scholarly journals Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium.

1994 ◽  
Vol 104 (5) ◽  
pp. 821-856 ◽  
Author(s):  
I Bezprozvanny ◽  
B E Ehrlich
Keyword(s):  
Ryanodine Receptor ◽  
Single Channel ◽  
Divalent Cations ◽  
Reversal Potential ◽  
Ca Channel ◽  
Ca Channels ◽  
Ca Current ◽  
Open Probability ◽  
Inositol 1,4,5 Trisphosphate ◽  
Conduction Properties

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.

Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells

1992 ◽  
Vol 262 (2) ◽  
pp. C453-C460 ◽  
Author(s):  
K. Naruse ◽  
D. S. McGehee ◽  
G. S. Oxford
Keyword(s):  
Intracellular Signaling ◽  
Single Channel ◽  
Outward Current ◽  
Transient Outward Current ◽  
Ca Channel ◽  
Ca Channels ◽  
Drg Neurons ◽  
Open Probability ◽  
K Channels ◽  
Drg Neuron

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

scholarly journals Nicotinic acid–adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor

2002 ◽  
Vol 367 (2) ◽  
pp. 423-431 ◽  
Author(s):  
Martin HOHENEGGER ◽  
Josef SUKO ◽  
Regina GSCHEIDLINGER ◽  
Helmut DROBNY ◽  
Andreas ZIDAR
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Nicotinic Acid ◽  
Ryanodine Receptor ◽  
Spatial Information ◽  
Single Channel ◽  
Reversal Potential ◽  
Open Probability ◽  
Release Channel

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca2+-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca2+-release from intracellular Ca2+ stores can be triggered by diffusible second messengers like InsP3, cyclic ADP-ribose or nicotinic acid—adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca2+-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca2+-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca2+-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC5030nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

Expression of single calcium channels in Xenopus oocytes after injection of mRNA from rat heart

1987 ◽  
Vol 253 (4) ◽  
pp. H985-H991 ◽  
Author(s):  
J. R. Moorman ◽  
Z. Zhou ◽  
G. E. Kirsch ◽  
A. E. Lacerda ◽  
J. M. Caffrey ◽  
...  
Keyword(s):  
Rat Heart ◽  
Single Channel ◽  
Reversal Potential ◽  
Mean Amplitude ◽  
High Threshold ◽  
Ca Channel ◽  
Ca Channels ◽  
Adult Rat ◽  
Average Current ◽  
Single Calcium Channels

Oocytes of Xenopus laevis, after microinjection with mRNA from rat heart, display typical high-threshold calcium (Ca) whole cell currents. To prepare to study structure-function relationships of the cardiac Ca channel molecule, we examined the fidelity of expression of biophysical and pharmacological properties at the molecular level. Cell-attached gigaseal recordings in five K-depolarized oocytes injected with adult rat heart mRNA showed single channel Ba currents with mean amplitude 1.3-1.5 pA at 0 mV, slope conductance 18-25 pS, and extrapolated reversal potential 57-68 mV. Openings were predominantly brief (mean 1.2 ms) but longer openings (mean 9 ms) were greatly enhanced in 10(-6) M BAY-K 8644, increasing the ensemble average current at 0 mV by more than fivefold. These features are typical of high-threshold cardiac Ca channels. In two patches from one injected oocyte, we saw multiple Ca channel conductances, as recently observed in other preparations. We conclude that X. laevis oocytes injected with adult rat heart mRNA produce high-threshold cardiac Ca channels with molecular properties identical to native cells.

Ca2+ influx through the osteoclastic plasma membrane ryanodine receptor

2002 ◽  
Vol 282 (5) ◽  
pp. F921-F932 ◽  
Author(s):  
Baljit S. Moonga ◽  
Sun Li ◽  
Jameel Iqbal ◽  
Robert Davidson ◽  
Vijai S. Shankar ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Divalent Cation ◽  
Single Channel ◽  
Divalent Cations ◽  
Reversal Potential ◽  
Basal Membrane ◽  
Ruthenium Red ◽  
Membrane Potentials ◽  
Excised Patches

We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a Ca2+ influx channel and as a divalent cation (Ca2+) sensor. Cytosolic Ca2+ measurements revealed Ca2+ influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca2+ change was, as expected, not seen in Ca2+-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (∼25 mV) ryanodine triggered extracellular Ca2+ influx that was blocked by Ni2+. In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective ∼60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential ( E rev) ∼0 mV]. In the presence of a Ba2+ gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ∼120 pS and E rev shifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca2+ channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab34. To demonstrate that extracellular Ca2+ sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic Ca2+ signals triggered by either Ni2+(5 mM) or Cd2+ (50 μM). Finally, intracellular application of antiserum Ab34 potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca2+ influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates Ca2+ influx but also functions as a sensor for extracellular divalent cations.

Carbachol modulates voltage sensitivity of calcium channels in bronchial smooth muscle of rats

1992 ◽  
Vol 263 (1) ◽  
pp. C69-C77 ◽  
Author(s):  
T. Kamishima ◽  
M. T. Nelson ◽  
J. B. Patlak
Keyword(s):  
Smooth Muscle ◽  
Channel Blocker ◽  
Single Channel ◽  
Membrane Depolarization ◽  
Bay K 8644 ◽  
Voltage Sensitivity ◽  
Ca Channel ◽  
Ca Channels ◽  
Open Probability ◽  
Voltage Dependent

The role of voltage-dependent Ca channels in carbachol (CCh)-induced contraction of rat bronchus was investigated. Membrane depolarization and BAY K 8644, a Ca channel opener, significantly enhanced CCh-induced contractions. Nisoldipine, an organic Ca channel blocker, significantly inhibited the contractions. Cadmium, an inorganic Ca channel blocker, completely inhibited maintained contractions caused by CCh. These results suggested that the voltage-dependent Ca channels play an important role in sustained cholinergic contractions. This hypothesis was tested further by investigating the properties of single Ca channels of rat bronchus smooth muscle cells. We used 10 mM Ba as the charge carrier and BAY K 8644 to increase open times. The single-channel conductance was 16.8 pS. Steady-state open probability (NP(o)) increased steeply with membrane depolarization (e-fold for 4 mV). The primary effect of CCh (10 microM) on Ca channels was to shift the membrane potential at which NP(o) was half maximal from -34 to -43 mV without changing the steepness factor or maximal NP(o). This CCh-induced increase in NP(o) was not caused by depolarization, because the single-channel current amplitude was unchanged by CCh. We conclude that one of the mechanisms by which CCh opens Ca channels of rat bronchus smooth muscle is by shifting the activation curve in the hyperpolarized direction.

scholarly journals Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology.

1985 ◽  
Vol 86 (1) ◽  
pp. 1-30 ◽  
Author(s):  
B P Bean
Keyword(s):  
Single Channel ◽  
Cardiac Cells ◽  
Fluctuation Analysis ◽  
Ca Channel ◽  
Ca Channels ◽  
Ca Current ◽  
Channel Current ◽  
Atrial Cells ◽  
Cell Recording ◽  
Ca Channel Current

Currents through Ca channels were recorded in single canine atrial cells using whole-cell recording with patch pipettes. Two components of Ca channel current could be distinguished. One ("Ifast") was present only if cells were held at negative potentials, was most prominent for relatively small depolarizations, and inactivated within tens of milliseconds. The other ("Islow"), corresponding to the Ca current previously reported in single cardiac cells, persisted even at relatively positive holding potentials, required stronger depolarizations for maximal current, and inactivated much more slowly. Both currents were unaffected by tetrodotoxin and both were reduced by Co. Ifast had the same size and kinetics when Ca was exchanged for Ba, while Islow was bigger and slower with Ba as the charge carrier. In isotonic BaCl2, fluctuation analysis showed that Ifast had a smaller single channel current than Islow. Islow was much more sensitive to block by nitrendipine than was Ifast; also, Islow, but not Ifast, was increased by the dihydropyridine drug BAY K8644. Isoproterenol produced large increases in Islow but had no effect on Ifast.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

scholarly journals Caffeine-induced inhibition of inositol(1,4,5)-trisphosphate-gated calcium channels from cerebellum.

1994 ◽  
Vol 5 (1) ◽  
pp. 97-103 ◽  
Author(s):  
I Bezprozvanny ◽  
S Bezprozvannaya ◽  
B E Ehrlich
Keyword(s):  
Lipid Bilayers ◽  
Inhibitory Action ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Single Channels ◽  
Ca Channels ◽  
Open Time ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insp3 Receptors ◽  
Intracellular Ca

Effects of the xanthine drug caffeine on inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels from canine cerebellum were studied using single channels incorporated into planar lipid bilayers. Caffeine, used widely as an agonist of ryanodine receptors, inhibited the activity of InsP3-gated Ca channels in a noncooperative fashion with half-inhibition at 1.64 mM caffeine. The frequency of channel openings was decreased more than threefold after addition of 5 mM caffeine; there was only a small effect on mean open time of the channels, and the single channel conductance was unchanged. Increased InsP3 concentration overcame the inhibitory action of caffeine, but caffeine did not reduce specific [3H]InsP3 binding to the receptor. The inhibitory action of caffeine on InsP3 receptors suggests that the action of caffeine on the intracellular Ca pool must be interpreted with caution when both ryanodine receptors and InsP3 receptors are present in the cell.

Ryanodine receptor dysfunction in porcine stunned myocardium

1997 ◽  
Vol 273 (2) ◽  
pp. H796-H804 ◽  
Author(s):  
C. Valdivia ◽  
J. O. Hegge ◽  
R. D. Lasley ◽  
H. H. Valdivia ◽  
R. Mentzer
Keyword(s):  
Coronary Artery ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Myocardial Stunning ◽  
Single Channel ◽  
Stunned Myocardium ◽  
Wall Thickening ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

Enhanced single-channel K+ current in arterial membranes from genetically hypertensive rats

1993 ◽  
Vol 264 (5) ◽  
pp. H1337-H1345 ◽  
Author(s):  
S. K. England ◽  
T. A. Wooldridge ◽  
W. J. Stekiel ◽  
N. J. Rusch
Keyword(s):  
Single Channel ◽  
Muscle Cells ◽  
Ca Channel ◽  
Ca Channels ◽  
Hypertensive Rats ◽  
Genetically Hypertensive Rats ◽  
K Current ◽  
Inside Out ◽  
Genetically Hypertensive ◽  
Arterial Muscle

Arterial smooth muscle from hypertensive rats shows an increased membrane permeability to K+ that depends on Ca2+ influx. To define the mechanism of this membrane alteration, we tested the hypothesis that Ca(2+)-dependent K+ current (IK(Ca)) is increased in arterial muscle membranes from genetically hypertensive rats. Single-channel K+ currents measured in cell-attached and inside-out aortic membrane patches from spontaneously hypertensive rats (SHR) were compared with those from normotensive Wistar-Kyoto rats (WKY). Inside-out patches from both rat strains showed a predominant 225 pS, Ca(2+)- and voltage-dependent K+ channel in symmetrical 145 mM KCl solutions, which was blocked by tetraethylammonium [concentration for half-maximal block (IC50) < or = 0.3 mM]. In cell-attached patches of aortic muscle cells bathed in physiological salt solution, this channel [IK(Ca) channel] showed a fivefold higher open-state probability (NPo) in SHR as compared with WKY. This increased NPo of SHR IK(Ca) channels in membranes of intact aortic muscle cells was not correlated with an altered membrane potential in current-clamped SHR myocytes or with changes in cytosolic free Ca2+ concentration in fura-2-loaded aortic muscle cells. However, inside-out aortic membrane patches from SHR showed more detected IK(Ca) channels per patch, a higher IK(Ca) channel NPo, and a greater total patch current than their WKY counterparts. Further analysis revealed a greater Ca2+ sensitivity of SHR than WKY IK(Ca) channels. These results suggest that IK(Ca) channel function is altered in isolated membrane patches of arterial muscle from genetically hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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