scholarly journals Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells.

1994 ◽  
Vol 104 (1) ◽  
pp. 107-121 ◽  
Author(s):  
C Mathes ◽  
S H Thompson
Keyword(s):  
Muscarinic Receptors ◽  
Current Density ◽  
Calcium Current ◽  
Time Course ◽  
Calcium Release ◽  
Calcium Influx ◽  
Neuroblastoma Cells ◽  
Reversal Potential ◽  
Calcium Currents ◽  
National Academy Of Sciences

The activation of muscarinic receptors in N1E-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value approximately 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in approximately 10 s after washing the agonist away. The current density is 0.11 +/- 0.08 pA/pF (mean +/- SD; n = 12). It is absent in zero-Ca++ saline and reduced by Mn++ and Ba++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 +/- 41 nM (n = 7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 +/- 0.20 pA/pF (n = 6). Thapsigargin activated current is reduced by Mn++ and Ba++ and increased by elevated external Ca++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 +/- 35 nM. The Ca++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R. Penner. 1993. Journal of Physiology. 465:359-386; Zweifach, A., and R. S. Lewis, 1993. Proceedings of the National Academy of Sciences, USA. 90:6295-6299).

scholarly journals The relationship between depletion of intracellular Ca2+ stores and activation of Ca2+ current by muscarinic receptors in neuroblastoma cells.

1995 ◽  
Vol 106 (5) ◽  
pp. 975-993 ◽  
Author(s):  
C Mathes ◽  
S H Thompson
Keyword(s):  
Muscarinic Receptors ◽  
Current Density ◽  
Time Course ◽  
Neuroblastoma Cells ◽  
Calcium Stores ◽  
Store Depletion ◽  
Selective Membrane ◽  
Current Activation ◽  
M1 Muscarinic ◽  
The Relationship

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilling. The time course of Ca2+ store depletion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca2+ current activation measured with nystatin patch voltage clamp. At the time of maximum current density (0.18 + .03 pA/pF; n = 48), the Ca2+ content of the IP3-releasable Ca2+ pool is reduced to 39 + 3% (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a minimum Ca2+ content in 15-30 s. The activation of Ca2+ current is delayed by 10-15 s after the beginning of Ca2+ release and continues to gradually increase for nearly 60 s, long after Ca2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the production and accumulation of a second messenger is the rate-limiting step in current activation. The time course of Ca2+ store depletion was also measured after adding thapsigargin to block intracellular Ca2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca2+ stores are depleted by > 90% and the Ca2+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca2+ buffer EGTA/AM (10 microM; 30 min) depletes IP3-releasable Ca2+ stores by between 25 and 50%, and it activates a voltage-independent inward current with properties similar to the current activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca(2+)-dependent inactivation.

scholarly journals Two components of voltage-dependent calcium influx in mouse neuroblastoma cells. Measurement with arsenazo III.

1986 ◽  
Vol 88 (2) ◽  
pp. 149-165 ◽  
Author(s):  
S R Bolsover
Keyword(s):  
Intracellular Calcium ◽  
Calcium Current ◽  
Calcium Influx ◽  
Neuroblastoma Cells ◽  
Calcium Currents ◽  
Arsenazo Iii ◽  
Optical Absorbance ◽  
Mouse Neuroblastoma ◽  
Calcium Indicator ◽  
Voltage Dependent

N1E-115 mouse neuroblastoma cells were injected with the calcium indicator dye arsenazo III. Optical absorbance changes during voltage-clamp depolarization were used to examine the properties of the two calcium currents present in these cells. The rapidly inactivating calcium current (Moolenar and Spector, 1979b, Journal of Physiology, 292:307-323) inactivates by a voltage-dependent mechanism. The slowly inactivating calcium current is dominant in raising intracellular calcium during depolarizations to greater than -20 mV. Lowering the extracellular calcium concentration affects the two calcium currents unequally, with the slowly inactivating current being reduced more. Intracellular calcium falls very slowly (tau greater than 1 min) after a depolarization. The rapidly inactivating calcium current is responsible for a calcium action potential under physiological conditions. In contrast, it is unlikely that the slowly inactivating calcium current has an important electrical role. Rather, its function may be to add a further increment of calcium influx over and above the calcium influx through the rapidly inactivating calcium channels.

Pharmacologically and Functionally Distinct Calcium Currents of Stomatogastric Neurons

1998 ◽  
Vol 79 (4) ◽  
pp. 2070-2081 ◽  
Author(s):  
Laura M. Hurley ◽  
Katherine Graubard
Keyword(s):  
Neuromuscular Junction ◽  
Calcium Channel ◽  
Calcium Current ◽  
Calcium Influx ◽  
Outward Current ◽  
Calcium Currents ◽  
High Threshold ◽  
Channel Blockers ◽  
Postsynaptic Potentials ◽  
Different Types

Hurley, Laura M. and Katherine Graubard. Pharmacologically and functionally distinct calcium currents of stomatogastric neurons. J. Neurophysiol. 79: 2070–2081, 1998. Previous studies have suggested the presence of different types of calcium channels in different regions of stomatogastric neurons. We sought to pharmacologically separate these calcium channel types. We used two different preparations from different regions of stomatogastric neurons to screen a range of selective calcium channel blockers. The two preparations were isolated cell bodies in culture, in which calcium current was measured directly, and isolated neuromuscular junction, in which synaptic transmission was the indirect assay for presynaptic calcium influx. The selective blockers were two different dihydropyridines, ω-Agatoxin IVA, and ω-Conotoxin GVIA. Cultured cell bodies possessed both high-threshold calcium current and calcium-activated outward current, similar to intact neurons. The calcium current had transient and maintained components, but both components had the same voltage dependence of activation and inactivation. Dihydropyridines at ≥10 μM blocked both high-threshold calcium current and calcium-activated outward current. Nanomolar doses of ω-Agatoxin IVA did not block calcium current, but micromolar doses did. ω-Conotoxin GVIA did not block either current. In contrast, at the neuromuscular junction, dihydropyridines reduced the amplitude of postsynaptic potentials by only a modest amount, whereas ω-Agatoxin IVA at doses as low as 64 nM reduced the amplitude of postsynaptic potentials almost entirely. These effects were presynaptic. ω-Conotoxin GVIA did not change the amplitude of postsynaptic potentials. The different pharmacological profiles of the two isolated preparations suggest that there are at least two different types of calcium channel in stomatogastric neurons and that ω-Agatoxin IVA and dihydropridines can be used to pharmacologically distinguish them.

scholarly journals Effect of changes in action potential shape on calcium currents and transmitter release in a calyx–type synapse of the rat auditory brainstem

1999 ◽  
Vol 354 (1381) ◽  
pp. 347-355 ◽  
Author(s):  
J. G. G. Borst ◽  
B. Sakmann
Keyword(s):  
Action Potential ◽  
Transmitter Release ◽  
Time Course ◽  
Calcium Influx ◽  
Auditory Brainstem ◽  
Calcium Currents ◽  
Medial Nucleus ◽  
Potential Shape ◽  
Presynaptic Calcium ◽  
Rat Brainstem

We studied the relation between the size of presynaptic calcium influx and transmitter release by making simultaneous voltage clamp recordings from presynaptic terminals, the calyces of Held and postsynaptic cells, the principal cells of the medial nucleus of the trapezoid body, in slices of the rat brainstem. Calyces were voltage clamped with different action potential waveforms. The amplitude of the excitatory postsynaptic currents depended supralinearly on the size of the calcium influx, in the absence of changes in the time–course of the calcium influx. This result is in agreement with the view thact at this synapse most vesicles are released by the combined action of multiple calcium channels.

Calcium currents in isolated canine airway smooth muscle cells

1988 ◽  
Vol 254 (6) ◽  
pp. C793-C801 ◽  
Author(s):  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Calcium Current ◽  
Reversal Potential ◽  
Physiological Saline ◽  
Muscle Cells ◽  
Calcium Currents ◽  
Airway Smooth Muscle Cells ◽  
Outward Currents ◽  
Rapid Inactivation

Canine tracheal smooth muscle cells were enzymatically dissociated, and individual myocytes were voltage clamped through use of the whole cell, patch-clamp method. Cells dialyzed with solutions high in potassium and bathed in physiological saline demonstrated brief inward currents, followed by large outward currents that inactivated very slowly. When outward currents were blocked, a voltage-activated inward current was observed that activated with depolarizations to voltages positive to -45 mV, with an apparent reversal potential greater than 110 mV, and a peak current at 15 mV. This current was identified as a calcium current on the basis of 1) its presence under conditions in which calcium was the only permeant cation, 2) the lack of a blocking effect of 2 microM tetrodotoxin, and 3) block of the current by Mn2+, Cd2+, and CO2+. Increases in external calcium concentration from 2 to 20 mM resulted in an increase in current amplitude and a shift of voltage activation toward more positive potentials. The current displayed a rapid inactivation phase with a time constant of 16-52 ms, which was well fit by a single exponential. Steady-state inactivation of the calcium current was sigmoidal, with a voltage of half inactivation of -21 mV in 20 mM Ca2+. The principle component of the calcium current was further identified as a transient current on the basis of its rapid inactivation, current-voltage characteristics, and relative insensitivity to dihydropyridine calcium channel blocking agents.

Increased calcium current in carotid body glomus cells following in vivo acclimatization to chronic hypoxia

1996 ◽  
Vol 76 (3) ◽  
pp. 1880-1886 ◽  
Author(s):  
S. C. Hempleman
Keyword(s):  
Carotid Body ◽  
Calcium Current ◽  
Chronic Hypoxia ◽  
Calcium Influx ◽  
Calcium Currents ◽  
Glomus Cell ◽  
Glomus Cells ◽  
Cell Calcium ◽  
Carotid Bodies

1. Rat pups were gestated and born in normoxia (inspired O2 pressure 149 mmHg) or chronic hypoxia (insured O2 pressure 80 mmHg) to test whether chronic hypoxia alters carotid body glomus cell calcium currents. Carotid bodies were removed from 5- to 8-day-old-pups under halothane anesthesia, at which time blood hematocrits averaged 52 +/- 1% (mean +/- SE) in the chronically hypoxic pups and 36 +/- 1% in the normoxic pups (P < 0.05). Glomus cells were then enzymatically isolated from the carotid bodies, and calcium currents were recorded with whole cell patch clamp. 2. Compared with normoxic glomus cells (n = 29), chronically hypoxic glomus cells (n = 32) superfused with 10 mM CaCl2 had larger peak calcium current (146 +/- 16 pA vs. 49 +/- 7 pA, P < 0.05), larger peak calcium current density (12.0 +/- 1.1 pA/pF vs. 7.3 +/- 1.0 pA/pF, P < 0.05), and larger membrane capacitance (12.1 +/- 0.9 pF vs. 7.5 +/- 0.6 pF, P < 0.05). 3. Threshold for calcium current activation was approximately -40 mV. Currents showed little inactivation during 45-ms test pulses and were half-inactivated by a steady holding voltage of -11 +/- 2 mV (n = 15). Currents were reduced 43 +/- 13% by 50 microM nifedipine (n = 6, P < 0.05), and were augmented with barium as the charge carrier. These properties suggest that glomus cell calcium current is carried in part through L-type channels, and that is is relatively resistant to steady-state inactivation. 4. Augmented calcium influx through voltage-gated channels in glomus cells from chronically hypoxic neonatal rats may increase carotid body excitability through increased stimulus-secretion coupling. Overall, acclimatization to chronic hypoxia is known to depress acute hypoxic ventilatory reflex responses in neonates. The observations reported here suggest that inhibition of ventilatory reflexes by chronic hypoxia in neonates occurs centrally rather than peripherally.

Inhibition of the Aplysia sensory neuron calcium current with dopamine and serotonin

2013 ◽  
Vol 110 (9) ◽  
pp. 2071-2081 ◽  
Author(s):  
Tyler W. Dunn ◽  
Wayne S. Sossin
Keyword(s):  
Calcium Current ◽  
Kinase Inhibitors ◽  
Calcium Influx ◽  
Receptor Activation ◽  
Calcium Currents ◽  
Src Tyrosine Kinase ◽  
Nociceptive Neurons ◽  
Readily Releasable Pool ◽  
G Protein Coupled ◽  
Calcium Secretion

The inhibition of Aplysia pleural mechanosensory neuron synapses by dopamine and serotonin through activation of endogenous dopaminergic and expressed 5-HT1Apl(a)/b receptors, respectively, involves a reduction in action potential-associated calcium influx. We show that the inhibition of synaptic efficacy is downstream of the readily releasable pool, suggesting that inhibition is at the level of calcium secretion coupling, likely a result of the changes in the calcium current. Indeed, the inhibitory responses directly reduce a CaV2-like calcium current in isolated sensory neurons. The inhibition of the calcium current is voltage independent as it is not affected by a strong depolarizing prepulse, consistent with other invertebrate CaV2 calcium currents. Similar to voltage-independent inhibition of vertebrate nociceptors, inhibition was blocked with Src tyrosine kinase inhibitors. The data suggest a conserved mechanism by which G protein-coupled receptor activation can inhibit the CaV2 calcium current in nociceptive neurons.

scholarly journals Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors.

1994 ◽  
Vol 103 (1) ◽  
pp. 125-147 ◽  
Author(s):  
J García ◽  
T Tanabe ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channel ◽  
Calcium Current ◽  
Calcium Release ◽  
Voltage Dependence ◽  
Calcium Transient ◽  
Calcium Currents ◽  
Calcium Transients ◽  
Charge Movement ◽  
The Relationship

In both skeletal and cardiac muscle, the dihydropyridine (DHP) receptor is a critical element in excitation-contraction (e-c) coupling. However, the mechanism for calcium release is completely different in these muscles. In cardiac muscle the DHP receptor functions as a rapidly-activated calcium channel and the influx of calcium through this channel induces calcium release from the sarcoplasmic reticulum (SR). In contrast, in skeletal muscle the DHP receptor functions as a voltage sensor and as a slowly-activating calcium channel; in this case, the voltage sensor controls SR calcium release. It has been previously demonstrated that injection of dysgenic myotubes with cDNA (pCAC6) encoding the skeletal muscle DHP receptor restores the slow calcium current and skeletal type e-c coupling that does not require entry of external calcium (Tanabe, Beam, Powell, and Numa. 1988. Nature. 336:134-139). Furthermore, injection of cDNA (pCARD1) encoding the cardiac DHP receptor produces rapidly activating calcium current and cardiac type e-c coupling that does require calcium entry (Tanabe, Mikami, Numa, and Beam. 1990. Nature. 344:451-453). In this paper, we have studied the voltage dependence of, and the relationship between, charge movement, calcium transients, and calcium current in normal skeletal muscle cells in culture. In addition, we injected pCAC6 or pCARD1 into the nuclei of dysgenic myotubes and studied the relationship between the restored events and compared them with those of the normal cells. Charge movement and calcium currents were recorded with the whole cell patch-clamp technique. Calcium transients were measured with Fluo-3 introduced through the patch pipette. The kinetics and voltage dependence of the charge movement, calcium transients, and calcium current in dysgenic myotubes expressing pCAC6 were qualitatively similar to the ones elicited in normal myotubes: the calcium transient displayed a sigmoidal dependence on voltage and was still present after the addition of 0.5 mM Cd2+ + 0.1 mM La3+. In contrast, the calcium transient in dysgenic myotubes expressing pCARD1 followed the amplitude of the calcium current and thus showed a bell shaped dependence on voltage. In addition, the transient had a slower rate of rise than in pCAC6-injected myotubes and was abolished completely by the addition of Cd2+ + La3+.

Activation of calcium release assessed by calcium release-induced inactivation of calcium current in rat cardiac myocytes

2004 ◽  
Vol 286 (2) ◽  
pp. C330-C341 ◽  
Author(s):  
Alexandra Zahradníková ◽  
Zuzana Kubalová ◽  
Jana Pavelková ◽  
Sándor Györke ◽  
Ivan Zahradník
Keyword(s):  
Cardiac Myocytes ◽  
Lipid Bilayers ◽  
Calcium Current ◽  
Calcium Release ◽  
Flash Photolysis ◽  
Ventricular Myocytes ◽  
Calcium Influx ◽  
Ryanodine Receptors ◽  
Test Pulse ◽  
Dyadic Space

In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current ( ICa). We used this Ca2+ release-dependent inactivation (RDI) of ICa as a local probe of sarcoplasmic reticulum Ca2+ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca2+ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak ICa during a test pulse. The negative correlation between peak ICa suppression and ICa inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca2+ ions injected during the prepulse and during the subsequent tail current suppressed peak ICa in the test pulse to a different extent. Quantitative analysis indicated that equal Ca2+ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca2+ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca2+ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus.

Sign in / Sign up

Export Citation Format

Share Document