ± 5.0 pA/pF (PG, n = 20 cells) vs. 53.9 ± 5.0 pA/pF (EG, n = 24)], Isus [11.3 ± 1.4 pA/pF (PG, n = 20) vs. 10.2 ± 1.0 pA/pF (EG, n = 24)], and IK1 [,1.6 ± 0.3 pA/pF (PG, n = 17) vs. ,2.0 ± 0.3 pA/pF (EG, n = 22)]. However, alcohol infusion resulted in a remarkable reduction of ICa,L current densities [,28.4 ± 1.8 pA/pF (PG, n = 20) vs. ,15.2 ± 1.4 pA/pF (EG, n = 22)] and INa [,75.4 ± 3.6 pA/pF (PG, n = 17) vs. ,35.4 ± 4.4 pA/pF (EG, n = 21)], respectively. Conclusion:, Sustained short-term ethanol infusion in rabbits alters atrial current densities. HHS might be favored by alcohol-induced atrial electrical remodeling. [source] Electrophysiological characterization of pathways for K+ uptake into growing and non-growing leaf cells of barleyPLANT CELL & ENVIRONMENT, Issue 12 2009VADIM VOLKOV ABSTRACT Potassium is a major osmolyte used by plant cells. The accumulation rates of K+ in cells may limit the rate of expansion. In the present study, we investigated the involvement of ion channels in K+ uptake using patch clamp technique. Ion currents were quantified in protoplasts of the elongation and emerged blade zone of the developing leaf 3 of barley (Hordeum vulgare L.). A time-dependent inward-rectifying K+ -selective current was observed almost exclusively in elongation zone protoplasts. The current showed characteristics typical of Shaker-type channels. Instantaneous inward current was highest in the epidermis of the emerged blade and selective for Na+ over K+. Selectivity disappeared, and currents decreased or remained the same, depending on tissue, in response to salt treatment. Net accumulation rates of K+ in cells calculated from patch clamp current,voltage curves exceeded rates calculated from membrane potential and K+ concentrations of cells measured in planta by factor 2.5,2.7 at physiological apoplastic K+ concentrations (10,100 mm). It is concluded that under these conditions, K+ accumulation in growing barley leaf cells is not limited by transport properties of cells. Under saline conditions, down-regulation of voltage-independent channels may reduce the capacity for growth-related K+ accumulation. [source] Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethraTHE JOURNAL OF PHYSIOLOGY, Issue 19 2008Gerard P. Sergeant Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca2+ waves. The purpose of this study was to investigate if this activity was influenced by Ca2+ uptake into mitochondria. Spontaneous Ca2+ waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 ,m) and antimycin A (5 ,m) abolished Ca2+ waves and increased basal Ca2+ levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 ,m) and CCCP (1 ,m). Spontaneous Ca2+ waves were not inhibited by the ATP synthase inhibitor oligomycin (1 ,m), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at ,60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 ,m) also inhibited STICS. Stimulation of Ca2+ uptake into mitochondria using the plant flavonoid kaempferol (10 ,m) induced a series of propagating Ca2+ waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca2+, but was not significantly affected by the IP3 receptor blocker 2-APB (100 ,m). These data suggest that spontaneous Ca2+ waves in urethral ICC are regulated by buffering of cytoplasmic Ca2+ by mitochondria. [source] Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004Juan Shi We investigated, by using the patch clamp technique, Ca2+ -mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 ,m)-activated TRPC6 currents (ITRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2o+) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2o+ solely inhibited TRPC7 currents (ITRPC7). Vigorous buffering of intracellular Ca2+ (Ca2i+) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2o+, disclosing fast potentiation (EC50: ,0.4 mm) and inhibition (IC50: ,4 mm) of ITRPC6 and fast inhibition (IC50: ,0.4 mm) of ITRPC7. This inhibition of ITRPC6 and ITRPC7 seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of ITRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of ITRPC6 or its acceleration by Ca2o+ probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 ,m), coexpression of Ca2+ -insesentive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2i+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of ITRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+,CaM-dependent and -independent mechanisms. [source] Determinants of activation kinetics in mammalian hyperpolarization-activated cation channelsTHE JOURNAL OF PHYSIOLOGY, Issue 1 2001Takahiro M. Ishii 1The structural basis for the different activation kinetics of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels was investigated with the whole-cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS,7). 2The activation time constant of HCN4 was about 40-fold longer than that of HCN1 when compared at ,100 mV. 3In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1-S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide-binding domain (CNBD) also significantly affected the activation kinetics. 4The S1 domain and S1-S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2-fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1-S2 region of HCN1 by that of HCN4 resulted in a 10, to 20-fold slowing. 5The results indicate that S1 and S1-S2, and S6-CNBD are the crucial components for the activation gating of HCN channels. [source] Antagonist effect of flufenamic acid on TRPM2 cation channels activated by hydrogen peroxideCELL BIOCHEMISTRY AND FUNCTION, Issue 4 2007Mustafa Naz Abstract The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+ -permeable cation channel that is activated by hydrogen peroxide (H2O2) as a consequence of oxidative stress although the channel activation by H2O2 appears to represent a cell-specific process in cells with endogenous expression of TRPM2. Flufenamic acid (FA) is a non-steroidal anti-inflammatory compound. Whether H2O2 activates or FA inhibits TRPM2 channels in Chinese hamster ovary (CHO) cell is currently unknown. Due to lack of known antogonists of this channel, we demonstrate in CHO cells that FA inhibits TRPM2 activated by extracellular H2O2. CHO cells were transfected with cDNA coding for TRPM2. Cells were studied with the conventional whole-cell patch clamp technique. The intracellular solution used EDTA (10,mM) as chelator for Ca2+ and heavy metal ions. H2O2 (10,mM) and FA (0.1,mM) were applied extracellularly. Non-selective cation currents were consistently induced by H2O2. The time cause of H2O2 effects was characterized by a delay of 2,5,min and a slow current induction to reach a plateau. The H2O2 - induced inward current was effectively inhibited by 0.1,mM FA applied extracellularly. In conclusion, we have demonstrated that FA is an effective antogonist of TRPM2 channels and H2O2activated currents in CHO cells. FA in CHO cells may be considered, at best, a starting point for the development of TRPM2 channel blockers. Copyright © 2006 John Wiley & Sons, Ltd. [source] | |||||||||||||