Temperature-sensitive intracellular Mg2+ block of L-type Ca2+ channels in cardiac myocytes

2002 ◽  
Vol 282 (3) ◽  
pp. H1092-H1101 ◽  
Author(s):  
Kaoru Yamaoka ◽  
Tsunetsugu Yuki ◽  
Kayoko Kawase ◽  
Makoto Munemori ◽  
Issei Seyama
Keyword(s):  
Cardiac Myocytes ◽  
Late Phase ◽  
Slow Phase ◽  
Temperature Sensitive ◽  
Patch Clamp Technique ◽  
Rapid Phase ◽  
Channel Current ◽  
Whole Cell Patch Clamp ◽  
Blocking Effects ◽  
Ca2 Channels

We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current ( I Ca) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTPi). The late phase of the I Ca increase (late I Ca) was suppressed by GTPi (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24°C, respectively) rather than lower temperatures (24 or 17.5°C, respectively). At pMg = 6.0, raising the temperature from 24 to 32°C evoked late I Ca with a Q10 of 14.5. Restoring the temperature to 24°C decreased I Ca with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I Ca (pMg = 5–6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I Ca. This effect of phosphorylation together with the inhibitory action of GTPi on Mg2+-dependent blocking of I Ca are common properties of mammalian and amphibian cardiomyocytes.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle

1992 ◽  
Vol 262 (3) ◽  
pp. C691-C700 ◽  
Author(s):  
F. Vogalis ◽  
N. G. Publicover ◽  
K. M. Sanders
Keyword(s):  
Time Course ◽  
Fast Component ◽  
Patch Clamp Technique ◽  
Similar Time ◽  
Whole Cell Patch Clamp ◽  
Gastric Smooth Muscle ◽  
Double Exponential ◽  
Circular Layer ◽  
Double Exponential Function ◽  
Ca2 Channels

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Ca2+ Currents in Central Insect Neurons: Electrophysiological and Pharmacological Properties

1997 ◽  
Vol 77 (1) ◽  
pp. 186-199 ◽  
Author(s):  
Dieter Wicher ◽  
Heinz Penzlin
Keyword(s):  
Periplaneta Americana ◽  
Low Voltage ◽  
Fast Time ◽  
Channel Blockers ◽  
Pharmacological Properties ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Blocking Effects ◽  
Dum Neurons ◽  
Maximal Peak

Wicher, Dieter, and Heinz Penzlin. Ca2+ currents in central insect neurons: electrophysiological and pharmacological properties. J. Neurophysiol. 77: 186–199, 1997. Ca2+ currents in dorsal unpaired median (DUM) neurons isolated from the fifth abdominal ganglion of the cockroach Periplaneta americana were investigated with the whole cell patch-clamp technique. On the basis of kinetic and pharmacological properties, two different Ca2+ currents were separated in these cells: mid/low-voltage-activated (M-LVA) currents and high-voltage-activated (HVA) currents. M-LVA currents had an activation threshold of −50 mV and reached maximal peak values at −10 mV. They were sensitive to depolarized holding potentials and decayed very rapidly. The decay was largely Ca2+ dependent. M-LVA currents were effectively blocked by Cd2+ median inhibiting concentration (IC50 = 9 μM), but they also had a remarkable sensitivity to Ni2+ (IC50 = 19 μM). M-LVA currents were insensitive to vertebrate LVA channel blockers like flunarizine and amiloride. The currents were, however, potently blocked by ω-conotoxin MVIIC (1 μM) and ω-agatoxin IVA (50 nM). The blocking effects of ω-toxins developed fast (time constant τ = 15 s) and were fully reversible after wash. HVA currents activated positive to −30 mV and showed maximal peak currents at +10 mV. They were resistant to depolarized holding potentials up to −50 mV and decayed in a less pronounced manner than M-LVA currents. HVA currents were potently blocked by Cd2+ (IC50 = 5 μM) but less affected by Ni2+ (IC50 = 40 μM). These currents were reduced by phenylalkylamines like verapamil (10 μM) and benzothiazepines like diltiazem (10 μM), but they were insensitive to dihydropyridines like nifedipine (10 μM) and BAY K 8644 (10 μM). Furthermore, HVA currents were sensitive to ω-conotoxin GVIA (1 μM). The toxin-induced reduction of currents appeared slowly (τ ∼ 120 s) and the recovery after wash was incomplete in most cases. The dihydropyridine insensitivity of the phenylalkylamine-sensitive HVA currents is a property the cockroach DUM cells share with other invertebrate neurons. Compared with Ca2+ currents in vertebrates, the DUM neuron currents differ considerably from the presently known types. Although there are some similarities concerning kinetics, the pharmacological profile of the cockroach Ca2+ currents especially is very different from profiles already described for vertebrate currents.

scholarly journals Differential Interactions of Na+ Channel Toxins with T-type Ca2+ Channels

2008 ◽  
Vol 132 (1) ◽  
pp. 101-113 ◽  
Author(s):  
Hui Sun ◽  
Diego Varela ◽  
Denis Chartier ◽  
Peter C. Ruben ◽  
Stanley Nattel ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Low Voltage ◽  
Parallel Evolution ◽  
Na Channel ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
The Right ◽  
Ca2 Channels

Two types of voltage-dependent Ca2+ channels have been identified in heart: high (ICaL) and low (ICaT) voltage-activated Ca2+ channels. In guinea pig ventricular myocytes, low voltage–activated inward current consists of ICaT and a tetrodotoxin (TTX)-sensitive ICa component (ICa(TTX)). In this study, we reexamined the nature of low-threshold ICa in dog atrium, as well as whether it is affected by Na+ channel toxins. Ca2+ currents were recorded using the whole-cell patch clamp technique. In the absence of external Na+, a transient inward current activated near −50 mV, peaked at −30 mV, and reversed around +40 mV (HP = −90 mV). It was unaffected by 30 μM TTX or micromolar concentrations of external Na+, but was inhibited by 50 μM Ni2+ (by ∼90%) or 5 μM mibefradil (by ∼50%), consistent with the reported properties of ICaT. Addition of 30 μM TTX in the presence of Ni2+ increased the current approximately fourfold (41% of control), and shifted the dose–response curve of Ni2+ block to the right (IC50 from 7.6 to 30 μM). Saxitoxin (STX) at 1 μM abolished the current left in 50 μM Ni2+. In the absence of Ni2+, STX potently blocked ICaT (EC50 = 185 nM) and modestly reduced ICaL (EC50 = 1.6 μM). While TTX produced no direct effect on ICaT elicited by expression of hCaV3.1 and hCaV3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni2+ (IC50 increased to 550 μM Ni2+ for CaV3.1 and 15 μM Ni2+ for CaV3.2); in contrast, 30 μM TTX directly inhibited hCaV3.3-induced ICaT and the addition of 750 μM Ni2+ to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni2+ alone. 1 μM STX directly inhibited CaV3.1-, CaV3.2-, and CaV3.3-mediated ICaT but did not enhance the ability of Ni2+ to block these currents. These findings provide important new implications for our understanding of structure–function relationships of ICaT in heart, and further extend the hypothesis of a parallel evolution of Na+ and Ca2+ channels from an ancestor with common structural motifs.

Possible contribution of long open state to noninactivating Ca2+ current in detrusor cells

1995 ◽  
Vol 269 (1) ◽  
pp. C48-C54 ◽  
Author(s):  
S. Nakayama ◽  
A. F. Brading
Keyword(s):  
Smooth Muscles ◽  
Activation Parameters ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Voltage Range ◽  
Channel Current ◽  
Whole Cell Patch Clamp ◽  
Dependent Inactivation ◽  
Window Current ◽  
Isolated Smooth Muscle Cells

The whole cell patch-clamp technique was used to measure Ca2+ current in isolated smooth muscle cells from guinea pig urinary bladder. Noniactivating Ca2+ channel current was modeled incorporating the long open state of the Ca2+ channel. When inactivation was examined over a wide voltage range, a completely U-shaped curve was obtained. Lack of inactivation at +80 mV could be attributed to the long open state induced by large depolarization as well as to minimal Ca2+ influx and Ca(2+)-dependent inactivation. Activation parameters were obtained by comparing the amplitudes of conditioned (by +80 mV, 5 s) and unconditioned test potentials. With the use of the activation curve and the U-shaped inactivation curve, a noninactivating current that peaks around +20 mV was obtained. This current is composed of a so-called “window” current and a persistent current brought about by the long open state. Differences in the voltage dependence of the development of the long open state in various smooth muscles, as well as differences in the equilibrium constant between open and inactivated states, could underlie the different patterns of contractile behavior that characterize smooth muscles.

Fentanyl Decreases Ca2+Currents in a Population of Capsaicin-responsive Sensory Neurons

2003 ◽  
Vol 98 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Thomas S. McDowell
Keyword(s):  
Neurotransmitter Release ◽  
Dorsal Root ◽  
Dorsal Root Ganglion Neurons ◽  
Patch Clamp Technique ◽  
Opioid Agonist ◽  
Excitatory Neurotransmitter ◽  
Nociceptive Neurons ◽  
Whole Cell Patch Clamp ◽  
Ganglion Neurons ◽  
Ca2 Channels

Background Neuraxial opioids produce analgesia in part by decreasing excitatory neurotransmitter release from primary nociceptive neurons, an effect that may be due to inhibition of presynaptic voltage-activated Ca2+ channels. The purpose of this study was to determine whether opioids decrease Ca2+ currents (I Ca ) in primary nociceptive neurons, identified by their response to the algogenic agent capsaicin. Methods I was recorded from acutely isolated rat dorsal root ganglion neurons using the whole cell patch clamp technique before, during, and after application of the micro -opioid agonist fentanyl (0.01-1 micro m). Capsaicin was applied to each cell at the end of the experiment. Results Fentanyl reduced I Ca in a greater proportion of capsaicin-responsive cells (62 of 106, 58%) than capsaicin-unresponsive cells (2 of 15, 13%; P &lt; 0.05). Among capsaicin-responsive cells, the decrease in I Ca was 38 +/- 3% (n = 36, 1 micro m) in fentanyl-sensitive cells just 7 +/- 1% (n = 15, 1 micro m; P &lt; 0.05) in fentanyl-insensitive cells. Among capsaicin-responsive cells, I Ca inactivated more rapidly in fentanyl-sensitive cells (tau, 52 +/- 4 ms, n = 22) than in fentanyl-insensitive cells (93 +/- 14 ms, n = 24; P &lt; 0.05). This was not due to differences in the types of Ca2+ channels expressed as the magnitudes of omega-conotoxin GVIA-sensitive (N-type), nifedipine-sensitive (L-type), and GVIA/nifedipine-resistant (primarily P-/Q-type) components of I Ca were similar. Conclusions The results show that opioid-sensitive Ca2+ channels are expressed by very few capsaicin-unresponsive neurons but by more than half of capsaicin-responsive neurons. The identity of the remaining capsaicin-responsive (and therefore presumed nociceptive) neurons that express opioid-insensitive Ca2+ channels is unknown but may represent a potential target of future non-opioid-based therapies for acute pain.

Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

2008 ◽  
Vol 294 (2) ◽  
pp. C572-C578 ◽  
Author(s):  
C. N. White ◽  
E. J. Hamilton ◽  
A. Garcia ◽  
D. Wang ◽  
K. K. M. Chia ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Cardiac Myocytes ◽  
Pump Current ◽  
No Synthase ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Patch Pipette ◽  
Pump Activity ◽  
Opposing Effects

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na+-K+ pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become “uncoupled” and preferentially synthesize O2·−, which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na+-K+ pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. l-Arginine (l-Arg; 500 μmol/l) increased DAF-2 DA fluorescence by 51% compared with control ( n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na+-K+ pump current ( Ip). Mean Ip of 0.35 ± 0.03 pA/pF ( n = 44) was increased to 0.48 ± 0.03 pA/pF ( n = 7, P < 0.05) by 10 μmol/l l-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1 H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O2·−-sensitive dye dihydroethidium compared with control ( n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured Ip with 100 μmol/l paraquat included in patch pipette solutions. This decreased Ip to 0.28 ± 0.03 pA/pF ( n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na+-K+ pump, whereas uncoupling of NOS causes O2·−-mediated pump inhibition.

Dietary cholesterol affects Na+-K+ pump function in rabbit cardiac myocytes

1997 ◽  
Vol 272 (4) ◽  
pp. H1680-H1689 ◽  
Author(s):  
D. F. Gray ◽  
P. S. Hansen ◽  
M. M. Doohan ◽  
L. C. Hool ◽  
H. H. Rasmussen
Keyword(s):  
Cardiac Myocytes ◽  
Serum Cholesterol ◽  
Dietary Cholesterol ◽  
Membrane Cholesterol ◽  
Patch Clamp Technique ◽  
Apparent Affinity ◽  
Pump Function ◽  
Whole Cell Patch Clamp

Alterations in membrane cholesterol induced in vitro can alter Na+-K+ pump function. Because dietary cholesterol can influence membrane cholesterol in vivo, we examined if dietary cholesterol is a determinant of Na+-K+ pump function. Rabbits were fed cholesterol-supplemented diets for 1-4 wk. Cardiac myocytes were then isolated, and Na+-K+ pump currents (Ip) were measured using the whole cell patch-clamp technique. When the Na+ concentration in the patch pipettes ([Na]pip) was 10 mM, a modest diet-induced increase in serum cholesterol was associated with stimulation of Ip; large increases in serum cholesterol were associated with inhibition. There was no effect of modest or large increases in serum cholesterol on Ip when [Na]pip was 80 mM. The [Na]pip-Ip relationship determined using seven different levels of [Na]pip from 0 to 80 mM indicated that a modest increase in serum cholesterol increased the apparent affinity of the pump for cytoplasmic Na+. In contrast, dietary cholesterol had no effect on the apparent affinity of the pump for extracellular K+. We conclude that cholesterol intake influences the sarcolemmal Na+-K+ pump. This may have clinical implications for cardiovascular function.

scholarly journals Heterologous expression of BI Ca2+ channels in dysgenic skeletal muscle.

1994 ◽  
Vol 104 (5) ◽  
pp. 985-996 ◽  
Author(s):  
B A Adams ◽  
Y Mori ◽  
M S Kim ◽  
T Tanabe ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Time Constant ◽  
Ionic Current ◽  
Ionic Currents ◽  
Rabbit Brain ◽  
Charge Movement ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell Patch Clamp ◽  
Ca2 Channels

We have examined the ability of BI (class A) Ca2+ channels, cloned from rabbit brain, to mediate excitation-contraction (E-C) coupling in skeletal muscle. Expression plasmids carrying cDNA encoding BI channels were microinjected into the nuclei of dysgenic mouse myotubes grown in primary culture. Ionic currents and intramembrane charge movements produced by the BI channels were recorded using the whole-cell patch-clamp technique. Injected myotubes expressed high densities of ionic BI Ca2+ channel current (average 31 pA/pF) but did not display spontaneous contractions, and only very rarely displayed evoked contractions. The expressed ionic current was pharmacologically distinguished from the endogenous L-type current of dysgenic skeletal muscle (Idys) by its insensitivity to the dihydropyridine antagonist (+)-PN 200-110. Peak BI Ca2+ currents activated with a time constant (tau a) of approximately 2 ms and inactivated with a time constant (tau h) of approximately 260 ms (20-23 degrees C). The time constant of inactivation (tau h) was not increased by substituting Ba2+ for Ca2+ as charge carrier, demonstrating that BI channels expressed in dysgenic myotubes do not undergo Ca(2+)-dependent inactivation. The average maximal Ca2+ conductance (Gmax) produced by the BI channels was quite large (approximately 534 S/F). In contrast, the average maximal charge movement (Qmax) produced in the same myotubes (approximately 2.7 nC/microF) was quite small, being barely larger than Qmax in control dysgenic myotubes (approximately 2.3 nC/microF). Thus, the ratio Gmax/Qmax for the BI channels was considerably higher than previously found for cardiac or skeletal muscle L-type Ca2+ channels expressed in the same system, indicating that neuronal BI Ca2+ channels exhibit a much higher open probability than these L-type Ca2+ channels.

Comparison of Ca2+ currents of peptidergic neurons developing differing morphology with time in culture.

1997 ◽  
Vol 200 (4) ◽  
pp. 723-733
Author(s):  
D E Meyers ◽  
I M Cooke
Keyword(s):  
Defined Medium ◽  
Small Cells ◽  
Neurosecretory System ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Mature Neurons ◽  
Average Current ◽  
Culturing Conditions ◽  
Ca2 Channels

The whole-cell patch-clamp technique was used to examine Ca2+ currents (ICa) in mature neurons cultured in defined medium and derived from the principal neurosecretory system of decapod crustaceans, the X-organ-sinus gland. After 1 day in culture, X-organ neurons of the crab Cardisoma carnifex showed vigorous outgrowth characterized either by the production of broad lamellipodia (veils) or, from smaller somata, a branching morphology. The neurons developing veils (veilers) had a large ICa (approximately 650 pA) and ICa current density (approximately 5 microA cm-2) while other types of neuron had little or no ICa. This distinction between the two types was still present after 5-6 days in culture. However, morphologies observed after additional outgrowth, when correlated with the ICa responses, allowed four groups to be distinguished: (1) veilers and (2) branching veilers, which developed from veilers and had a similar ICa density (approximately 3 microA cm-2); and, developing from the 1 day branchers, (3) spiny branchers or (4) small cells (ICa density approximately 0.8 microA cm-2). Immunoreactivity indicative of the presence of crustacean hyperglycemic hormone was found in all veilers and branching veilers tested, while moltinhibiting hormone reactivity, when observed, was seen in cells having a robust ICa density (&gt; or = 1.2 microA cm-2). Normalized average current-voltage curves for each morphological group were examined for changes with increasing time in culture. The curves were consistent with the ICa being produced by a population of high-voltage-activated Ca2+ channels whose properties are biophysically indistinguishable and unaffected by time in culture. The averaged peak current did not change, despite an increase in neuronal surface area as outgrowth proceeded, and this resulted in a reduction of ICa density. This indicated that net addition of Ca2+ channels did not match the addition of new membrane under our culturing conditions.

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