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Reinhold Penner - One of the best experts on this subject based on the ideXlab platform.
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Calcium release activated Calcium Current icrac is a direct target for sphingosine
Journal of Biological Chemistry, 1998Co-Authors: Chris Mathes, Andrea Fleig, Reinhold PennerAbstract:Whole cell patch-clamp recordings were made to study the regulation of the store-operated Calcium release-activated Calcium Current (ICRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits ICRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, including N,Ndimethylsphingosine, DL-threo-dihydrosphingosine, and N-acetylsphingosine (C2-ceramide) also block ICRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block ICRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit ICRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits ICRAC, and this inhibition can be antagonized by reducing sphingosine production with L-cycloserine, suggesting that ICRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocks ICRAC, suggesting that the sphingomyelin pathway is involved in ICRAC regulation.
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the store operated Calcium Current icrac nonlinear activation by insp3 and dissociation from Calcium release
Cell, 1997Co-Authors: Anant B Parekh, Andrea Fleig, Reinhold PennerAbstract:Patch-clamp experiments aimed at determining the relationship between intracellular Ca2+ release and activation of store-operated Calcium Current I(CRAC) reveal that both agonist and InsP3-mediated activation of I(CRAC) are highly nonlinear, occurring over a narrow concentration range. Ca2+ release and Ca2+ influx can be dissociated, as they possess differential sensitivities to InsP3: low concentrations induce substantial Ca2+ release without any activation of I(CRAC), whereas micromolar concentrations of InsP3 are required to activate Ca2+ influx. This suggests functionally distinct stores controlling Ca2+ release and influx and enables cells to switch between sources of Ca2+ to fit best their Current needs.
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Calcium release activated Calcium Current in rat mast cells
The Journal of Physiology, 1993Co-Authors: Markus Hoth, Reinhold PennerAbstract:1. Whole-cell patch clamp recordings of membrane Currents and fura-2 measurements of free intracellular Calcium concentration ([Ca2+]i) were used to study the biophysical properties of a Calcium Current activated by depletion of intracellular Calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward Calcium release-activated Calcium Current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of Calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out Calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward Current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward Current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward Currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the Calcium-sensitive fluorescence (390 nm excitation) with the Calcium Current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated Calcium Currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The Current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the Current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Kurt G Beam - One of the best experts on this subject based on the ideXlab platform.
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Calcium transients associated with the T type Calcium Current in myotubes.
The Journal of general physiology, 1994Co-Authors: Jesús García, Kurt G BeamAbstract:Immature skeletal muscle cells, both in vivo and in vitro, express a high density of T type Calcium Current and a relatively low density of the dihydropyridine receptor, the protein thought to function as the Islow Calcium channel and as the voltage sensor for excitation-contraction coupling. Although the role of the voltage sensor in eliciting elevations of myoplasmic, free Calcium (Calcium transients) has been examined, the role of the T type Current has not. In this study we examined Calcium transients associated with the T type Current in cultured myotubes from normal and dysgenic mice, using the whole cell configuration of the patch clamp technique in conjunction with the Calcium indicator dye Fluo-3. In both normal and dysgenic myotubes, the T type Current was activated by weak depolarizations and was maximal for test pulses to approximately -20 mV. In normal myotubes that displayed T type Calcium Current, the Calcium transient followed the amplitude and the integral of the Current at low membrane potentials (-40 to -20 mV) but not at high potentials, where the Calcium transient is caused by SR Calcium release. The amplitude of the Calcium transient for a pulse to -20 mV measured at 15 ms after depolarization represented, on average, 4.26 +/- 0.68% (n = 19) of the maximum amplitude of the Calcium transient elicited by strong, 15-ms test depolarizations. In dysgenic myotubes, the Calcium transient followed the integral of the Calcium Current at all test potentials, in cells expressing only T type Current as well as in cells possessing both T type Current and the L type Current Idys. Moreover, the Calcium transient also followed the amplitude and time course of Current in dysgenic myotubes expressing the cardiac, DHP-sensitive Calcium channel. Thus, in those cases where the transient appears to be a consequence of Calcium entry, it has the same time course as the integral of the Calcium Current. Inactivation of the T type Calcium Current with 1-s prepulses, or block of the Current by the addition of amiloride (0.3-1.0 mM) caused a reduction in the Calcium transient which was similar in normal and dysgenic myotubes. To allow calculation of expected changes of intracellular Calcium in response to influx, myotubes were converted to a roughly spherical shape (myoballs) by adding 0.5 microM colchicine to culture dishes of normal cells. Calcium Currents and Calcium transients recorded from myoballs were similar to those in normal myotubes.(ABSTRACT TRUNCATED AT 250 WORDS)
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Measurement of Calcium Transients and Slow Calcium Current in Myotubes
The Journal of general physiology, 1994Co-Authors: Jesús García, Kurt G BeamAbstract:The purpose of this study was to characterize excitation-contraction (e-c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the Calcium indicator dye Fluo-3 to study the Calcium transients and slow Calcium Currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow Calcium Current. The slow Calcium Current had a threshold for activation of about 0 mV; the peak amplitude of the Current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The Calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong (> or = 50 mV) that the amplitude of the slow Calcium Current became very small. Thus, the slow Calcium Current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular Calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the Calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger Calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of Calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the Calcium transient by mechanisms that appeared to be unrelated to the block of Current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large Calcium releases and becomes more efficient when the size of the release is reduced.
N. S. Virgo - One of the best experts on this subject based on the ideXlab platform.
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Acetylcholine reduces the slow Calcium Current in embryonic skeletal muscle cells in culture
Pflugers Archiv : European journal of physiology, 1993Co-Authors: Frances Moody-corbett, N. S. VirgoAbstract:Xenopus skeletal muscle cells when grown in culture develop a slow inward Calcium Current that is sensitive to dihydropyridines. Acetylcholine (ACh, 10 μM) applied through a puffer pipette caused a large inward Current in these cells (at the holding potential) through the nicotinic receptor channels and reduced the inward Calcium Current (during a step depolarization to 0 mV). After the ACh application was discontinued the holding Current rapidly returned to pre-ACh levels (20 s) whereas the Calcium Current showed a slow, partial recovery to pre-ACh levels. Outward potassium Current was also reduced during the application of ACh but recovered completely after ACh was discontinued. The effect of ACh on the Calcium Current was not mimicked by muscarine (100 μM) and was absent when 10 μg/ml α-bungarotoxin was added to the bath suggesting that the decrease in Calcium Current was mediated by Current through the nicotinic receptor.
Gary Matthews - One of the best experts on this subject based on the ideXlab platform.
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ultrafast exocytosis elicited by Calcium Current in synaptic terminals of retinal bipolar neurons
Neuron, 1996Co-Authors: Steven Mennerick, Gary MatthewsAbstract:Abstract Using high resolution capacitance measurements, we have characterized an ultrafast component of transmitter release in ribbon-type synaptic terminals of retinal bipolar neurons. During depolarization, capacitance increases to a plateau of ∼30 fF with a time constant of ∼1.5 ms. When not limited by activation kinetics of Calcium Current, the small pool is depleted even faster, with a time constant of 0.5 ms. After the ultrafast pool is depleted, capacitance rises with a slower time constant of ∼300 ms. EGTA (5 mM) depresses the slower capacitance rise but leaves the ultrafast phase intact. BAPTA (5 mM) depresses both components of exocytosis. With paired-pulse stimulation, the ultrafast pool recovers from depletion with a time constant of ∼4 s. The ultrafast component may represent fusion of docked vesicles at the base of the synaptic ribbon, while the slower component represents more distal vesicles on the ribbon.
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Calcium-dependent inactivation of Calcium Current in synaptic terminals of retinal bipolar neurons
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996Co-Authors: H Von Gersdorff, Gary MatthewsAbstract:Giant synaptic terminals (approximately 10 micrometer diameter) of bipolar neurons from goldfish retina were used to directly investigate Calcium-dependent inactivation of presynaptic Calcium Current. During sustained depolarization, Calcium Current was initially constant for a period lasting up to several hundred milliseconds and then it declined exponentially. The duration of the initial delay was shorter and the rate of inactivation was faster with larger Calcium Current. The fastest time constant of inactivation (in the range of 2–5 sec) was observed under weak Calcium buffering conditions. Inactivation was attenuated when external Ca2+ was replaced with Ba2+ and when terminals were dialyzed with high concentrations of internal BAPTA. Elevation of intracellular Calcium concentration ([Ca2+]i) by application of the Calcium ionophore ionomycin or by dialysis with pipette solutions containing buffered elevated [Ca2+] produced inactivation of Calcium Current. The rate of recovery from inactivation was not determined by the recovery of [Ca2+]i to baseline after a stimulus. The results demonstrate that the presynaptic Calcium Current in bipolar neurons is inactivated by elevated [Ca2+]i, but the inactivation is approximately 100-fold slower than previously described Calcium-dependent inactivation in other types of cells.
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Substance P modulates Calcium Current in retinal bipolar neurons.
Visual neuroscience, 1992Co-Authors: George S. Ayoub, Gary MatthewsAbstract:Retinal bipolar cells are non-spiking interneurons that relay information from photoreceptors to amacrine and ganglion cells. In turn, bipolar cells receive extensive synaptic feedback from amacrine cells, some of which contain neuropeptides, including substance P. We have examined the effect of substance P on single bipolar neurons isolated from goldfish retina and find that substance P (0.1-1 nM) produced a voltage-dependent inhibition of Calcium Current in these cells. The inhibition was strongest at negative potentials, with the peak suppression occurring at -20 to -30 mV; at potentials positive to 0 mV, there was little effect on Calcium Current. Thus, the net effect was to shift the voltage range of activation of Calcium Current toward more positive potentials. The inhibition of Calcium Current by substance P required GTP in the patch pipette and was blocked by internal GDP-beta-S. Similar effects on Calcium Current were observed with somatostatin and metenkephalin, which are also found in amacrine cells.
Mauro Robello - One of the best experts on this subject based on the ideXlab platform.
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inactivation of voltage dependent Calcium Current in an insulinoma cell line
European Biophysics Journal, 1994Co-Authors: Carla Marchetti, C Amico, Daniela Podesta, Mauro RobelloAbstract:We have studied the mechanism of Ca Current inactivation in the β-cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of Current and tail Current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum Current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (τh1 ≈ 12 ms and τh2 ≈ 60 ms) and a very slowly inactivating component (τ > 1 s). The faster component (τh1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type Current, which deactivates more slowly (τ ≈ 3–5 ms) than the other components (τ ≈ 0.2–0.3 ms). The intermediate component (τh2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) Current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 μM) did not affect the LVA Current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA Current and by 33 ± 12% the long lasting component. We suggest that three components of the Calcium Current can be resolved in HIT cells and the main target of DHPs is a HVA Current, which inactivates faster than the DHP-resistant HVA component and does so primarily through Calcium influx.
