Functional sensitivity of the native skeletal Ca2+-release channel to divalent cations and the Mg–ATP complex

1992 ◽  
Vol 70 (3) ◽  
pp. 394-402 ◽  
Author(s):  
Eric Rousseau ◽  
Janet Pinkos ◽  
Diane Savaria
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Divalent Cation ◽  
Single Channel ◽  
Adenine Nucleotides ◽  
Buffer System ◽  
Open Probability ◽  
Release Channel ◽  
Gating Mechanism

Sarcoplasmic reticulum (SR) vesicles, prepared from rabbit skeletal muscle, were characterized by functional and binding assays and incorporated into planar lipid bilayers. Single-channel activity was recorded in an asymmetric calcium buffer system and studied under voltage clamp conditions. Under these experimental conditions, a large conductance (100 pS in 50 mM Ca2+trans) divalent cation selective channel displaying high ruthenium red and low Ca2+ sensitivity was identified. This pathway has been previously described as the Ca2+-release channel of the SR of skeletal muscle. We now report that in the presence of a Mg–ATP complex, the Ca2+ sensitivity of the open probability of this channel is increased. Furthermore, we show that micromolar cis Sr2+ concentrations also activated the Ca2+-release channel. The open probability of the Sr2+-activated channel was increased in the presence of a 2 mM Mg–ATP complex and adenine nucleotides on the cytoplasmic face of the Ca2+-release channel. These results were confirmed by isotopic flux measurements using passively 45Ca2+-loaded vesicles. In the latter case, the presence of extra vesicular AMP-PCP (the nonhydroly sable ATP analog) enhanced the percentage of 45Ca2+ release induced either by Ca2+ or Sr2+ activation. In conclusion our findings emphasize the fact that the divalent cation activation of the Ca2+-release channel may be induced by Ca2+ and Sr2+, but not by Ba2+, in the presence of adenine nucleotides. Furthermore, they support the view that in situ Ca2+ and Mg–ATP complexes are involved in modulating the gating mechanism of this specific pathway.Key words: Ca2+ release, sarcoplasmic reticulum, planar lipid bilayer, strontium.

scholarly journals Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation by Ca2+ and ATP and modulation by Mg2+.

1986 ◽  
Vol 88 (5) ◽  
pp. 573-588 ◽  
Author(s):  
J S Smith ◽  
R Coronado ◽  
G Meissner
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Calcium Release ◽  
Single Channel ◽  
Ca Channel ◽  
Channel Measurements ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Release Channel

A high-conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy-density skeletal muscle sarcoplasmic reticulum (SR) fractions into planar lipid bilayers of the Mueller-Rudin type. cis Ca2+ in the range of 2-950 microM increased open probability (Po) in single channel records without affecting open event lifetimes. Millimolar ATP was found to be as good as or better than Ca2+ in activation; however, both Ca2+ and ATP were required to fully activate the channel, i.e., to bring Po = 1. Exponential fits to open and closed single channel lifetimes suggested that the channel may exist in many distinct states. Two open and two closed states were identified when the channel was activated by either Ca2+ or ATP alone or by Ca2+ plus nucleotide. Mg2+ was found to permeate the SR Ca channel in a trans-to-cis direction such that iMg2+/iCa2+ = 0.40. cis Mg2+ was inhibitory and in single channel recordings produced an unresolvable flickering of Ca- and nucleotide-activated channels. At nanomolar cis Ca2+, 4 microM Mg2+ completely inhibited nucleotide-activated channels. In the presence of 2 microM cis Ca2+, the nucleotide-activated macroscopic Ba conductance was inhibited by cis Mg2+ with an IC50 equal to 1.5 mM.

Mechanisms of Pi regulation of the skeletal muscle SR Ca2+ release channel

2000 ◽  
Vol 278 (3) ◽  
pp. C601-C611 ◽  
Author(s):  
Edward M. Balog ◽  
Bradley R. Fruen ◽  
Patricia K. Kane ◽  
Charles F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Adenine Nucleotides ◽  
Muscle Fibers ◽  
Open Probability ◽  
Release Channel ◽  
High Concentrations ◽  
Channel Open Time ◽  
Stimulation Of

Inorganic phosphate (Pi) accumulates in the fibers of actively working muscle where it acts at various sites to modulate contraction. To characterize the role of Pi as a regulator of the sarcoplasmic reticulum (SR) calcium (Ca2+) release channel, we examined the action of Pi on purified SR Ca2+ release channels, isolated SR vesicles, and skinned skeletal muscle fibers. In single channel studies, addition of Pi to the cis chamber increased single channel open probability ( P o; 0.079 ± 0.020 in 0 Pi, 0.157 ± 0.034 in 20 mM Pi) by decreasing mean channel closed time; mean channel open times were unaffected. In contrast, the ATP analog, β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP), enhanced P o by increasing single channel open time and decreasing channel closed time. Pi stimulation of [3H]ryanodine binding by SR vesicles was similar at all concentrations of AMP-PCP, suggesting Pi and adenine nucleotides act via independent sites. In skinned muscle fibers, 40 mM Pi enhanced Ca2+-induced Ca2+ release, suggesting an in situ stimulation of the release channel by high concentrations of Pi. Our results support the hypothesis that Pi may be an important endogenous modulator of the skeletal muscle SR Ca2+ release channel under fatiguing conditions in vivo, acting via a mechanism distinct from adenine nucleotides.

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.

Modification of the gating of the cardiac sarcoplasmic reticulum Ca(2+)-release channel by H2O2 and dithiothreitol

1994 ◽  
Vol 267 (3) ◽  
pp. H1010-H1016 ◽  
Author(s):  
A. Boraso ◽  
A. J. Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Single Channel ◽  
Inhibitory Response ◽  
Dependent Manner ◽  
Open Probability ◽  
Release Channel ◽  
Concentration Dependent Manner ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Gating Mechanism ◽  
Cytosolic Side

The effect of hydrogen peroxide (H2O2) on the sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel has been investigated under voltage-clamp conditions after incorporation of native membrane vesicles into planar phospholipid bilayers. In the presence of micromolar activating calcium concentrations on the cytosolic side of the membrane, H2O2 (3-5 mM) increased open probability of the channels. H2O2 did not affect the conductance of the channel or the response to activating compounds, such as ATP and caffeine. H2O2 did not alter the inhibitory response to magnesium or the modification of channels by ryanodine. At subactivating calcium concentrations (approximately 45 pM) on the cytosolic side of the membrane, 5 mM H2O2 was still able to open the channel. Analysis of single-channel open and closed lifetimes suggested that H2O2 had a direct effect on the gating mechanism of the channel. Open probability of the SR Ca(2+)-release channel is reduced by millimolar concentrations of dithiothreitol, a sulfhydryl-protecting compound, in a concentration-dependent manner. In conclusion, it is probable that H2O2 activates the SR Ca(2+)-release channel via an oxidation of cysteine thiol groups in the channel protein.

Ethanol enhances caffeine-induced Ca2+-release channel activation in skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 272 (2) ◽  
pp. C622-C627 ◽  
Author(s):  
T. Oba ◽  
M. Koshita ◽  
M. Yamaguchi
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Skeletal Muscles ◽  
Single Channel ◽  
Channel Activity ◽  
Electrical Parameters ◽  
Open Probability ◽  
Release Channel ◽  
Channel Current ◽  
Open Time

When sarcoplasmic reticulum (SR) vesicles prepared from frog skeletal muscles were actively loaded with Ca2+, pretreatment of the SR with 2.2 mM (0.01%) ethanol for 30 s significantly potentiated 5 mM caffeine-induced release of Ca2+ from 16.7 +/- 3.7 nmol/mg protein in control without ethanol to 28.0 +/- 2.6 nmol/mg (P < 0.05, n = 5). Ethanol alone caused no release of Ca2+ from the SR. Exposure of the Ca2+-release channel, incorporated into planar lipid bilayers, to 2 mM caffeine significantly increased open probability (Po) and mean open time, but unitary conductance was not affected. Ethanol (2.2 mM) enhanced caffeine-induced Ca2+-release channel activity, with Po reaching 3.02-fold and mean open time 2.85-fold the values in the absence of ethanol. However, ethanol alone did not affect electrical parameters of single-channel current, over a concentration range of 2.2 mM (0.01%) to 217 mM (1%). The synergistic action of ethanol and caffeine on the channel activity could be attributable to enhancement of caffeine-induced release of Ca2+ from the SR vesicles in the presence of ethanol.

A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

1996 ◽  
Vol 270 (6) ◽  
pp. C1675-C1686 ◽  
Author(s):  
J. I. Kourie ◽  
D. R. Laver ◽  
G. P. Ahern ◽  
A. F. Dulhunty
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Chloride Channel ◽  
Single Channel ◽  
Single Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Single Channel Activity ◽  
Maximum Conductance

A Ca(2+)-activated Cl- channel is described in sarcoplasmic reticulum (SR) enriched vesicles of skeletal muscle incorporated into lipid bilayers. Small chloride (SCl) channels (n = 20) were rapidly and reversibly activated when cis- (cytoplasmic) [Ca2+] was increased above 10(-7) M, with trans-(luminal) [Ca2+] at either 10(-3) or 10(-7) M. The open probability of single channels increased from zero when cis-[Ca2+] was 10(-7) M to 0.61 +/- 0.12 when [Ca2+] was 10(-4) M. High- and low-conductance levels in single-channel activity were activated at different cis-[Ca2+]. Channel openings to the maximum conductance, 65-75 pS (250/50 mM Cl-, cis/ trans), were active when cis-[Ca2+] was increased above 5 x 10(-6) M. In contrast to the maximum conductance, channel openings to submaximal levels between 5 and 40 pS were activated at a lower cis-[Ca2+] and dominated channel activity between 5 x 10(-7) and 5 x 10(-6) M. Activation of SCl channels was Ca2+ specific and not reproduced by cytoplasmic Mg2+ concentrations of 10(-3) M. We suggest that the SCl channel arises in the SR membrane. The Ca2+ dependence of this channel implies that it is active at [Ca2+] achieved during muscle contraction.

Ryanodine modifies conductance and gating behavior of single Ca2+ release channel

1987 ◽  
Vol 253 (3) ◽  
pp. C364-C368 ◽  
Author(s):  
E. Rousseau ◽  
J. S. Smith ◽  
G. Meissner
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Ruthenium Red ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Release Channel ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling

Ryanodine affects excitation-contraction coupling in skeletal and cardiac muscle by specifically interacting with the sarcoplasmic reticulum (SR) Ca2+ release channel. The effect of the drug at the single channel level was studied by incorporating skeletal and cardiac SR vesicles into planar lipid bilayers. The two channels were activated by micromolar free Ca2+ and millimolar ATP and inhibited by Mg2+ and ruthenium red. Addition of micromolar concentrations of ryanodine decreased about twofold the unit conductance of the Ca2+- and ATP-activated skeletal and cardiac channels. A second effect of ryanodine was to increase the open probability (Po) of the channels in such a way that Po was close to unity under a variety of activating and inactivating conditions. The effects of ryanodine were long lasting in that removal of ryanodine by perfusion did not return the channels into their fully conducting state.

scholarly journals Interdomain Interactions within Ryanodine Receptors Regulate Ca2+ Spark Frequency in Skeletal Muscle

2001 ◽  
Vol 119 (1) ◽  
pp. 15-32 ◽  
Author(s):  
Alexander Shtifman ◽  
Christopher W. Ward ◽  
Takeshi Yamamoto ◽  
Jianli Wang ◽  
Beth Olbinski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Laser Scanning ◽  
Muscle Fibers ◽  
Mammalian Skeletal Muscle ◽  
Open Probability ◽  
Release Channel ◽  
Pronounced Increase ◽  
Open Time ◽  
Interdomain Interactions

DP4 is a 36-residue synthetic peptide that corresponds to the Leu2442-Pro2477 region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca2+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618–11625). We have investigated the effects of DP4 on local SR Ca2+ release events (Ca2+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca2+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca2+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca2+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca2+ release channel(s) generating the sparks. DP4 also increased [3H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca2+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca2+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg17 to Cys17 replacement (DP4mut), which corresponds to the Arg2458-to-Cys2458 mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg2+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg2+-free RyR(s), thus promoting channel opening and production of Ca2+ sparks.

scholarly journals Interactions of a Reversible Ryanoid (21-Amino-9α-Hydroxy-Ryanodine) with Single Sheep Cardiac Ryanodine Receptor Channels

1998 ◽  
Vol 112 (1) ◽  
pp. 55-69 ◽  
Author(s):  
Bhavna Tanna ◽  
William Welch ◽  
Luc Ruest ◽  
John L. Sutko ◽  
Alan J. Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Single Channel ◽  
Single Channels ◽  
Open Probability ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Channel Protein ◽  
High Affinity ◽  
Conductance State

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 82 (2) ◽  
pp. 447-452 ◽  
Author(s):  
Terence G. Favero ◽  
, Anthony C. Zable ◽  
, David Colter ◽  
Jonathan J. Abramson
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Single Channel ◽  
Channel Activity ◽  
Release Channel ◽  
Muscle Lactate ◽  
Tension Development ◽  
Contraction Coupling ◽  
Single Channel Activity ◽  
High Concentrations

Favero, Terence G., Anthony C. Zable, David Colter, and Jonathan J. Abramson. Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 82(2): 447–452, 1997.—Sarcoplasmic reticulum (SR) Ca2+-release channel function is modified by ligands that are generated during about of exercise. We have examined the effects of lactate on Ca2+- and caffeine-stimulated Ca2+ release, [3H]ryanodine binding, and single Ca2+-release channel activity of SR isolated from rabbit white skeletal muscle. Lactate, at concentrations from 10 to 30 mM, inhibited Ca2+- and caffeine-stimulated [3H]ryanodine binding to and inhibited Ca2+- and caffeine-stimulated Ca2+ release from SR vesicles. Lactate also inhibited caffeine activation of single-channel activity in bilayer reconstitution experiments. These findings suggest that intense muscle activity, which generates high concentrations of lactate, will disrupt excitation-contraction coupling. This may lead to decreases in Ca2+ transients promoting a decline in tension development and contribute to muscle fatigue.

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