Delayed rectification in single cells isolated from guinea pig sinoatrial node

1992 ◽  
Vol 262 (3) ◽  
pp. H921-H925 ◽  
Author(s):  
J. M. Anumonwo ◽  
L. C. Freeman ◽  
W. M. Kwok ◽  
R. S. Kass
Keyword(s):  
Guinea Pig ◽  
Sinoatrial Node ◽  
Single Cells ◽  
Equilibrium Potential ◽  
Delayed Rectifier ◽  
Experimental Conditions ◽  
Whole Cell ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Delayed Rectification

We have studied delayed rectifier K+ currents (IK) in cells isolated from the sinoatrial node (SAN) region of the guinea pig. Using whole cell patch-clamp procedures, we measured the voltage dependence of IK activation and IK kinetics and the IK equilibrium potential in 4.8 mM extracellular K concentration solutions. Experiments were designed to contrast properties of guinea pig SAN IK with those of IK recorded from SAN cells of the rabbit. We find that guinea pig SAN IK differs from IK recorded from single rabbit SAN cells in its activation threshold, and in the absence of inactivation of whole cell currents recorded over a wide voltage range. These results, along with the relative insensitivity of guinea pig SAN IK to E-4031 and lanthanum, suggest that under our experimental conditions, a strongly rectifying IK component (IK,r) is not the major component of delayed rectification in the guinea pig SAN, as it appears to be in SAN cells of the rabbit.

Whole cell patch-clamp study of putative vasomotor neurons isolated from the rostral ventrolateral medulla

1998 ◽  
Vol 274 (4) ◽  
pp. R1099-R1110 ◽  
Author(s):  
Janusz Lipski ◽  
Yoshinori Kawai ◽  
Jianguo Qi ◽  
Alison Comer ◽  
Joe Win
Keyword(s):  
Pressure Control ◽  
Ventrolateral Medulla ◽  
Delayed Rectifier ◽  
High Threshold ◽  
Experimental Conditions ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Current Clamp Mode ◽  
Upper Thoracic ◽  
Clamp Study

A distinct subpopulation of neurons in the rostral and ventrolateral part of the medulla oblongata (RVL) plays a key role in controlling sympathetic vasomotor tone. To characterize these neurons under conditions in which all cell-to-cell interactions are eliminated, RVL neurons were acutely dissociated from 13- to 19-day old rats. Cells projecting to the upper thoracic segments were retrogradely labeled with fluorescent beads. Fifty-two percent (17/33) of examined spinally projecting neurons were catecholaminergic, as demonstrated by single-cell reverse transcription-polymerase chain reaction or immunocytochemistry. No spontaneous (capacitive) spikes were revealed in the tight seal cell-attached configuration. Whole cell recordings were made from 54 spinally projecting neurons using Cs+- or K+-containing pipettes. No spontaneous firing was observed in current-clamp mode with K+-based pipettes (membrane potential, −61.5 ± 2.3 mV). Step depolarizations (300- or 400-ms pulses, up to 100 pA) evoked regular firing or one to four spikes. Several voltage-gated currents, resembling the transient and persistent Na+, delayed rectifier and low- and high-threshold Ca2+, were revealed in voltage-clamp mode. These results show that isolated spinally projecting RVL neurons display no pacemaker-like activity. Because data from the literature indicate that these neurons are capable of generating such activity under different experimental conditions, the factors responsible for different behavior need to be determined. Dissociated RVL neurons provide a useful new model for studying biophysical and other properties of neurons involved in blood pressure control.

Low-Threshold L-type Calcium Channels in Rat Dopamine Neurons

2004 ◽  
Vol 91 (3) ◽  
pp. 1450-1454 ◽  
Author(s):  
P. Durante ◽  
C. G. Cardenas ◽  
J. A. Whittaker ◽  
S. T. Kitai ◽  
R. S. Scroggs
Keyword(s):  
Ganglion Cells ◽  
Oscillatory Potentials ◽  
Dopamine Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Whole Cell ◽  
Voltage Range ◽  
Channel Current ◽  
Whole Cell Patch Clamp ◽  
P Type ◽  
Dorsal Root Ganglion Cells

Ca2+ channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 μM), ω-conotoxin GVIA (Ctx, 1 μM), or ω-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca2+ channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca2+ channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca2+ channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca2+-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca2+ channels may exhibit different thresholds for activation in different types of neurons.

scholarly journals The effect of C-type natriuretic peptide on delayed rectifier potassium currents in gastric antral circular myocytes of the guinea-pig

2008 ◽  
pp. 55-62
Author(s):  
HY Xu ◽  
X Huang ◽  
M Yang ◽  
J-B Sun ◽  
L-H Piao ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Phosphodiesterase Inhibitor ◽  
Potassium Currents ◽  
Inhibitory Effect ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dependent Protein ◽  
Gastric Myocytes

C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch-clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [I(K (V))] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1+/-1.6 % (n=7, P<0.05), 78.4+/-2.6 % (n=10, P<0.01) and 67.7+/-2.3 % (n=14, P<0.01), at concentrations of 0.01 micromol/l, 0.1 micromol/l and 1 micromol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 micromol/l LY83583, a guanylate cyclase inhibitor, the 1 ?micromol/l CNP-induced inhibition of I(K (V)) was significantly impaired but when the cells were preincubated with 0.1 micromol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 micromol/l CNP-induced inhibition of I(K (V)) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on I(K (V)). CNP-induced inhibition of I(K (V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibits the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig.

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

2005 ◽  
Vol 289 (2) ◽  
pp. C425-C436 ◽  
Author(s):  
Bok Hee Choi ◽  
Jung-Ah Park ◽  
Kyung-Ryoul Kim ◽  
Ggot-Im Lee ◽  
Yong-Tae Lee ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cytochalasin B ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk− cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC50 of 4.2 μM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC50 of 1.4 μM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 μM/s and 7.5 s−1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 μM) also inhibited an ultrarapid delayed rectifier K+ current ( IK,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous IK,ur in a cytoskeleton-independent manner as an open-channel blocker.

scholarly journals Properties of single-channel and whole cell Cl− currents in guinea pig detrusor smooth muscle cells

2019 ◽  
Vol 316 (5) ◽  
pp. C698-C710 ◽  
Author(s):  
Viktor Yarotskyy ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Niflumic Acid ◽  
Detrusor Smooth Muscle ◽  
Whole Cell ◽  
Whole Cell Patch Clamp

Multiple types of Cl− channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl− channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl− channels in freshly isolated guinea pig DSM cells. The recorded single Cl− channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of −41.5 mV, which is close to the ECl of −65 mV, confirming preferential permeability to Cl−. The Cl− channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~−20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl− with I−, Br−, or methanesulfonate (MsO−), resulting in anionic permeability sequence: Cl−>Br−>I−>MsO−. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl− current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl− channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

scholarly journals Modulation of Calcium Channels in Human Erythroblasts by Erythropoietin

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 92-100 ◽  
Author(s):  
Joseph Y. Cheung ◽  
Xue-Qian Zhang ◽  
Krister Bokvist ◽  
Douglas L. Tillotson ◽  
Barbara A. Miller
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Erythropoietin Receptor ◽  
Single Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Open Time ◽  
High Doses

Abstract Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i ) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (−50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 ± 0.75 to 11.15 ± 1.80 ms) and open probability (from 0.26 ± 0.06 to 2.56 ± 0.59%) of this Ba2+-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.

L-364,373 (R-L3) enantiomers have opposite modulating effects on IKs in mammalian ventricular myocytes

2013 ◽  
Vol 91 (8) ◽  
pp. 586-592 ◽  
Author(s):  
Claudia Corici ◽  
Zsófia Kohajda ◽  
Attila Kristóf ◽  
András Horváth ◽  
László Virág ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Guinea Pig ◽  
Action Potential ◽  
Right Ventricular ◽  
Action Potential Duration ◽  
Action Potentials ◽  
Ventricular Arrhythmias ◽  
Ventricular Myocytes ◽  
Delayed Rectifier ◽  
Whole Cell Patch Clamp

Activators of the slow delayed rectifier K+ current (IKs) have been suggested as promising tools for suppressing ventricular arrhythmias due to prolongation of repolarization. Recently, L-364,373 (R-L3) was nominated to activate IKs in myocytes from several species; however, in some studies, it failed to activate IKs. One later study suggested opposite modulating effects from the R-L3 enantiomers as a possible explanation for this discrepancy. Therefore, we analyzed the effect of the RL-3 enantiomers on IKs in ventricular mammalian myocytes, by applying standard microelectrode and whole-cell patch-clamp techniques at 37 °C. We synthesized 2 substances, ZS_1270B (right) and ZS_1271B (left), the 2 enantiomers of R-L3. In rabbit myocytes, ZS_1270B enhanced the IKs tail current by approximately 30%, whereas ZS_1271B reduced IKs tails by 45%. In guinea pig right ventricular preparations, ZS_1270B shortened APD90 (action potential duration measured at 90% repolarization) by 12%, whereas ZS_1271B lengthened it by approximately 15%. We concluded that R-L3 enantiomers in the same concentration range indeed have opposite modulating effects on IKs, which may explain why the racemic drug R-L3 previously failed to activate IKs. ZS_1270B is a potent IKs activator, therefore, this substance is appropriate to test whether IKs activators are ideal tools to suppress ventricular arrhythmias originating from prolongation of action potentials.

scholarly journals Modulation of Calcium Channels in Human Erythroblasts by Erythropoietin

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 92-100 ◽  
Author(s):  
Joseph Y. Cheung ◽  
Xue-Qian Zhang ◽  
Krister Bokvist ◽  
Douglas L. Tillotson ◽  
Barbara A. Miller
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Erythropoietin Receptor ◽  
Single Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Open Time ◽  
High Doses

Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i ) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (−50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 ± 0.75 to 11.15 ± 1.80 ms) and open probability (from 0.26 ± 0.06 to 2.56 ± 0.59%) of this Ba2+-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.

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