Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum

doi:10.1016/0143-4160(88)90032-2

Cell Calcium

Volume 9, Issue 1, February 1988, Pages 1-7

https://doi.org/10.1016/0143-4160(88)90032-2 Get rights and content

Abstract

Ryanodine modulates Ca²⁺ permeability in isolated terminal cisternae of sarcoplasmic reticulum, suggesting that it is a specific ligand for the calcium release channel. Our laboratory has purified the ryanodine receptor and demonstrated it to be equivalent to the feet structures, which are involved in the junctional association of the transverse tubule with the terminal cisternae. Recently, Smith, Coronado and Meissner have incorporated sarcoplasmic reticulum into bilayers and found a high conductivity channel (∼ 100 pS) which has a number of characteristics expected of the Ca²⁺ release channels in SR. We now find that the high conductivity channel in the bilayer is sensitive to ryanodine. Low concentrations of ryanodine (sub μM): (1) lock the channels in an open state; (2) prevent the action of ruthenium red (μM) to completely close the channel; and (3) much higher concentrations of ryanodine (300μM) close the channel. In these three respects ryanodine acts similarly on the channel in the bilayer as in vesicles. Further, the bilayer studies provide new insight into the action of ryanodine on the channel in that: (1) ryanodine locks the channel in the open state, but the conductivity is reduced to about 40%; (2) ryanodine prevents ruthenium red from closing the channel, although there is a further decrease in the open current. These studies provide support that the high conductivity calcium channel in sarcoplasmic reticulum is involved in excitation-contraction coupling. By the same token the pharmacological action of ryanodine is pinpointed to the calcium release channel.

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Cited by (81)

The TMEM16A blockers benzbromarone and MONNA cause intracellular Ca2+-release in mouse bronchial smooth muscle cells
2023, European Journal of Pharmacology
We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCC_inhA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1–10 μM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period. Benzbromarone (1 μM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 μM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 μM) and CaCC_inhA01 (10 μM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 μM) had no effect on carbachol-induced contractions.
Confocal imaging revealed that benzbromarone (0.3 μM), MONNA (1 μM) and CaCC_inhA01 (10 μM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 μM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 μM) blocked the ability of benzbromarone (0.3 μM) to increase calcium, while tetracaine (100 μM) reversibly reduced the rise in calcium induced by benzbromarone.
We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect.
Structural Basis for Gating and Activation of RyR1
2016, Cell
Citation Excerpt :
We attribute these features to one ryanodine molecule averaged over four symmetrically equivalent binding sites. Besides locking the channel in a partially open state, even a single ryanodine molecule is likely to occlude part of the pore, consistent with reduced conductance in the presence of ryanodine (Nagasaki and Fleischer, 1988). This putative ryanodine position is directly adjacent to Q4933 (Figure 6B), mutation of which specifically reduces ryanodine binding without affecting channel function (Fessenden et al., 2001).
The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca²⁺) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca²⁺, ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca²⁺ alone induces conformational changes in the cytoplasmic assembly (“priming”), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.
Structures of the colossal RyR1 calcium release channel
2016, Current Opinion in Structural Biology
Citation Excerpt :
The eponymous RyR-ligand ryanodine exhibits peculiar effects on RyR function. At low concentrations (nm–∼10 μm), ryanodine locks the channel in a state of unitary open probability but reduced single channel conductance, while at higher concentrations the same ligand blocks the channel, locking it in a non-conducting state [46]. Structural analysis of RyR1 in the presence of Ca2+ and ryanodine gives some insight as to the provenance of both effects.
Ryanodine receptors (RyRs) are intracellular cation channels that mediate the rapid and voluminous release of Ca²⁺ from the sarcoplasmic reticulum (SR) as required for excitation–contraction coupling in cardiac and skeletal muscle. Understanding of the architecture and gating of RyRs has advanced dramatically over the past two years, due to the publication of high resolution cryo-electron microscopy (cryoEM) reconstructions and associated atomic models of multiple functional states of the skeletal muscle receptor, RyR1. Here we review recent advances in our understanding of RyR architecture and gating, and highlight remaining gaps in understanding which we anticipate will soon be filled.
Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells
2015, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
Similarly we did not consider the function of mitochondria as putative Ca2 + stores or sources in our model, since pretreatment of the cells with Ru360, a cell-permeable mitochondrial Ca2 + uptake blocker [57], did not inhibit Ca2 + oscillations (data not shown). With the aim to investigating the role of RyRs in mesothelial cell Ca2 + oscillations, cells were exposed to a high concentration of ryanodine (50 μM); ryanodine fully closes RyRs at micromolar concentrations [58]. In most cells ryanodine did not affect serum-induced Ca2 + oscillations (Fig. 2D), but in some cases transiently and slightly reduced the frequency of oscillations together with a short-lasting lowering of basal ccyt.
Brief changes in the cytosolic and intra-organellar Ca^2 + concentration serve as specific signals for various physiological processes. In mesothelial cells lining the surface of internal organs and the walls of body cavities, a re-entry in the cell cycle (G₀–G₁ transition) evoked by serum re-administration induces long-lasting Ca^2 + oscillations with a slowly decreasing frequency. Individual mesothelial cells show a wide range of different oscillatory patterns within a single, supposedly homogenous cell population. Changes in the cytoplasmic Ca^2 + concentration (c_cyt) show baseline oscillatory patterns i.e., discrete Ca^2 + transients starting from a constant basal c_cyt level. The ER Ca^2 + concentration (c_ER) displays a sawtooth wave at a semi-depleted ER state; the minimum level is reached just briefly after the maximal value for c_cyt. These oscillations depend on plasmalemmal Ca^2 + influx and on the inositol trisphosphate concentration [InsP₃]; the Ca^2 + influx is a crucial determinant of the oscillation frequency. Partial blocking of SERCA pumps modifies the oscillation frequency in both directions, i.e. increasing it in some cells and lowering it in others. Current mathematical models for Ca^2 + oscillations mostly fail to reproduce two experimentally observed phenomena: the broad range of interspike intervals and constant basal c_cyt levels between two Ca^2 + spikes. Here we developed a new model based on – and fitted to – Ca^2 + recordings of c_cyt and c_ER recorded in primary mouse mesothelial cells. The model allowed for explaining many features of experimentally observed Ca^2 + oscillations. We consider this model to be suitable to simulate various types of InsP₃ receptor-based baseline Ca^2 + oscillations.
3,5-Di-t-butyl catechol is a potent human ryanodine receptor 1 activator, not suitable for the diagnosis of malignant hyperthermia susceptibility
2012, Pharmacological Research
Citation Excerpt :
To assess whether DTCAT released Ca2+ mostly from ryanodine sensitive stores, myotubes were incubated for 25 min in 50 μM ryanodine to block the SR Ca2+ release channel. In fact ryanodine, at nanomolar concentrations, activates the Ca2+ release channel but blocks it at micromolar concentrations [14]. The initial activation of the ryanodine receptor 1 leads to an increase of [Ca2+]i and activation of Ca2+ influx [15].
3,5-Di-t-butyl catechol (DTCAT) releases Ca²⁺ from rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Hence, it is a candidate for use as a substitute for halothane or caffeine in the in vitro contracture test for the diagnosis of susceptibility to malignant hyperthermia (MH).
To characterize the effect of DTCAT at cell level, Ca²⁺ release experiments were performed on cultured, human skeletal muscle myotubes using the fluorescent Ca²⁺ indicator fura2-AM. DTCAT was also assayed in the in vitro contracture test on human skeletal muscle bundles obtained from individuals diagnosed susceptible (MHS), normal (MHN) or equivocal for halothane (MHEH) and compared to the standard test substances caffeine and halothane.
DTCAT increased, in a concentration-dependent manner and with a higher efficacy as compared to caffeine, the free, intracellular Ca²⁺ levels of cultured MHN and MHS skeletal muscle myotubes. This effect was similar in both types of myotubes and involved the release of Ca²⁺ from SR stores as well as Ca²⁺-influx from the extracellular space. Inhibition of ryanodine receptors either with ryanodine or with ruthenium red markedly reduced DTCAT-induced increase in intracellular Ca²⁺ concentration while abolishing that induced by caffeine. In MHN skeletal muscle bundles, DTCAT induced contractures with an EC₅₀ value of 160 ± 91 μM. However, the sensitivity of MHS or MHEH muscles to DTCAT was similar to that of MHN muscles.
In conclusion, DTCAT is not suitable for the diagnosis of MH susceptibility due to its failure to discriminate between MHN and MHS muscles.
F281, synthetic agonist of the sigma-2 receptor, induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells
2009, Cell Calcium
We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca²⁺]_i. In particular, CB-64D induces a Ca²⁺ response while PB28 supresses Ca²⁺ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC₅₀ values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca²⁺ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca²⁺-entry through the channels activated by store depletion was also observed. After the increase in [Ca²⁺]_i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.

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Cell Calcium

Abstract

References (28)

Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle

J. Biol. Chem.

Identification and characterization of the high affinity [³H]-ryanodine receptor of the junctional sarcoplasmic reticulum Ca²⁺ release channel

J. Biol. Chem.

Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum

Biophys. J.

Single channel and ⁴⁵Ca²⁺ flux measurements of the cardiac sarcoplasmic reticulum calcium channel

Biophys. J.

Inhibition of calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles

J. Biol. Chem.

Mechanisms of action of ryanodine on cardiac sarcoplasmic reticulum

Biochim. Biophys. Acta

High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca²⁺-activated protease

J. Biol. Chem.

Rapid filtration measurements of Ca²⁺ release from cisternal sarcoplasmic reticulum vesicles

FEBS Letters

Ryanodine activation and inhibition of the Ca²⁺ release channel of sarcoplasmic reticulum

J. Biol. Chem.

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

J. Biol. Chem.

Calcium release from the sarcoplasmic reticulum

Physiol. Rev.

Mechanisms of Ca²⁺ release from sarcoplasmic reticulum of skeletal muscle

Physiol. Rev.

Cited by (81)

The TMEM16A blockers benzbromarone and MONNA cause intracellular Ca<sup>2+</sup>-release in mouse bronchial smooth muscle cells

Structural Basis for Gating and Activation of RyR1

Structures of the colossal RyR1 calcium release channel

Characterization and modeling of Ca<sup>2+</sup> oscillations in mouse primary mesothelial cells

3,5-Di-t-butyl catechol is a potent human ryanodine receptor 1 activator, not suitable for the diagnosis of malignant hyperthermia susceptibility

F281, synthetic agonist of the sigma-2 receptor, induces Ca<sup>2+</sup> efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum

Abstract

J. Biol. Chem.

J. Biol. Chem.

Biophys. J.

Biophys. J.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

FEBS Letters

J. Biol. Chem.

J. Biol. Chem.

Calcium release from the sarcoplasmic reticulum

Physiol. Rev.

Mechanisms of Ca2+ release from sarcoplasmic reticulum of skeletal muscle

Physiol. Rev.

Mechanisms of Ca²⁺ release from sarcoplasmic reticulum of skeletal muscle