Acute effects of repetitive depolarization on sodium current in chick myocytes

1991 ◽  
Vol 260 (6) ◽  
pp. H1810-H1818
Author(s):  
M. R. Gold ◽  
G. R. Strichartz
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Complete Recovery ◽  
Acute Effects ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

Layer-Specific Properties of the Persistent Sodium Current in Sensorimotor Cortex

2006 ◽  
Vol 95 (6) ◽  
pp. 3460-3468 ◽  
Author(s):  
P. Aracri ◽  
E. Colombo ◽  
M. Mantegazza ◽  
P. Scalmani ◽  
G. Curia ◽  
...  
Keyword(s):  
Sensorimotor Cortex ◽  
Time Course ◽  
Pyramidal Neurons ◽  
Sodium Current ◽  
Peak Amplitude ◽  
Persistent Sodium Current ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
Steady State Inactivation ◽  
Layer V

We evaluated the characteristics of the persistent sodium current ( INaP) in pyramidal neurons of layers II/III and V in slices of rat sensorimotor cortex using whole cell patch-clamp recordings. In both layers, INaP began activating around −60 mV and was half-activated at −43 mV. The INaP peak amplitude and density were significantly higher in layer V. The voltage-dependent INaP steady-state inactivation occurred at potentials that were significantly more positive in layer V ( V1/2: −42.3 ± 1.1 mV) than in layer II/III ( V1/2: −46.8 ± 1.6 mV). In both layers, a current fraction corresponding to about 25% of the maximal peak amplitude did not inactivate. The time course of INaP inactivation and recovery from inactivation could be fitted with a biexponential function. In layer V pyramidal neurons the faster time constant of development of inactivation had variable values, ranging from 158.0 to 1,133.8 ms, but it was on average significantly slower than that in layer II/III (425.9 ± 80.5 vs. 145.8 ± 18.2 ms). In both layers, INaP did not completely inactivate even with very long conditioning depolarizations (40 s at −10 mV). Recovery from inactivation was similar in the two layers. Layer V intrinsically bursting and regular spiking nonadapting neurons showed particularly prolonged depolarized plateau potentials when Ca2+ and K+ currents were blocked and slower early phase of INaP development of inactivation. The biexponential kinetics characterizing the time-dependent inactivation of INaP in layers II/III and V indicates a complex inactivating process that is incomplete, allowing a residual “persistent” current fraction that does not inactivate. Moreover, our data indicate that INaP has uneven inactivation properties in pyramidal neurons of different layers of rat sensorimotor cortex. The higher current density, the rightward shifted voltage dependency of inactivation as well the slower kinetics of inactivation characterizing INaP in layer V with respect to layer II/III pyramidal neurons may play a significant role in their ability to fire recurrent action potential bursts, as well in the high susceptibility to generate epileptic events.

scholarly journals CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1858
Author(s):  
Waheed Shabbir ◽  
Nermina Topcagic ◽  
Mohammed Aufy ◽  
Murat Oz
Keyword(s):  
A549 Cells ◽  
Na Channel ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Knock Down ◽  
Whole Cell Patch Clamp ◽  
Glycosylation Sites ◽  
Epithelial Na Channel

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle

1992 ◽  
Vol 262 (3) ◽  
pp. C691-C700 ◽  
Author(s):  
F. Vogalis ◽  
N. G. Publicover ◽  
K. M. Sanders
Keyword(s):  
Time Course ◽  
Fast Component ◽  
Patch Clamp Technique ◽  
Similar Time ◽  
Whole Cell Patch Clamp ◽  
Gastric Smooth Muscle ◽  
Double Exponential ◽  
Circular Layer ◽  
Double Exponential Function ◽  
Ca2 Channels

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

scholarly journals Irbesartan prevents sodium channel remodeling in a canine model of atrial fibrillation

2018 ◽  
Vol 19 (1) ◽  
pp. 147032031875526 ◽  
Author(s):  
Xuewen Wang ◽  
Guangping Li
Keyword(s):  
Atrial Fibrillation ◽  
Sodium Current ◽  
Renin Angiotensin System ◽  
Canine Model ◽  
Atrial Pacing ◽  
Chain Reaction ◽  
Patch Clamp Technique ◽  
Angiotensin System ◽  
Whole Cell Patch Clamp ◽  
Polymerase Chain

Introduction: Activation of the renin-angiotensin system (RAS) plays an important role in atrial electrical remodeling (AER). The purpose of the present study was to evaluate the effects of irbesartan on cardiac sodium current (INa) in a canine model of atrial fibrillation. Materials and methods: Eighteen dogs were randomized into sham, pacing or pacing+irbesartan groups ( n = 6 in each group). The dogs in the pacing and irbesartan group were paced at 500 bpm for two weeks. Irbesartan (60 mg·kg−1·d−1) was administered orally in the pacing+irbesartan groups. INa was recorded using the whole-cell patch clamp technique from canine atrial myocytes. The expressions of cardiac Na+ channels (Nav1.5) mRNA were semi-quantified by reverse transcription-polymerase chain reaction. Results: Our results showed that INa density and Nav1.5 mRNA expression in the pacing group decreased significantly ( p < 0.05 vs. sham). However, rapid atrial pacing had no effects on the half-activation voltage (V1/2act) and half-inactivation voltage (V1/2inact) of INa ( p > 0.05 vs. sham). Irbesartan significantly increased INa densities and gene expression and hyperpolarized V1/2act without concomitant changes in V1/2inact. Conclusions: Irbesartan significantly increased INa densities, which contributed to improving intra-atrial conduction and prevented the induction and promotion of AF in atrial pacing dogs.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons

1996 ◽  
Vol 76 (5) ◽  
pp. 3415-3424 ◽  
Author(s):  
K. S. Wilcox ◽  
R. M. Fitzsimonds ◽  
B. Johnson ◽  
M. A. Dichter
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Hippocampal Slices ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dissociated Cell Culture ◽  
Cultured Hippocampal Neurons

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

Abstract 17193: Inhibiting Late Sodium Current Attenuates Isoproterenol-Induced Transient Inward Current and Delayed Afterdepolarizations in Ventricular Myocytes

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yejia Song ◽  
Nesrine El-Bizri ◽  
Sridharan Rajamani ◽  
Luiz Belardinelli
Keyword(s):  
Ventricular Myocytes ◽  
Sodium Current ◽  
Selective Inhibition ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Cardiac Tissues ◽  
Inward Current ◽  
Transient Inward Current ◽  
Whole Cell Patch Clamp ◽  
Series Of Experiments

Introduction: The β-adrenergic agonist isoproterenol (ISO) is known to induce the arrhythmogenic transient inward current (I Ti ) and delayed afterdepolarization (DAD) via a stimulation of L-type Ca 2+ current. Recent studies found that ISO-induced DADs in cardiac tissues are inhibited by GS967, a selective blocker of the late Na + current (I NaL ). Thus, we hypothesize that I NaL contributes to the actions of ISO, and selective inhibition of this current will reduce ISO-induced I Ti and DADs. Methods: Transmembrane currents and action potentials of rabbit and guinea pig (GP) ventricular myocytes were recorded using the whole-cell patch-clamp technique. ISO (0.1 μM), GS967 (1 μM) and the Na + channel blocker tetrodotoxin (TTX, 3 μM) were used in the experiments. Results: In rabbit myocytes, application of ISO caused an increase in the amplitude of I NaL from -0.10±0.03 to -0.32±0.04 pA/pF (n = 17, p < 0.05). The ISO-stimulated I NaL was inhibited by GS967 and TTX. In one series of experiments, ISO increased the I NaL from -0.14±0.04 to -0.35±0.06 pA/pF, and GS967 applied in the presence of ISO reduced the current to -0.14±0.03 pA/pF (n = 9, p < 0.05). In another series of experiments, the amplitude of I NaL was increased by ISO from -0.17±0.08 to -0.41±0.09 pA/pF, and was decreased to -0.09±0.08 pA/pF when TTX was applied with ISO (n = 5, p < 0.05). Application of ISO also induced I Ti and DADs. GS967 applied in the presence of ISO inhibited the amplitude of I Ti by 52±6%, from -1.79±0.30 to -0.87±0.16 pA/pF (n = 8, p < 0.05). Consistent with the inhibition of I Ti , GS967 suppressed the amplitude of ISO-induced DADs by 56±12%, from 6.54±1.59 to 3.22±1.27 mV (n = 5, p < 0.05). Similarly, in GP myocytes ISO-induced I Ti and DADs were decreased by GS967 from -1.14±0.21 to -0.73±0.16 pA/pF (n = 7, p < 0.05) and from 7.16±0.59 to 4.67±0.24 mV (n = 5, p < 0.05), respectively. Conclusions: An increased I NaL is likely to contribute to the proarrhythmic effects of ISO in cardiac myocytes. GS967 significantly attenuated ISO-induced I NaL , I Ti and DADs, suggesting that inhibiting this current could be an effective strategy to antagonize the arrhythmogenic actions of β-adrenergic stimulation.

scholarly journals Contribution of L-type Ca2+current to electrical activity in sinoatrial nodal myocytes of rabbits

1999 ◽  
Vol 276 (3) ◽  
pp. H1064-H1077 ◽  
Author(s):  
E. Etienne Verheijck ◽  
Antoni C. G. van Ginneken ◽  
Ronald Wilders ◽  
Lennart N. Bouman
Keyword(s):  
Action Potential ◽  
Calcium Current ◽  
Sodium Current ◽  
Delayed Rectifier ◽  
Current Clamp ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Delayed Rectifier Current ◽  
Diastolic Depolarization ◽  
Recovery From Inactivation

The role of L-type calcium current ( I Ca,L) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit, with the use of the amphotericin-perforated patch-clamp technique. Nifedipine, at a concentration of 5 μM, was used to block I Ca,L. At this concentration, nifedipine selectively blocked I Ca,L for 81% without affecting the T-type calcium current ( I Ca,T), the fast sodium current, the delayed rectifier current ( I K), and the hyperpolarization-activated inward current. Furthermore, we did not observe the sustained inward current. The selective action of nifedipine on I Ca,L enabled us to determine the activation threshold of I Ca,L, which was around −60 mV. As nifedipine (5 μM) abolished spontaneous activity, we used a combined voltage- and current-clamp protocol to study the effects of I Ca,L blockade on repolarization and diastolic depolarization. This protocol mimics the action potential such that the repolarization and subsequent diastolic depolarization are studied in current-clamp conditions. Nifedipine significantly decreased action potential duration at 50% repolarization and reduced diastolic depolarization rate over the entire diastole. Evidence was found that recovery from inactivation of I Ca,L occurs during repolarization, which makes I Ca,L available already early in diastole. We conclude that I Ca,L contributes significantly to the net inward current during diastole and can modulate the entire diastolic depolarization.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Desensitization to acetylcholine in single sinoatrial node cells isolated from rabbit hearts

1992 ◽  
Vol 263 (6) ◽  
pp. H1779-H1789 ◽  
Author(s):  
H. Honjo ◽  
I. Kodama ◽  
W. J. Zang ◽  
M. R. Boyett
Keyword(s):  
Potassium Current ◽  
Sinoatrial Node ◽  
Chronotropic Effect ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Chronotropic Response ◽  
Negative Chronotropic Effect ◽  
Whole Cell Patch Clamp ◽  
Sinoatrial Node Cells ◽  
Continuous Presence

The negative chronotropic effect of acetylcholine (ACh) on the sinoatrial node fades in the continuous presence of ACh as a result of desensitization. We have investigated the mechanism underlying desensitization in single rabbit sinoatrial node cells using the whole cell patch clamp technique. The negative chronotropic effect resulting from the injection of a constant hyperpolarizing current faded. ACh activated an inwardly rectifying potassium current (iK,ACh), which faded in the continuous presence of ACh. ACh had no effect on “basal” L-type calcium current (iCa), but ACh decreased iCa, which had been potentiated by isoprenaline. This effect did not fade during a 2-min exposure to ACh. ACh decreased the hyperpolarization-activated current (i(f)). This effect again did not fade. These results suggest that desensitization of the negative chronotropic response to ACh is, in part, the result of the membrane hyperpolarization and, in part, the result of the fade of iK,ACh. These results also suggest that, whereas the activation of potassium current by ACh rapidly fades, the effects resulting from the inhibition of adenylate cyclase do not.

Cl- current in IMCD cells activated by hypotonicity: time course, ATP dependence, and inhibitors

1996 ◽  
Vol 271 (3) ◽  
pp. F552-F559 ◽  
Author(s):  
K. A. Volk ◽  
C. Zhang ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Time Course ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Renal Medulla ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The hypertonic environment of the renal medulla can change rapidly according to the state of hydration of the animal. We used primary cultures of rat inner medullary collecting duct (IMCD) cells to investigate the characteristics of Cl- currents activated by an acute reduction in osmolarity (ICl(osm)). Using the whole cell patch-clamp technique, we identified an outwardly rectifying current that decayed slowly at strongly depolarizing voltages. The onset of ICl(osm) began 6.7 min after the fall in bath osmolarity, a delay longer than reported in other cell types. Hypotonicity did not induce an increase in intracellular Ca2+ concentration, and activation of ICl(osm) did not require the presence of Ca2+. Intracellular ATP was needed to evoke ICl(osm) when the hypotonic stimulus was modest (50 mosmol/l or less) but was not necessary when the stimulus was stronger (100 mosmol/ l). ICl(osm) was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by tamoxifen or glibenclamide. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid produced a voltage-dependent block. Acute reduction in osmolarity using cells grown on filters did not induce a Cl- secretory current. The ICl(osm) of IMCD cells appears to be on the basolateral membrane and displays some unique features.

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