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.

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.

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 The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

1998 ◽  
Vol 330 (1) ◽  
pp. 559-564 ◽  
Author(s):  
C. Edwin THROWER ◽  
J. A. Edward LEA ◽  
P. Alan DAWSON
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Artificial Bilayers

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors

1998 ◽  
Vol 274 (4) ◽  
pp. C914-C921 ◽  
Author(s):  
Toshiharu Oba ◽  
Tatsuya Ishikawa ◽  
Mamoru Yamaguchi
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptors ◽  
Reversal Potential ◽  
Skinned Fibers ◽  
Open Probability ◽  
Release Channel ◽  
Channel Activation ◽  
Channel Current ◽  
Open Time ◽  
Luminal Side

The mechanism underlying H2O2-induced activation of frog skeletal muscle ryanodine receptors was studied using skinned fibers and by measuring single Ca2+-release channel current. Exposure of skinned fibers to 3–10 mM H2O2 elicited spontaneous contractures. H2O2 at 1 mM potentiated caffeine contracture. When the Ca2+-release channels were incorporated into lipid bilayers, open probability ( P o) and open time constants were increased on intraluminal addition of H2O2 in the presence of cis catalase, but unitary conductance and reversal potential were not affected. Exposure to cis H2O2 at 1.5 mM failed to activate the channel in the presence of trans catalase. Application of 1.5 mM H2O2 to the transside of a channel that had been oxidized by cis p-chloromercuriphenylsulfonic acid (pCMPS; 50 μM) still led to an increase in P o, comparable to that elicited by trans 1.5 mM H2O2 without pCMPS. Addition of cis pCMPS to channels that had been treated with or without trans H2O2 rapidly resulted in high P o followed by closure of the channel. These results suggest that oxidation of luminal sulfhydryls in the Ca2+-release channel may contribute to H2O2-induced channel activation and muscle contracture.

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.

Metabolic end products inhibit sarcoplasmic reticulum Ca2+ release and [3H]ryanodine binding

1995 ◽  
Vol 78 (5) ◽  
pp. 1665-1672 ◽  
Author(s):  
T. G. Favero ◽  
A. C. Zable ◽  
M. B. Bowman ◽  
A. Thompson ◽  
J. J. Abramson
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Single Channel ◽  
Oxygen Species ◽  
Channel Activity ◽  
Open Probability ◽  
Release Channel ◽  
Tension Development ◽  
Contraction Coupling ◽  
End Products ◽  
Decreasing Ph

Sarcoplasmic reticulum (SR) Ca2+ release channel function is modified by ligands (Mg2+, Ca2+, ATP, and H+) that are generated during a bout of exercise. We have examined the effects of changing intracellular metabolites on Ca2+ release, [3H]ryanodine binding, and single-Ca2+ release channel activity of SR isolated from white rabbit skeletal muscle. Increasing Mg2+ (from 0 to 4 mM) and decreasing pH (7.1–6.5) inhibited SR Ca2+ release and [3H]-ryanodine binding. In addition, increasing lactate concentrations from 2 to 20 mM inhibited [3H]ryanodine binding to SR vesicles, inhibited SR Ca2+ release, and decreased the single-channel open probability. These findings suggest that intracellular modifications that disrupt excitation-contraction coupling and decrease Ca2+ transients will promote a decline in tension development and contribute to muscle fatigue. In addition, we show that hydrogen peroxide induces Ca2+ release and increases [3H]ryanodine binding to its receptor, suggesting that reactive oxygen species produced during exercise may compromise muscle function by altering the normal gating of the SR Ca2+ release channel.

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.

Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I K,ACh Channel Activity in Atrial Tachycardia Remodeling

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Niels Voigt ◽  
Ange Maguy ◽  
Yung-Hsin Yeh ◽  
Xiao-Yan Qi ◽  
Ursula Ravens ◽  
...  
Keyword(s):  
Atrial Tachycardia ◽  
Single Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Direct Demonstration ◽  
Open Time ◽  
Therapy Target ◽  
Channel Properties ◽  
Constitutively Active

Background: Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I K,ACh that increases susceptibility to AF, direct demonstration of dysregulated I K,ACh channel function is lacking. We studied AT effects on single I K,ACh channel activity in dog atria. Methods: I K,ACh channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min −1 ) dogs. Results : AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification were observed in CTL and AT (Figure ). AT produced prominent agonist-independent I K,ACh activity due to 7-fold increased opening frequency (f o ) and 10-fold increased open probability (P o ) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I K,ACh activation (10 μm carbachol, CCh), f o was 38% lower, open time constant 25% higher, and P o and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f o and P o by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of I K,ACh. Conclusions : AT produces prominent agonist-independent constitutive single-channel I K,ACh activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I K,ACh , as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I K,ACh channels in AT-remodeling and support their interest as a potential novel AF-therapy target.

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