Effects of cannabinoids on endogenous K+ and Ca2+ currents in HEK293 cells

Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and anandamide (1 μM) decreased the calcium current by 53.1 ± 2.6, 47.5 ± 1.2, and 38.8 ± 3.1%, respectively, after transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current was not changed after application of these agonists, but the inward rectifier was increased by 94.0 ± 3.6, 83.7 ± 5.1, and 63.0 ± 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These results suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells, and that some expressed receptors may be constitutively active.Key words: cannabinoids, WIN 55,212-2, anandamide, methanandamide, inverse agonists.

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Ginsenoside Rb1 exerts antiarrhythmic effects by inhibiting INa and ICaL in rabbit ventricular myocytes

Scientific Reports ◽

10.1038/s41598-019-57010-9 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Zhipei Liu ◽

Lv Song ◽

Peipei Zhang ◽

Zhenzhen Cao ◽

Jie Hao ◽

...

Keyword(s):

Ion Channels ◽

Action Potential ◽

Potassium Current ◽

Ischemia Reperfusion ◽

Calcium Currents ◽

Delayed Rectifier ◽

Dependent Manner ◽

Ginsenoside Rb1 ◽

Patch Clamp Technique ◽

High Calcium

AbstractGinsenoside Rb1 exerts its pharmacological action by regulating sodium, potassium and calcium ion channels in the membranes of nerve cells. These ion channels are also present in cardiomyocytes, but no studies have been reported to date regarding the effects of Rb1 on cardiac sodium currents (INa), L-type calcium currents (ICaL) and action potentials (APs). Additionally, the antiarrhythmic potential of Rb1 has not been assessed. In this study, we used a whole-cell patch clamp technique to assess the effect of Rb1 on these ion channels. The results showed that Rb1 inhibited INa and ICaL, reduced the action potential amplitude (APA) and maximum upstroke velocity (Vmax), and shortened the action potential duration (APD) in a concentration-dependent manner but had no effect on the inward rectifier potassium current (IK1), delayed rectifier potassium current (IK) or resting membrane potential (RMP). We also designed a pathological model at the cellular and organ level to verify the role of Rb1. The results showed that Rb1 abolished high calcium-induced delayed afterdepolarizations (DADs), depressed the increase in intracellular calcium ([Ca2+]i), relieved calcium overload and protected cardiomyocytes. Rb1 can also reduce the occurrence of ventricular premature beats (VPBs) and ventricular tachycardia (VT) in ischemia-reperfusion (I-R) injury.

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The development of voltege-dependent ionic conductances In murine spinal cord neurones in culture

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y88-217 ◽

1988 ◽

Vol 66 (10) ◽

pp. 1328-1336 ◽

Cited By ~ 14

Author(s):

C. Krieger ◽

T. A. Sears

Keyword(s):

Spinal Cord ◽

Sodium Current ◽

Potassium Conductance ◽

Calcium Currents ◽

Delayed Rectifier ◽

Patch Clamp Technique ◽

Calcium Dependent ◽

Ionic Conductances ◽

Voltage Dependent

The development of voltage-dependent ionic conductances of foetal mouse spinal cord neurones was examined using the whole-cell patch-clamp technique on neurones cultured from embryos aged 10–12 days (E10–E12) which were studied between the first day in vitro (V1) to V10. A delayed rectifier potassium conductance (IK) and a leak conductance were observed in neurones of E10.V1, E11, V1, and E12, V1 as well as in neurones cultured for longer periods. A rapidly activating and inactivating potassium conductance (IA) was seen in neurones from E11, V2 and E12, V1 and at longer times in vitro. A tetrodotoxin (TTX) sensitive sodium-dependent inward current was observed in neurones of E11 and E12 from V1 onwards. Calcium-dependent conductances were not detectable in these neurones unless the external calcium concentration was raised 10- to 20-foid and potassium conductances were blocked. Under these conditions calcium currents could be observed as early as E11, V3 and E12, V2 and at subsequent times in vitro. The pattern of development of voltage-dependent ionic conductances in murine spinal neurones is such that initially leak and potassium currents are present followed by sodium current and subsequently calcium current.

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Mechanisms of IhERG/IKr Modulation by α1-Adrenoceptors in HEK293 Cells and Cardiac Myocytes

Cellular Physiology and Biochemistry ◽

10.1159/000453180 ◽

2016 ◽

Vol 40 (6) ◽

pp. 1261-1273 ◽

Cited By ~ 3

Author(s):

Janire Urrutia ◽

Aintzane Alday ◽

Mónica Gallego ◽

L. Layse Malagueta-Vieira ◽

Ivan Arael Aréchiga-Figueroa ◽

...

Keyword(s):

Ventricular Fibrillation ◽

Cardiac Myocytes ◽

Torsades De Pointes ◽

Hek293 Cells ◽

Herg Channel ◽

Delayed Rectifier ◽

Patch Clamp Technique ◽

K Current ◽

Herg Channels ◽

Intracellular Pathway

Background: The rapid delayed rectifier K+ current (IKr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces IKr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates IKr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP2-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. IKr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of IhERG and IKr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP2 hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated IKr channel phosphorylation and subsequent IKr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and IKr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

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Properties of two types of calcium channels in clonal pituitary cells.

The Journal of General Physiology ◽

10.1085/jgp.87.1.161 ◽

1986 ◽

Vol 87 (1) ◽

pp. 161-182 ◽

Cited By ~ 151

Author(s):

D R Matteson ◽

C M Armstrong

Keyword(s):

Calcium Currents ◽

Gh3 Cells ◽

Pituitary Cells ◽

Patch Clamp Technique ◽

Barium Ions ◽

Channel Current ◽

Inward Currents ◽

A Cell ◽

Cell Variant ◽

Sodium And Potassium

The calcium currents of GH3 cells have been studied using the whole cell variant of the patch-clamp technique. Under conditions that eliminate sodium and potassium currents, we observed inward currents that activated within a few milliseconds, and deactivated with two time constants, approximately 150 microseconds and 3 ms at -80 mV, 18-20 degrees C. The components are called FD and SD (fast deactivating and slow deactivating). Both components are calcium currents, and are greatly reduced when magnesium is substituted for most of the calcium in the bath. In addition to (a) their different rates of deactivation, the two components differ in a number of other properties. (b) The SD component inactivates almost completely, with a time constant of 23 ms at 20 mV, 19 degrees C. The FD component, on the other hand, shows little or no sign of inactivation, and is almost the same in amplitude from 10 to 100 ms. The components thus seem quite independent of each other, and must arise from two independent sets of channels. (c) The FD channels activate more rapidly than SD at 20 mV, by a factor of approximately 2 as is shown in several ways. (d) In 10 Ca or 10 Ba, the activation curve for SD channels is approximately 20 mV more negative than for FD or Na channels. (e) FD channels conduct barium ions more effectively than calcium by a ratio of approximately 2. (f) FD channels "wash out" within minutes after the patch electrode breaks into a cell, whereas SD channel current remains relatively stable. It is argued that SD channels, because of their negative activation threshold, are involved in electrical events near threshold, and that FD channels are best suited for calcium injection once a spike has been initiated.

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Zacopride Exerts an Antiarrhythmic Effect by Specifically Stimulating the Cardiac Inward Rectifier Potassium Current in Rabbits: Exploration of a New Antiarrhythmic Strategy

Current Pharmaceutical Design ◽

10.2174/1381612826666200701135508 ◽

2020 ◽

Vol 26 (44) ◽

pp. 5746-5754

Author(s):

Yuanyuan Lin ◽

Junhu Li ◽

Baozhong Zhu ◽

Qinghua Liu ◽

Xiaojie Bai ◽

...

Keyword(s):

Potassium Current ◽

Inward Rectifier ◽

Antiarrhythmic Effect ◽

Delayed Rectifier ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Inward Rectifier Potassium Current ◽

Antiarrhythmic Effects

Background: Zacopride, a potent antagonist of 5-HT3 receptors and an agonist of 5-HT4 receptors, is a gastrointestinal prokinetic agent. In a previous study, we discovered that zacopride selectively stimulated the inward rectifier potassium current (IK1) in the rat and that agonizing IK1 prevented or eliminated aconitine-induced arrhythmias in rats. Objective: Our aims were to confirm that the antiarrhythmic effects of zacopride are mediated by selectively enhancing IK1 in rabbits. Methods: The effects of zacopride on the function of the main ion channels were investigated using a whole-cell patch-clamp technique in rabbits. Effects of zacopride on cardiac arrhythmias were also explored experimentally both in vivo and in vitro. Results: Zacopride moderately enhanced cardiac IK1 but had no apparent action on voltage-gated sodium current (INa), L- type calcium current (ICa-L), sodium-calcium exchange current (INa/Ca), transient outward potassium current (Ito), or delayed rectifier potassium current (IK) in rabbits. Zacopride also had a marked antiarrhythmic effect in vivo and in vitro. We proved that the resting membrane potential (RMP) was hyperpolarized in the presence of 1 μmol/L zacopride, and the action potential duration (APD) at 90% repolarization (APD90) was shortened by zacopride (0.1-10 μmol/L) in a concentration- dependent manner. Furthermore, zacopride at 1 μmol/L significantly decreased the incidence of drug-induced early afterdepolarization (EAD) in rabbit ventricular myocytes. Conclusion: Zacopride is a selective agonist of rabbit cardiac IK1 and that IK1 enhancement exerts potential antiarrhythmic effects.

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