Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor.

Ca2+ regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca2+-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln3+) as probes to investigate the Ca2+ sensors of RyR, because they specifically bind to Ca2+-binding proteins and they are impermeable to the channel. Eu3+'s and Sm3+'s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 µM Ca2+, Ln3+ potently inhibited RyR's open probability (Kd Eu3+ = 167 ± 5 nM and Kd Sm3+ = 63 ± 3 nM), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Ln3+ acts as an agonist of both Ca2+-binding sites. More importantly, the voltage-dependent characteristics of Ln3+'s action led to the conclusion that the activating Ca2+ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca2+] from 50 µM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site and support our vestibular Ca2+ sensor-model further.

4-chloro-orto-cresol activates ryanodine receptor more selectively and potently than 4-chloro-meta-cresol.

In this study we performed the comprehensive pharmacological analysis of two stereoisomers of 4-chloro-meta-cresol (4CMC), a popular ryanodine receptor (RyR) agonist used in muscle research. Experiments investigating the Ca2+-releasing action of the isomers demonstrated that the most potent isomer was 4-chloro-orto-cresol (4COC) (EC50 = 55 ± 14 µM), although 3-chloro-para-cresol (3CPC) was more effective, as it was able to induce higher magnitude of Ca2+ flux from isolated terminal cisterna vesicles. Nevertheless, 3CPC stimulated the hydrolytic activity of the sarcoplasmic reticulum ATP-ase (SERCA) with an EC50 of 91 ± 17 µM, while 4COC affected SERCA only in the millimolar range (IC50 = 1370 ± 88 µM). IC50 of 4CMC for SERCA pump was 167 ± 8 µM, indicating that 4CMC is not a specific RyR agonist either, as it activated RyR in a similar concentration (EC50 = 121 ± 20 µM). Our data suggest that the use of 4COC might be more beneficial than 4CMC in experiments, when Ca2+ release should be triggered through RyRs without influencing SERCA activity.

Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor.

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala(8)]MCa, [Ala(19)]MCa, [Ala(20)]MCa, [Ala(22)]MCa, [Ala(23)]MCa, and [Ala(24)]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical (24)Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys(20), Lys(22), Arg(23), Arg(24), and Lys(8). Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.

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.

Troponin I converts the skeletal muscle ryanodine receptor into a rectifying calcium release channel.

The goal of our present studies has been to find novel ryanodine receptor (RyR1) interacting polypeptides that modulate the channel activity from the luminal side of RyR1. Using K(+) as charge carrier for recording of single channel events here we demonstrate a very unexpected observation that troponin I substantially alters RyR's gating behavior, and that RyR1 in association with troponin I becomes a rectifying Ca(2+) release channel. Troponin I rapidly locks the RyR1 in a non-conducting state only at a negative holding potential, and only when applied to the luminal side; switching to a positive holding potential results in the channel returning to its original activity, immediately. A hypothesis is proposed to account for how an intraluminally located, positively charged molecule might function as a RyR1 regulator under physiological conditions.

Effect of thymol on calcium handling in mammalian ventricular myocardium.

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.

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).

Alterations in the calcium homeostasis of skeletal muscle from postmyocardial infarcted rats.

In chronic heart failure, skeletal muscles develop a weakness that is not associated to an impaired circulatory function but rather to alterations in the skeletal muscle fibers themselves. To understand these changes, the steps in excitation-contraction coupling of rats that underwent a left anterior coronary artery occlusion were studied. About 24 weeks after the myocardial infarction, neither the total amount nor the voltage dependence of intramembrane charge were altered. In contrast, calcium release from the sarcoplasmic reticulum was considerably suppressed, and its voltage dependence shifted toward more positive voltages. Elementary calcium-release events showed altered morphology as the relative proportion of embers increased. Calcium sparks were smaller in amplitude and had larger time-to-peak values. Isolated ryanodine receptors (RyR) displayed an unusual rectification with increased single-channel conductance at positive (cis vs trans) voltages. In addition, the bell-shaped calcium dependence of channel activity was broader, with a slight shift of activation to lower and a larger shift in inactivation to higher calcium concentrations. These data indicate that the number of channels that open during a calcium-release event is decreased and that RyR function is altered; thus, calcium-release is suppressed after a myocardial infarction. These observations give an explanation for the impaired skeletal muscle function in these animals.

Modulation of sarcoplasmic reticulum function by Na+/K+ pump inhibitors with different toxicity: digoxin and PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride].

OBJECTIVE: To gain some insight on the lesser arrhythmogenic properties of PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride] compared with digoxin, we compared modulation of intracellular Ca2+ dynamics by the two agents. METHODS: SERCA (sarcoplasmic reticulum Ca2+-ATPase) activity and Ca2+ leak rate were measured in sarcoplasmic reticulum (SR) vesicles from guinea pig ventricles. Membrane current, intracellular Ca2+, and twitch amplitude were evaluated in guinea pig ventricular myocytes with or without blockade of the Na+/Ca2+ exchanger. RESULTS: In SR vesicles, PST2744 (30-300 nM), but not digoxin, increased SERCA activity; digoxin only (> or =0.1 nM) increased SR Ca2+ leak. In myocytes with blocked Na+/Ca2+ exchanger, Ca2+ reloading of caffeine-depleted SR was enhanced by PST2744 and slightly inhibited by digoxin. In myocytes with functioning Na+/Ca2+ exchanger, both agents increased diastolic Ca2+, SR Ca2+ content, the gain of Ca2+-induced Ca2+ release, the rate of cytosolic Ca2+ decay, twitch amplitude, and relaxation rate. Consistent with the observations in SR vesicles, the effects on SR Ca2+ content and Ca2+ decay rate were significantly larger for PST2744 than for digoxin. CONCLUSIONS: In isolated SR vesicles, PST2744 and digoxin directly affected SR function in opposite ways; this could be reproduced in myocytes during Na+/Ca2+ exchanger blockade. Under physiological conditions (functioning Na+/Ca2+ exchanger), the two agents affected Ca2+ dynamics in the same direction, as expected by their Na+/K+ pump inhibition; however, differential SR modulation was still expressed by quantitative differences. Thus, the more favorable inotropy-to-toxicity ratio previously described for PST2744 appears to be associated with direct SERCA stimulation and/or lack of enhancement of Ca2+ leak.

Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of maurocalcine in skeletal muscle cells.

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.