Heavy Sarcoplasmic Reticulum Research Articles

Shotgun proteomic analysis of sarcoplasmic reticulum preparations from rabbit skeletal muscle

To obtain a comprehensive understanding of proteins involved in excitation-contraction coupling, a catalog of proteins from sarcoplasmic reticulum (SR) membrane fractions of New Zealand white rabbit skeletal muscle was analyzed by an optimized shotgun proteomic method. Light and heavy SR membrane fractions were obtained by nonlinear sucrose gradient centrifugation and separated by 1DE followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ) Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 483 proteins were identified from both of the two independent SR preparations. Proteins involved in calcium release unit complex, including ryanodine receptor 1, dihydropyridine receptor, calmodulin, triadin, junctin, and calsequestrin, were all detected, which offered validation for this protein identification method. Rigorous bioinformatics analysis was performed. Protein pI value, molecular weight range, hydrophobicity index, and transmembrane region were calculated using bioinformatics softwares. Eighty-three proteins were classified as hydrophobic proteins and 175 proteins were recognized as membrane proteins. Based on the proteomic analysis results, we found as the first time that not only transverse tubule but also mitochondrion physically connected to SR. The complete mapping of these proteomes may help in the elucidation of the process of excitation-contraction coupling and excitation-metabolism coupling.

Functional Properties of SR Cl- and K+ Channels during Postnatal Development of Cardiac Muscle

In adult (AD) heart, the sarcoplasmic reticulum (SR) contains Cl- & K+ channels presumably involved in controlling RyR-mediated SR Ca2+ release. These channels provide a countercurrent mechanism that attenuates the drop in Ca2+ driving force across the SR membrane, thereby preventing early termination of Ca2+ release. We showed that in newborn (NB), Ca2+ sparks occur with similar frequency than in AD but have shorter duration and smaller amplitude, implying an early termination of Ca2+ release. Although the functional properties of SR Cl- & K+ channels have been thoroughly described in AD, little is known about their presence and their role in NB. Consequently, we first tested the hypothesis that the early termination of Ca2+ release in NB coincides with absence/low density of SR Cl- & K+ channels at this stage. To this end, the heavy microsomal fraction was obtained from 5-days-old NB and AD rat hearts and SR Cl- & K+ channels were reconstituted into artificial planar lipid bilayers. Our results indicate that Cl- & K+ channels can be reconstituted from NB heavy SR microsomes with a similar success rate (number of SR channel incorporations / total number of bilayers) than in AD (∼0.2 for Cl- channels & ∼0.1 for K+ channels). Thus, an alternative mechanism would imply that in NB, smaller counterion fluxes result from different functional properties of SR Cl- & K+ channels. This assumption was tested by measuring their unitary conductance, open probability, and voltage dependence. The results in NB channels revealed no significant differences in any of these parameters in comparison to AD. Thus, we concluded that SR Cl- & K+ channels do not contribute to the developmental changes of Ca2+ release in NB cardiomyocytes. Supported by AHA-0655656Z to RMA.

Sevoflurane modulation of Ca2+ regulation in skeletal muscle sarcoplasmic reticulum vesicles from young and mature rabbits

Developmental differences in splice variants of the two key sarcoplasmic reticulum (SR) calcium regulatory proteins, ryanodine (RyR1), and sarcoendoplasmic reticulum calcium pump (SERCA1) have been linked to various neuromuscular disorders, but not malignant hyperthermia (MH). However, it is unclear whether an age-related difference in volatile anesthetic-mediated SR calcium function exists that could add to our current understanding of the clinical presentation of MH syndrome and provide insight into molecular mechanisms for general anesthesia that may have other physiologic and/or pathophysiologic significance. Therefore, the effects of sevoflurane on intracellular calcium regulation in isolated SR membrane vesicles from the skeletal muscle of healthy young rabbits were compared to their adult counterpart using an established in vitro model with the assumption that exposure to sevoflurane would elicit a weaker response in the young SR. Through dual wavelength spectroscopy of Ca(2+): Arsenazo III difference absorbance, the effects of sevoflurane on SR Ca(2+) uptake rate and release in heavy and light fraction SR membrane vesicles isolated from the white muscle of anesthetized, postweaned (age = 6 weeks, n = 5) and adult (age = 6 months, n = 5) male New Zealand rabbits were examined. The adult group showed a 50% increase in Ca(2+) uptake rate from control at both subclinical and clinically relevant anesthetic concentrations, whereas in the SR from the younger animals, Ca(2+) uptake rate was not altered by any concentration of sevoflurane. The sensitivity of both the low and high affinity Ca(2+)-binding sites on RyR1 was increased by sevoflurane to the same extent in the SR vesicles from the young and mature adult rabbits. Interestingly, a greater potency of sevoflurane for the high affinity-binding site was identified, and this was independent of age. These findings suggest that the sensitivity of the SR to sevoflurane-mediated Ca(2+) uptake may be increased with maturity, while an analogous developmental effect on RyR1 is less probable. Nonetheless, this study shows for the first time that a potent inhalational agent such as sevoflurane can influence the high affinity SR calcium-binding site by lowering the extraluminal concentration of calcium necessary to trigger calcium release. While this may not be of consequence when inhaled anesthetics are administered to normal children or adults, it may have life-threatening consequences in carriers of RyR1 mutations.

SRP-35 A Putative NAD(P)H Binding Protein Of Skeletal Muscle Sarcoplasmic Reticulum Membrane

SRP-35 (sarcoplasmic reticulum protein of 35 kDa) is a newly identified integral membrane protein constituent of skeletal muscle sarcoplasmic reticulum. We have deduced the primary structure of the protein from cDNA clones isolated from mouse and rat skeletal muscle cDNA libraries. Primary sequence prediction analysis indicates that the NH2- terminal sequence of SRP-35 encompasses a transmembrane spanning segment or a signal sequence. In addition, SRP-35 is homologous to proteins belonging to the short-chain dehydrogenase/reductases family. Members of this protein family has two domains: the first involved in binding the nucleotide co-factor NAD(P)H, the second responsible for the catalysis of the substrate. SRP35 contains only a putative NAD(P)H binding site. Analysis of tissue distribution of SRP-35 by western blot analysis with affinity purified Ab shows that SRP-35 expression is specific for skeletal muscle since our Ab did not stain any protein in other tissues including heart, brain, liver, kidney, lung, spleen and stomach. Immunohistochemistry of primary cultured mouse myotubes transfected with SRP-35 EGFP construct indicates that SRP-35 is distributed on sarco(endo)plasmic reticulum membranes. Staining of western blot of sarcoplasmic reticulum membrane subfractions isolated from adult mouse skeletal muscle revealed that SRP-35 is associated with heavy sarcoplasmic reticulum. In addition, we found that SRP-35 is an integral membrane protein since it was extracted neither by NaCO3 nor by high salt treatment of isolated sarcoplasmic reticulum membrane, but was solubilised by non-ionic detergent such as CHAPS, DDM and DHPC. We propose that , SRP-35 protein might provide the co-factor to enzymes involved in the generation of local reactive oxygen species within cellular subdomains

Iron (II) Modulation of the Cardiac Ryanodine Receptor (RyR2)

Cardiomyopathies and arrhythmias are major causes of mortality in chronic iron overload. There is evidence that iron overload impairs cardiomyocytes Ca2+ homeostasis (Baptista-Hon et al, 2005). However its molecular substrates remain unknown. Cardiac ryanodine receptors (RyR2) dysfunction is implicated in several diseases where Ca2+ homeostasis is lost. We therefore wanted to investigate RyR2 role as a potential target for iron-induced cardiomyopathies.We isolated heavy sarcoplasmic reticulum (HSR) vesicles containing RyR2 from sheep hearts. RyR2 were reconstituted into L-α-phosphatidylethanolamine (PE) bilayers. Unitary currents were measured under voltage-clamp with 250mM Cs+ as the charge carrier and 10μM activating Ca2+. High affinity [3H]ryanodine binding of the native vesicles was detected by liquid scintillation counting. Non-specific binding determined by incubations with 100x cold ryanodine.Fe2+ reduced RyR2 open probability and conductance in a dose dependent manner. Lifetime analysis revealed 5 shut times components and 3 open times components in control. Fe2+ caused an extra-shut component. Furthermore, there was a dose dependent shift in the open time constants towards the faster components. The binding assays revealed a [Fe2+] dependent, co-operative reduction in [3H]ryanodine binding to HSR vesicles. Preliminary data of [3H]ryanodine binding in increasing [Ca2+] showed a rightward shift in the presence of Fe2+.The results presented here show for the first time that Fe2+ is a potent inhibitor of RyR2. The mechanism of this inhibition may be due to competition with Ca2+ for RyR2 activation sites. Suppression of RyR2 activity by Fe2+ may therefore be one of the mechanisms involved in iron-induced cardiomyopathies.ReferencesBaptista-Hon, D, Diaz, M. E., and Elliot, A. C. Acute exposure to iron (II) alters calcium handling in isolated rat ventricular myocytes. Journal of Molecular and Cellular Cardiology 39, 179. 2005.

Loss of the Calmodulin-Dependent Inhibition of the RyR1 Calcium Release Channel upon Oxidation of Methionines in Calmodulin

The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of [3H]ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, in which calcium-activated CaM acts to enhance the calcium sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially weaken the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not significantly affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as part of a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress observed during biological aging. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response that enhances the duration of calcium transients to promote enhanced contractility.

Effect of natural phenol derivatives on skeletal type sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor

The effect of natural phenol derivatives was studied on skeletal type sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine receptor. The majority of the tested derivatives exerted inhibitory effect on the Ca(2+)-ATPase with an ascending sequence in regard to their effectiveness (IC(50)): cineole (3.33 mM) < ortho-vanillin (IC(50 )=1.13 mM) < 4-methyl-2-nitrophenol (1104 microM) < vanillin (525 microM) < thymol (224 microM) < carvacrol (162 microM). In two cases biphasic characteristic was observed: trans-anethole and meta-anisaldehyde first caused activation followed by inhibition (with IC(50)-s of 141 and 1903 microM respectively) as their concentration was increased. In some cases (cineole, ortho-vanillin, meta-anisaldehyde) total inhibition of Ca(2+)-ATPase could not be reached as the result of the limited solubility of these drugs. Para-anisaldehyde and 6-amino-meta-cresol did not show any effect up to 3 mM. In Ca(2+) release experiments drugs were applied on heavy sarcoplasmic reticulum vesicles isolated from skeletal muscle and actively loaded with calcium. Only thymol and carvacrol were able to evoke Ca(2+) release with EC(50) values of 158 +/- 16 and 211 +/- 55 microM respectively. Furthermore the effect of thymol and carvacrol was tested on the isolated ryanodine receptor incorporated into artificial lipid bilayer. Both drugs activated the RyR when applied in concentrations identical to their EC(50) values. These observations show that small differences in the structure of phenol derivatives sometimes have little impact on their effect on the sarcoplasmic reticulum Ca(2+)-ATPase or ryanodine receptor (thymol and carvacrol) whereas in certain cases they can completely abolish a particular effect (para- and meta-anisaldehyde).

Thermogenic activity of Ca 2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca 2+ channel

The sarcoplasmic reticulum Ca 2+ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca 2+ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca 2+ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg 2+ or ruthenium red, conditions that close the ryanodine Ca 2+ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH cal values of ATP hydrolysis increased significantly. Neither Mg 2+ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca 2+ channel is opened or closed.

Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein

The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [(35)S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H(2)O(2) and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [(35)S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H(2)O(2) and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to approximately 75 and approximately 50% of control, respectively. In addition, the effect of H(2)O(2) was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.

Arrhythmogenesis in Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2(R4496C+/-)). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2(R4496C+/-) myocytes (P=0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2(R4496C+/-) myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 micromol/L) but not by K201 (1 micromol/L or 10 micromol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2(R4496C+/-) mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6 interaction both in WT and RyR2(R4496C+/-) either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2(R4496C+/-) myocytes and ventricular arrhythmias in RyR2(R4496C+/-) mice; and (3) RyR2-FKBP12.6 interaction in RyR2(R4496C+/-) is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex.

Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters

Whether the density of sarcoplasmic reticulum (SR) calcium release channels/ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of [3H]-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes. Experiments utilized young (120 days) and older adult (300 days) hamsters. [3H]-ryanodine binding site density did not change with age in crude homogenate preparations, although total heart protein concentration increased significantly with age. In contrast, the density of [3H]-ryanodine binding sites decreased markedly in heavy SR membranes purified from older hearts. These results show that demonstration of age-related changes in cardiac ryanodine receptor density depends upon the preparation used. Furthermore, the increase in total ventricular protein with age suggests that normalization of data by membrane protein should be used with caution in studies of aging heart.

Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.

Effects ofS-Glutathionylation andS-Nitrosylation on Calmodulin Binding to Triads and FKBP12 Binding to Type 1 Calcium Release Channels

This study shows that the combination of glutathione (GSH) plus hydrogen peroxide (H2O2) promotes the S-glutathionylation of ryanodine receptor type 1 (RyR1) Ca2+ release channels, and confirms their joint S-glutathionylation and S-nitrosylation by S-nitrosoglutathione (GSNO). In addition, we show that 35S-labeled 12-kDa FK506-binding protein ([35S]FKBP12) bound with a Kd of 13.1 nM to RyR1 present in triads or heavy sarcoplasmic reticulum vesicles; RyR1 S-nitrosylation by NOR-3 or GSNO, but not S-glutathionylation, specifically increased by four- to fivefold this Kd value. RyR1 redox modifications also increased the Kd of [35S]calmodulin binding to triads without affecting Bmax. RyR1 S-glutathionylation (induced by GSH plus H2O2) or RyR1 S-nitrosylation (produced by NOR-3) increased by approximately six- or twofold, respectively, the Kd of apocalmodulin (apoCaM) or Ca2+-calmodulin (CaCaM) binding to triads. Likewise, the combined S-glutathionylation and S-nitrosylation of RyR1 induced by GSNO increased by fourfold the Kd of CaCaM binding to triads and abolished apoCaM binding. As both FKBP12 and CaCaM inhibit RyR1, decreased FKBP12 binding to RyR1 and/or decreased CaCaM binding to either RyR1 or dihydropyridine receptor in triad preparations may cause the reported enhanced activation of Ca2+-induced Ca2+ release kinetics mediated by S-glutathionylation/S-nitrosylation. We discuss possible consequences of these redox modifications on RyR1-mediated Ca2+ release in physiological or pathological conditions.

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca2 + -ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca2+ release, and the light fraction enriched in Ca(2+)-ATPase (SERCA), an enzyme responsible for Ca2+ transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca(2+)- ATPase activity and Ca(2+)-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca(2+)-uptake and Ca(2+)-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.

Effect of gadolinium on the ryanodine receptor/sarcoplasmic reticulum calcium release channel of skeletal muscle

The effect of gadolinium ions on the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptor (RyR1) was studied using heavy SR (HSR) vesicles and RyR1 isolated from rabbit fast twitch muscle. In the [(3)H]ryanodine binding assay, 5 microM Gd(3+) increased the K(d) of the [(3)H]ryanodine binding of the vesicles from 33.8 nM to 45.6 nM while B(max), referring to the binding capacity, was not affected significantly. In the presence of 18 nM[(3)H]ryanodine and 100 microM free Ca(2+), Gd(3+) inhibited the binding of the radiolabeled ryanodine with an apparent K(d) value of 14.7 microM and a Hill coefficient of 3.17. In (45)Ca(2+) experiments the time constant of (45)Ca(2+) efflux from HSR vesicles increased from 90.9 (+/- 11.1) ms to 187.7 (+/- 24.9) ms in the presence of 20 microM gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic (cis) (IC(50) = 5.65 +/- 0.33 microM, n(Hill) = 4.71) and the luminal (trans) side (IC(50) = 5.47 +/- 0.24 microM, n(Hill) = 4.31). The degree of inhibition on the cis side didn't show calcium dependency in the 100 microM to 1 mM Ca(2+) concentration range which indicates no competition with calcium on its regulatory binding sites. When Gd(3+) was applied at the trans side, EGTA was present at the cis side to prevent the binding of Gd(+3) to the cytoplasmic calcium binding regulatory sites of the RyR1 if Gd(3+) accidentally passed through the channel. The inhibition of the channel did not show any voltage dependence, which would be the case if Gd(3+) exerted its effect after getting to the cis side. Our results suggest the presence of inhibitory binding sites for Gd(3+) on both sides of the RyR1 with similar Hill coefficients and IC(50) values.

Channelling of substrate promiscuity of the skeletal-muscle ADP-ribosyl cyclase isoform.

The novel Ca2+-mobilizing second messengers cADPr (cyclic ADP-ribose) and NAADP (nicotinic acid-adenine dinucleotide phosphate) are both synthesized by ADP-ribosyl cyclases. Using HSR (heavy sarcoplasmic reticulum) fractions from rabbit skeletal muscle, NAADP-induced Ca2+ release was observed. In the present paper, we show in HSR membranes the formation of authentic cADPr, cGDPr (cyclic GDP-ribose) and NAADP. The cyclization reaction to form cADPr and cGDPr as well as the base-exchange reaction to form NAADP were strictly dependent on pH. Although the formation of cGDPr is optimized at pH 6, the synthesis of NAADP was most pronounced at a pH below 5. A novel regulation mechanism is provided for nicotinic acid, the co-substrate for NAADP synthesis. Nicotinic acid had virtually no influence on the cyclization reaction, but increased the affinity of NADP at an acidic pH and had the opposite effect at alkaline pH. Nicotinamide, the side product of cADPr synthesis, is an inhibitor of the cyclization reaction (IC50, 0.7+/-0.1 mM) and was 30-fold more potent at suppressing the base-exchange reaction. Although the synthesis of NAADP was highly sensitive to nicotinamide inhibition, this was not via a competition with the nicotinic-acid-binding site. In contrast with the ecto-ADP-ribosyl cyclase (CD38), the cyclization and base-exchange reaction of the skeletal muscle isoform was inhibited by Cu2+ and Zn2+, while other bivalent cations such as Ca2+, Mg2+ and Mn2+ had virtually no effect. These findings allow for the prediction of a novel ADP-ribosyl cyclase isoform in skeletal muscle HSR, other than CD38. Hence the enzymic prerequisite for cADPr- and NAADP-mediated Ca2+ signalling is present.

Activation of the sheep cardiac Ca 2+ release channel by simple heteroaromatics

The broad range of ligands known to modulate ryanodine receptor activity includes a class of heteroaromatic compounds displaying relatively poor efficacy. Greater understanding of the physicochemical properties that predispose these molecules to interaction with the channel should facilitate the rational design of more potent analogues. To this end we are examining the structure–activity relationship for simple heteroaromatic compounds. Efficacy is assessed by the ability to stimulate [ 3H]ryanodine binding to heavy sarcoplasmic reticulum vesicles. The propensity to activate the channel requires notably little chemical functionality and is associated with the capacity for charge-transfer complex formation in conjunction with steric bulk.

Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 μM and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 ± 0.03 to 0.22 ± 0.04 at 30 μM. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 ± 0.01 vs. 0.70 ± 0.01, but increased in duration, 56 ± 1 vs. 79 ± 1 ms, by 30 μM thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

Effects of eicosapentaenoic acid on cardiac SR Ca(2+)-release and ryanodine receptor function.

n-3 polyunsaturated fatty acids (PUFAs) can prevent life-threatening arrhythmias but the mechanisms responsible have not been established. There is strong evidence that part of the antiarrhythmic action of PUFAs is mediated through inhibition of the Ca(2+)-release mechanism of the sarcoplasmic reticulum (SR). It has also been shown that PUFAs activate protein kinase A (PKA) and produce effects in the cardiac cell similar to beta-adrenergic stimulation. We have investigated whether the inhibitory effect of PUFAs on the Ca(2+)-release mechanism is caused by direct inhibition of the SR Ca(2+)-release channel/ryanodine receptor (RyR) or requires activation of PKA. Experiments in intact cells under voltage-clamp show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce the frequency of spontaneous waves of Ca(2+)-release while increasing SR Ca(2+) content even when PKA activity is inhibited with H-89. This suggests that the EPA-induced inhibition of SR Ca(2+)-release is not dependent on activation of PKA. Consistent with this, single-channel studies demonstrate that EPA (10-100 microM), but not saturated fatty acids, reduce the open probability (Po) of the cardiac RyR incorporated into phospholipid bilayers. EPA also inhibited the binding of [3H]ryanodine to isolated heavy SR. Our results indicate that direct inhibition of RyR channel gating by PUFAs play an important role in the overall antiarrhythmic properties of these compounds.

Calmodulin Oxidation and Methionine to Glutamine Substitutions Reveal Methionine Residues Critical for Functional Interaction with Ryanodine Receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.