scholarly journals Cadmium block of squid calcium currents. Macroscopic data and a kinetic model.

1991 ◽  
Vol 98 (4) ◽  
pp. 751-770 ◽  
Author(s):  
R H Chow
Keyword(s):  
Transmembrane Potential ◽  
Calcium Currents ◽  
Entry Rate ◽  
Whole Cell Patch Clamp ◽  
Slow Channel ◽  
Voltage Dependent ◽  
A Site ◽  
Ca2 Channels ◽  
Final Block ◽  
Kinetic Features

The mechanism of Cd2+ block of Ca2+ currents (ICa) was explored in squid neurons using whole-cell patch clamp. Control currents activated sigmoidally, more rapidly at more positive potentials, and did not inactivate significantly. External Cd2+ up to 250 microM reduced ICa reversibly. For small depolarizations, the current for a step of 10 ms increased to a maintained value, resembling the control; but for Vm greater than 0 mV, the increase was followed by a decrease, as Cd2+ block became greater. Final block was greater for larger depolarizations. At 0 mV the half-blocking concentration was 125 microM. Tail currents, measured as channels close, had an initial "hook" when recorded in Cd2+: currents increased transiently, then decreased. This suggests that Cd2+ escapes from some channels, which then conduct briefly before closing. Analysis of tail currents shows that Cd2+ does not slow channel closing. The data can be explained if Cd2+ is a permeant blocker of Ca2+ channels and if channels can close when occupied by Cd2+. Cd2+ permeates the channels, but binds transiently to a site in the pore, obstructing the passage of other ions (e.g., Ca2+). Dwell time depends on the transmembrane potential, becoming shorter for more negative internal potentials. A five-state model was used to simulate the steady-state and kinetic features. It combines a Hodgkin-Huxley type m2 gating scheme and a one-site Woodhull ionic blockage model for a permeant blocker and includes a closed blocked state. To fit the data, the binding site for Cd2+ had to be near the outer end of the pore, with a well depth of -12.2 RT, and with a barrier at each end of the pore. The model predicts that the Cd2+ entry rate is nearly voltage independent, but the exit rate is steeply voltage dependent (e-fold/17 mV). Analysis further suggests that the channel closes at a normal rate with Cd2+ in the pore.

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)

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1989 ◽  
Vol 61 (3) ◽  
pp. 467-477 ◽  
Author(s):  
D. E. Meyers ◽  
J. L. Barker
Keyword(s):  
Patch Clamp ◽  
Hippocampal Neurons ◽  
Calcium Currents ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Cells Cultured ◽  
In Cells

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard “run-down” of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the “high-voltage-activated” Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150–450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

Low-voltage activated calcium currents increase in basal forebrain neurons from aged rats

1995 ◽  
Vol 74 (2) ◽  
pp. 876-887 ◽  
Author(s):  
D. Murchison ◽  
W. H. Griffith
Keyword(s):  
Time Course ◽  
Low Voltage ◽  
Age Groups ◽  
Medial Septum ◽  
Calcium Currents ◽  
Fischer 344 ◽  
Diagonal Band ◽  
Whole Cell Patch Clamp ◽  
Age Related ◽  
Voltage Dependent

1. Whole cell patch-clamp recordings were made of low-voltage-activated (LVA) calcium (Ca2+) currents using 2 mM barium (Ba2+) as charge carrier. Acutely dissociated neurons from medial septum (MS) and the nucleus of the diagonal band (nDB) were examined in young adult (1–3 mo) and aged (24–26 mo) Fischer 344 rats. 2. Most neurons in both age groups displayed LVA currents: 84% of young cells (110/131) and 87% in aged cells (62/71). Using cell capacitance as an indication of cell size, aged cells were significantly smaller (P < 0.05; 15.4 +/- 0.6 pF; mean +/- SE) than young cells (18.0 +/- 0.5 pF), although a single distribution of cell sizes was present in both populations. 3. The LVA currents were enhanced in cells from aged animals. When LVA currents were studied without activation of high voltage activated currents, the current density (pA/pF) was significantly (P < 0.05) increased at negative potentials in aged neurons (young: 4.92 +/- 0.35 pA/pF; Aged: 5.92 +/- 0.45 pA/pF, at a prepulse potential of -110 mV). No change in voltage-dependent activation or inactivation was seen. The time course of recovery from inactivation also was unchanged. 4. Kinetic parameters of LVA currents were compared in both age groups. No age-related difference in time-dependent activation or inactivation was observed. A single distribution of decay time constants of LVA currents was present in both age groups. 5. These results show that MS/nDB cells maintain robust LVA currents and have increased current densities in very old rats. An increased LVA current in the aged neurons suggests that their ability to fire rhythmically or in bursts is retained or enhanced and that the resulting increase in intracellular Ca2+ may contribute to an altered Ca2+ homeostasis.

Multiple calcium currents in a thyroid C-cell line: biophysical properties and pharmacology

1991 ◽  
Vol 260 (6) ◽  
pp. C1253-C1263 ◽  
Author(s):  
B. A. Biagi ◽  
J. J. Enyeart
Keyword(s):  
Cell Line ◽  
Time Constant ◽  
Calcium Currents ◽  
C Cells ◽  
Patch Clamp Technique ◽  
C Cell ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Rat Thyroid ◽  
Ca2 Channels

The whole cell version of the patch-clamp technique was used to characterize voltage-gated Ca2+ channels in the calcitonin-secreting rat thyroid C-cell line 6-23 (clone 6). Three types of Ca2+ channels could be distinguished based on differences in voltage dependence, kinetics, and pharmacological sensitivity. T-type current was half-maximal at -31 mV, showed steady-state voltage-dependent inactivation that was half-maximal at -57 mV, inactivated with a voltage-dependent time constant that reached a minimum of 20 ms at potentials positive to -20 mV, and deactivated with a single time constant of approximately 2 ms at -80 mV. Reactivation of inactivated channels occurred with a time constant of 1.26 s at -90 mV. T current was selectively blocked by Ni2+ at concentrations between 5 and 50 microM. La3+ and Y3+ blocked the T current at 10- to 20-fold lower concentrations. Dihydropyridine-sensitive L-type current was half-maximal at a test potential of -3 mV and was approximately doubled in size when Ba2+ replaced Ca2+ as the charge carrier. Unlike L-type Ca2+ current in many cells, this current in C-cells displayed little Ca(2+)-dependent inactivation. N-type current was composed of inactivating and sustained components that were inhibited by omega-conotoxin. The inactivating component was half-maximal at +9 mV and could be fitted by two exponentials with time constants of 22 and 142 ms. A slow inactivation of N current with a time constant of 24.9 s was observed upon switching the holding potential from -80 to -40 mV. These results demonstrate that, similar to other neural crest derived cells, thyroid C-cells express multiple Ca2+ channels, including one previously observed only in neurons.

Local Anesthetics Modulate Neuronal Calcium Signaling through Multiple Sites of Action

2003 ◽  
Vol 98 (5) ◽  
pp. 1139-1146 ◽  
Author(s):  
Fang Xu ◽  
Zayra Garavito-Aguilar ◽  
Esperanza Recio-Pinto ◽  
Jin Zhang ◽  
Thomas J. J. Blanck
Keyword(s):  
Local Anesthetics ◽  
K Channel ◽  
Na Channels ◽  
Channel Blockade ◽  
K Channels ◽  
Voltage Dependent ◽  
Sites Of Action ◽  
A Site ◽  
Ca2 Channels

Background Local anesthetics (LAs) are known to inhibit voltage-dependent Na+ channels, as well as K+ and Ca2+ channels, but with lower potency. Since cellular excitability and responsiveness are largely determined by intracellular Ca2+ availability, sites along the Ca2+ signaling pathways may be targets of LAs. This study was aimed to investigate the LA effects on depolarization and receptor-mediated intracellular Ca2+ changes and to examine the role of Na+ and K+ channels in such functional responses. Methods Effects of bupivacaine, ropivacaine, mepivacaine, and lidocaine (0.1-2.3 mm) on evoked [Ca2+](i) transients were investigated in neuronal SH-SY5Y cell suspensions using Fura-2 as the intracellular Ca2+ indicator. Potassium chloride (KCl, 100 mm) and carbachol (1 mm) were individually or sequentially applied to evoke increases in intracellular Ca2+. Coapplication of LA and Na+/K+ channel blockers was used to evaluate the role of Na+ and K+ channels in the LA effect on the evoked [Ca2+](i) transients. Results All four LAs concentration-dependently inhibited both KCl- and carbachol-evoked [Ca2+](i) transients with the potency order bupivacaine &gt; ropivacaine &gt; lidocaine &gt;/= mepivacaine. The carbachol-evoked [Ca2+](i) transients were more sensitive to LAs without than with a KCl prestimulation, whereas the LA-effect on the KCl-evoked [Ca2+](i) transients was not uniformly affected by a carbachol prestimulation. Na+ channel blockade did not alter the evoked [Ca2+](i) transients with or without a LA. In the absence of LA, K+ channel blockade increased the KCl-, but decreased the carbachol-evoked [Ca2+](i) transients. A coapplication of LA and K+ channel blocker resulted in larger inhibition of both KCl- and carbachol-evoked [Ca2+](i) transients than by LA alone. Conclusions Different and overlapping sites of action of LAs are involved in inhibiting the KCl- and carbachol-evoked [Ca2+](i) transients, including voltage-dependent Ca2+ channels, a site associated with the caffeine-sensitive Ca2+ store and a possible site associated with the IP(3)-sensitive Ca2+ store, and a site in the muscarinic pathway. K+ channels, but not Na+ channels, seem to modulate the evoked [Ca2+](i) transients, as well as the LA-effects on such responses.

scholarly journals N-Arachidonoyl l-Serine, a Putative Endocannabinoid, Alters the Activation of N-Type Ca2+ Channels in Sympathetic Neurons

2008 ◽  
Vol 100 (2) ◽  
pp. 1147-1151 ◽  
Author(s):  
Juan Guo ◽  
Damian J. Williams ◽  
Stephen R. Ikeda
Keyword(s):  
G Protein ◽  
Sympathetic Neurons ◽  
G Protein Coupled Receptors ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Nervous System Function ◽  
Ganglion Neurons ◽  
G Protein Coupled ◽  
Hyperpolarizing Shift ◽  
Ca2 Channels

The effect of N-arachidonoyl l-serine (ARA-S), a recently discovered lipoamino acid found in the CNS, on N-type Ca2+ channels of rat sympathetic ganglion neurons was determined using whole cell patch clamp. Application of ARA-S produced a rapid and reversible augmentation of Ca2+ current that was voltage dependent and resulted from a hyperpolarizing shift in the activation curve. ARA-S did not influence G protein modulation of Ca2+ channels and appeared to act independently of G-protein-coupled receptors. These findings provide a foundation for investigating possible roles for ARA-S in nervous system function.

Stimulation of voltage-dependent Ca2+ channels by NO at rat myenteric neurons

2007 ◽  
Vol 293 (4) ◽  
pp. G886-G893 ◽  
Author(s):  
Mabruka Sitmo ◽  
Matthias Rehn ◽  
Martin Diener
Keyword(s):  
Enteric Neurons ◽  
Channel Blockers ◽  
No Donor ◽  
Inward Current ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp ◽  
Target Organs ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Ca2 Channels

The aim of the present study was to characterize the action of the neurotransmitter NO on rat myenteric neurons. A NO donor such as GEA 3162 (10−4 mol/l) induced an increase in the intracellular Ca2+ concentration as indicated by an increase in the fura 2 ratio in ganglia loaded with this Ca2+-sensitive fluorescent dye. The effect of GEA 3162 was strongly reduced in the absence of extracellular Ca2+, suggesting an influx of Ca2+ from the extracellular space evoked by NO. A similar nearly complete inhibition was observed in the presence of Ca2+ channel blockers such as Ni2+ (5 × 10−4 mol/l) or nifedipine (10−6 mol/l). Whole cell patch-clamp recordings confirmed the activation of voltage-dependent Ca2+ channels, measured as inward current carried by Ba2+, by the NO donor. The peak Ba2+-carried inward current increased from −100 ± 19 to −185 ± 34 pA in the presence of sodium nitroprusside (10−4 mol/l). The consequence was a hyperpolarization of the membrane, which was blocked by intracellular Cs+ and thus most probably reflects the activation of Ca2+-dependent K+ channels. Furthermore, at least two subtypes of NO synthases, NOS-1 (neuronal form) and NOS-3 (endothelial form), were found as transcripts in mRNA isolated from the rat myenteric ganglia. The expression of these NO synthases was confirmed immunohistochemically. These observations suggest that NO, released from nitrergic neurons within the enteric nervous system, not only affects target organs such as smooth muscle cells in the gut but has in addition profound effects on the enteric neurons themselves, the key players in the regulation of many gastrointestinal functions.

High-Voltage-Activated Calcium Currents in Neurons Acutely Isolated From the Ventrobasal Nucleus of the Rat Thalamus

1997 ◽  
Vol 77 (1) ◽  
pp. 465-475 ◽  
Author(s):  
Paul J. Kammermeier ◽  
Stephen W. Jones
Keyword(s):  
High Voltage ◽  
Low Voltage ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Protein Activation ◽  
Whole Cell Patch Clamp ◽  
G Protein Activation ◽  
Channel Blocking ◽  
Voltage Dependent ◽  
Ventrobasal Nucleus

Kammermeier, Paul J. and Stephen W. Jones. High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. J. Neurophysiol. 77: 465–475, 1997. We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to −20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 μM) reversibly blocked 33 ± 1% (mean ± SE), and ω-conotoxin GVIA (1 μM) irreversibly blocked 25 ± 5%. The current resistant to DHPs and ω-conotoxin GVIA was inhibited almost completely by ω-conotoxin MVIIC (90 ± 5% at 3–5 μM) and was partially inhibited by ω-agatoxin IVA (54 ± 4% block at 1 μM). We conclude that there are at least four main HVA currents in thalamic neurons: N current, L current, and two ω-conotoxin MVIIC-sensitive currents that differ in their sensitivity to ω-agatoxin IVA. We also examined modulation of HVA currents by strong depolarization and by G protein activation. Long (∼1 s), strong depolarizations elicited large, slowly deactivating tail currents, which were sensitive to DHP antagonists. With guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) in the intracellular solution, brief (∼20 ms), strong depolarization produced a voltage-dependent facilitation of the current (44 ± 5%), compared with cells with GTP (22 ± 7%) or guanosine 5′-O-(2-thiodiphosphate) (7 ± 4%). However, the HVA current was inhibited only weakly by 100 μM acetylcholine (8 ± 4%). Effects of the γ-aminobutyric acid-B agonist baclofen were variable (3–39% inhibition, n = 12, at 10–50 μM).

Sphingolipid actions on sodium and calcium currents of rat ventricular myocytes

1996 ◽  
Vol 270 (2) ◽  
pp. C645-C649 ◽  
Author(s):  
K. Yasui ◽  
P. Palade
Keyword(s):  
Positive Charge ◽  
Ventricular Myocytes ◽  
Inhibitory Effect ◽  
Calcium Currents ◽  
Rat Ventricular Myocytes ◽  
Bulk Surface ◽  
Hydrocarbon Chains ◽  
Voltage Dependent ◽  
Ca2 Channels ◽  
Net Positive Charge

Sphingosine, an endogenous phospholipid known to produce significant decreases in myoplasmic Ca2+ transients, was shown to have a pronounced inhibitory effect on inward Na+ and L-type Ca2+ currents in rat ventricular myocytes. Sphingosine action was accompanied by a slowing of inactivation of both kinds of current. Both sphingosine and sphingosylphosphorylcholine (SPC) caused depolarizing shifts in the activation curves for the two channels. In tests on Ca2+ currents, sphingosine neither showed high affinity for inactivated states nor exhibited any use dependence. The mechanism of the blocking action of sphingosine does not appear to involve effects on bulk surface charge or, at least for Ca2+ channels, a voltage-dependent block. Instead, the results appear most consistent with an effect of sphingosine on channel gating. The shift in the voltage dependence of channel activation by sphingosine and SPC appears likely to be a feature of both the hydrocarbon chains and the net positive charge of these amphiphiles.

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