Modulation of CaV2.3 Calcium Channel Currents by Eugenol

2008 ◽  
Vol 87 (2) ◽  
pp. 137-141 ◽  
Author(s):  
G. Chung ◽  
J.N. Rhee ◽  
S.J. Jung ◽  
J.S. Kim ◽  
S.B. Oh
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Analgesic Effect ◽  
Molecular Mechanisms ◽  
Expression System ◽  
Calcium Currents ◽  
Analgesic Agent ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Channel Currents

Eugenol, a natural congener of capsaicin, is a routine analgesic agent in dentistry. We have recently demonstrated the inhibition of CaV2.2 calcium channel and sodium channel currents to be molecular mechanisms underlying the analgesic effect of eugenol. We hypothesized that CaV2.3 channels are also modulated by eugenol and investigated its mode of action using the whole-cell patch-clamp technique in a heterologous expression system. Eugenol inhibited calcium currents in the E52 cell line, stably expressing the human CaV2.3 calcium channels, where TRPV1 is not endogenously expressed. The extent of current inhibition was not significantly different between naïve E52 cells and TRPV1-expressing E52 cells, suggesting no involvement of TRPV1. In contrast, TRPV1 activation is prerequisite for the inhibition of CaV2.3 calcium channels by capsaicin. The results indicate that eugenol has mechanisms distinct from those of capsaicin for modulating CaV2.3 channels. We suggest that inhibition of CaV2.3 channels by eugenol might contribute to its analgesic effect.

Characterization of voltage-dependent calcium currents in mouse motoneurons

1992 ◽  
Vol 68 (1) ◽  
pp. 85-92 ◽  
Author(s):  
M. Mynlieff ◽  
K. G. Beam
Keyword(s):  
Voltage Dependence ◽  
Low Voltage ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Maximal Amplitude ◽  
Time To Peak ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Channel Currents

1. Calcium channel currents were measured with the whole-cell patch clamp technique in cultured, identified mouse motoneurons. Three components of current were operationally defined on the basis of voltage dependence, kinetics, and pharmacology. 2. Test potentials to -50 mV or greater (10 mM external Ca2+) elicited a low-voltage activated T-type current that was transient (decaying to baseline in less than 200 ms) and had a relatively slow time to peak (20-50 ms). A 1-s prepulse to -45 mV produced approximately half-maximal inactivation of this T current. 3. Two high-voltage activated (HVA) components of current (1 transient and 1 sustained) were activated by test potentials to -20 mV or greater (10 mM external Ca2+). A 1-s prepulse to -35 mV produced approximately half-maximal inactivation of the transient component without affecting the sustained component. 4. When Ba2+ was substituted for Ca2+ as the charge carrier, activation of the HVA components was shifted in the hyperpolarizing direction, and the relative amplitude of the transient HVA component was reduced. 5. Amiloride (1-2 mM) caused a reversible, partial block of the T current without affecting the HVA components. 6. The dihydropyridine agonist isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3- pyridine-carboxylate [(+)-SDZ 202-791, 100 nM-1 microM)] shifted the activation of the sustained component of HVA current to more negative potentials and increased its maximal amplitude. Additionally, (+)-SDZ 202-791 caused the appearance of a slowed component of tail current.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inhibition of L-type calcium-channel activity by thapsigargin and 2,5-t-butylhydroquinone, but not by cyclopiazonic acid

1994 ◽  
Vol 302 (1) ◽  
pp. 147-154 ◽  
Author(s):  
E J Nelson ◽  
C C R Li ◽  
R Bangalore ◽  
T Benson ◽  
R S Kass ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Cyclopiazonic Acid ◽  
Calcium Currents ◽  
Pituitary Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Low Concentrations ◽  
Ic50 Values

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+]i). Higher concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.

Functional Expression of L-, N-, P/Q-, and R-Type Calcium Channels in the Human NT2-N Cell Line

2000 ◽  
Vol 84 (6) ◽  
pp. 2933-2944 ◽  
Author(s):  
Torben R. Neelands ◽  
Anthony P. J. King ◽  
Robert L. Macdonald
Keyword(s):  
Calcium Channel ◽  
Cell Line ◽  
Functional Expression ◽  
Calcium Currents ◽  
Channel Blockers ◽  
Patch Clamp Technique ◽  
Teratocarcinoma Cell ◽  
Inactivation Curve ◽  
Whole Cell ◽  
Channel Currents

The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at −80 mV, barium currents ( I Bas) were evoked by voltage commands to above −35 mV that peaked at +5 mV. When holding potentials were reduced to −20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I Ba was fit with a Boltzmann curve ( V 1/2 = −43.3 mV; slope = −17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I Bas were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 μM nifedipine (19 vs. 25%), 10 μM conotoxin GVIA (27 vs. 25%), 10 μM conotoxin MVIIC (15 vs. 16%), and 1.75 μM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 μM cadmium. In the presence of nifedipine, GVIA, and MVIIC, ∼35% of current remained, which was reduced further by SNX-482 (7–14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).

scholarly journals A novel calcium current in dysgenic skeletal muscle.

1989 ◽  
Vol 94 (3) ◽  
pp. 429-444 ◽  
Author(s):  
B A Adams ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Charge Carrier ◽  
Calcium Channel ◽  
Calcium Current ◽  
Primary Cultures ◽  
High Sensitivity ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.

scholarly journals MicroRNA-204 Is Necessary for Aldosterone-Stimulated T-Type Calcium Channel Expression in Cardiomyocytes

2018 ◽  
Vol 19 (10) ◽  
pp. 2941 ◽  
Author(s):  
Riko Koyama ◽  
Tiphaine Mannic ◽  
Jumpei Ito ◽  
Laurence Amar ◽  
Maria-Christina Zennaro ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Calcium Currents ◽  
Neonatal Rat ◽  
Messenger Rnas ◽  
Screening Assay ◽  
Rat Cardiomyocytes ◽  
Ventricular Cardiomyocytes ◽  
Channel Expression ◽  
Beating Frequency

Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.

CALCIUM CHANNEL CURRENTS IN NEURONES FROM LOCUST (SCHISTOCERCA GREGARIA) THORACIC GANGLIA

1993 ◽  
Vol 177 (1) ◽  
pp. 201-221 ◽  
Author(s):  
H. A. Pearson ◽  
G. Lees ◽  
D. Wray
Keyword(s):  
Calcium Channel ◽  
Single Channel ◽  
Schistocerca Gregaria ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Transient Component ◽  
Thoracic Ganglia ◽  
Steady State Inactivation ◽  
Inward Currents ◽  
Channel Currents

1. Using the patch-clamp technique, Ca2+ channel currents were recorded from neurones freshly isolated from the thoracic ganglia of the desert locust Schistocerca gregaria. 2. In solutions containing 10 mmol l-1 Ba2+ we observed high-voltage-activated whole-cell inward currents with sustained and transient components, both of which had similar steady-state inactivation properties. 3. Substitution of Ca2+ for Ba2+ was found to reduce whole-cell currents, whereas removal of monovalent cations had no effect. 4. Cd2+ (1 mmol l-1) completely blocked the whole-cell current, but at 10 micromolar preferentially inhibited the sustained component without affecting the transient component. 5. Verapamil (1 micromolar) inhibited both current components but appeared to be more selective for the sustained component, whereas nitrendipine (1 micromolar) had no effect on either component. 6. A single-channel recording suggested that the transient component was carried by a low- conductance channel. 7. Certain compounds with insecticidal action (ryanodine, S-bioallethrin, deltamethrin and avermectin) did not affect calcium channel currents in these cells. 8. These data suggest that there are two types of Ca2+ channels present in locust neurones. These channel types have properties differing from the T-, L- and N-type channels found in vertebrates and, furthermore, were not targets for the insecticides we tested.

Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca2+ channel currents

2000 ◽  
Vol 279 (3) ◽  
pp. C603-C610 ◽  
Author(s):  
Sayaka Mitarai ◽  
Muneshige Kaibara ◽  
Katsusuke Yano ◽  
Kohtaro Taniyama
Keyword(s):  
Membrane Potential ◽  
Protein Kinase ◽  
Time Constant ◽  
Kinase Inhibitor ◽  
Slow Component ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Inward Currents ◽  
Inactivation Process ◽  
Channel Currents

We investigated the inactivation process of macroscopic cardiac L-type Ca2+ channel currents using the whole cell patch-clamp technique with Na+ as the current carrier. The inactivation process of the inward currents carried by Na+ through the channel consisted of two components >0 mV. The time constant of the faster inactivating component (30.6 ± 2.2 ms at 0 mV) decreased with depolarization, but the time constant of the slower inactivating component (489 ± 21 ms at 0 mV) was not significantly influenced by the membrane potential. The inactivation process in the presence of isoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currents and attenuated the effects of isoproterenol. In the presence of cAMP (500 μM), the inactivation process consisted of a single slow component. We propose that the faster inactivating component represents a kinetic of the dephosphorylated or partially phosphorylated channel, and phosphorylation converts the kinetics into one with a different voltage dependency.

Opioid Receptor-Mediated Inhibition of ω-Conotoxin GVIA-Sensitive Calcium Channel Currents in Rat Intracardiac Neurons

1998 ◽  
Vol 79 (2) ◽  
pp. 753-762 ◽  
Author(s):  
David J. Adams ◽  
Carlo Trequattrini
Keyword(s):  
Calcium Channel ◽  
Opioid Receptor ◽  
Voltage Dependence ◽  
Neonatal Rat ◽  
Patch Clamp Technique ◽  
Receptor Agonists ◽  
Channel Current ◽  
Opioid Receptor Agonists ◽  
Channel Currents ◽  
Intracardiac Neurons

Adams, David J. and Carlo Trequattrini. Opioid receptor-mediated inhibition of ω-conotoxin GVIA-sensitive calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 79: 753–762, 1998. Modulation of depolarization-activated ionic conductances by opioid receptor agonists was investigated in isolated parasympathetic neurons from neonatal rat intracardiac ganglia by using the whole cell perforated patch clamp technique. Met-enkephalin (10 μM) altered the action potential waveform, reducing the maximum amplitude and slowing the rate of rise and repolarization but the afterhyperpolarization was not appreciably altered. Under voltage clamp, 10 μM Met-enkephalin selectively and reversibly inhibited the peak amplitude of high-voltage–activated Ca2+ channel currents elicited at 0 mV by ∼52% and increased three- to fourfold the time to peak. Met-enkephalin had no effect on the voltage dependence of steady-state inactivation but shifted the voltage dependence of activation to more positive membrane potentials whereby stronger depolarization was required to open Ca2+ channels. Half-maximal inhibition of Ba2+ current ( I Ba) amplitude was obtained with 270 nM Met-enkephalin or Leu-enkephalin. The opioid receptor subtype selective agonists, DAMGO and DADLE, but not DPDPE, inhibited I Ba and were antagonized by the opioid receptor antagonists, naloxone and naltrindole with IC50s of 84 nM and 1 μM, respectively. The κ-opioid receptor agonists, bremazocine and dynorphin A, did not affect Ca2+ channel current amplitude or kinetics. Taken together, these data suggest that enkephalin-induced inhibition of Ca2+ channels in rat intracardiac neurons is mediated primarily by the μ-opioid receptor type. Addition of Met-enkephalin after exposure to 300 nM ω-conotoxin GVIA, which blocked ∼75% of the total Ca2+ channel current, failed to cause a further decrease of the residual current. Met-enkephalin inhibited the ω-conotoxin GVIA-sensitive but not the ω-conotoxin-insensitive I Ba in rat intracardiac neurons. Dialysis of the cell with a GTP-free intracellular solution or preincubation of the neurons in Pertussis toxin (PTX) abolished the attenuation of I Ba by Met-enkephalin, suggesting the involvement of a PTX-sensitive Gprotein in the signal transduction pathway. The activation of μ-opioid receptors and subsequent inhibition of N-type Ca2+ channels in the soma and terminals of postganglionic intracardiac neurons is likely to inhibit the release of ACh and thereby regulate vagal transmission to the mammalian heart.

Voltage-dependent calcium channels in ventricular cells of rainbow trout: effect of temperature changes in vitro

2000 ◽  
Vol 278 (6) ◽  
pp. R1524-R1534 ◽  
Author(s):  
Catherine S. Kim ◽  
Mary D. Coyne ◽  
Judith K. Gwathmey
Keyword(s):  
Rainbow Trout ◽  
Calcium Channels ◽  
Temperature Sensitivity ◽  
Calcium Currents ◽  
Temperature Dependency ◽  
Patch Clamp Technique ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

Voltage-dependent calcium channels (VDCC) in ventricular myocytes from rainbow trout ( Oncorhynchus mykiss) were investigated in vitro using the perforated patch-clamp technique, which maintains the integrity of the intracellular milieu. First, we characterized the current using barium as the charge carrier and established the doses of various pharmacological agents to use these agents in additional studies. Second, we examined the current at several physiological temperatures to determine temperature dependency. The calcium currents at 10°C (acclimation temperature) were identified as l-type calcium currents based on their kinetic behavior and response to various calcium channel agonists and antagonists. Myocytes were chilled (4°C) and warmed (18 and 22°C), and the response of VDCC to varying temperatures was observed. There was no significant dependency of the current amplitude and kinetics on temperature. Amplitude decreased 25–36% at 4°C (Q10 ∼1.89) and increased 18% at 18°C (Q10 ∼1.23) in control, Bay K8644 (Bay K)-, and forskolin-enhanced currents. The inactivation rates (τi) did not demonstrate a temperature sensitivity for the VDCC (Q10 1.23–1.92); Bay K treatment, however, increased temperature sensitivity of τi between 10 and 18°C (Q10 3.98). The low Q10 values for VDCC are consistent with a minimal temperature sensitivity of trout myocytes between 4 and 22°C. This low-temperature dependency may provide an important role for sarcolemmal calcium channels in adaptation to varying environmental temperatures in trout.

Calcium Currents in Retrogradely Labeled Pyramidal Cells From Rat Sensorimotor Cortex

2000 ◽  
Vol 83 (4) ◽  
pp. 2349-2354 ◽  
Author(s):  
Ansalan Stewart ◽  
Robert C. Foehring
Keyword(s):  
Pyramidal Neurons ◽  
Pyramidal Cells ◽  
Cell Types ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Multiple Populations ◽  
Whole Cell Patch Clamp ◽  
The Mean ◽  
P Type

Our previous studies of calcium (Ca2+) currents in cortical pyramidal cells revealed that the percentage contribution of each Ca2+ current type to the whole cell Ca2+ current varies from cell to cell. The extent to which these currents are modulated by neurotransmitters is also variable. This study was directed at testing the hypothesis that a major source of this variability is recording from multiple populations of pyramidal cells. We used the whole cell patch-clamp technique to record from dissociated corticocortical, corticostriatal, and corticotectal projecting pyramidal cells. There were significant differences between the three pyramidal cell types in the mean percentage of L-, P-, and N-type Ca2+ currents. For both N- and P-type currents, the range of percentages expressed was small for corticostriatal and corticotectal cells as compared with cells which project to the corpus callosum or to the general population. The variance was significantly different between cell types for N- and P-type currents. These results suggest that an important source of the variability in the proportions of Ca2+ current types present in neocortical pyramidal neurons is recording from multiple populations of pyramidal cells.

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