Desensitization of the skeletal muscle ryanodine receptor: evidence for heterogeneity of calcium release channels.

Ca release channels from the junctional sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle were incorporated into the lipid bilayer membrane, and the inactivation kinetics of the channel were studied at large membrane potentials. The channels conducting Cs currents exhibited a characteristic desensitization that is both ligand and voltage dependent: 1) with a test pulse to -100 mV (myoplasmic minus luminal SR), the channel inactivated with a time constant of 3.9 s; 2) the inactivation had an asymmetric voltage dependence; it was only observed at voltages more negative than -80 mV; and 3) repetitive tests to -100 mV usually led to immobilization of the channel, which could be recovered by a conditioning pulse to positive voltages. The apparent desensitization was seen in approximately 50% of the experiments, with both the native Ca release channel (in the absence of ryanodine) and the ryanodine-activated channel (1 microM ryanodine). The native Ca release channels revealed heterogeneous gating with regard to activation by ATP and binding to ryanodine. Most channels had high affinity to ATP activation (average open probability (po) = 0.55, 2 mM ATP, 100 microM Ca), whereas a small portion of channels had low affinity to ATP activation (po = 0.11, 2 mM ATP, 100 microM Ca), and some channels bound ryanodine faster (< 2 min), whereas others bound much slower (> 20 min). The faster ryanodine-binding channels always desensitized at large negative voltages, whereas those that bound slowly did not show apparent desensitization. The heterogeneity of the reconstituted Ca release channels is likely due to the regulatory roles of other junctional SR membrane proteins on the Ca release channel.     

Coupled gating between individual cardiac ryanodine calcium release channels

In order to study interactions between ryanodine receptor calcium release (RyR2) channels during excitation-contraction coupling in cardiac muscle, we used bilayer lipid membrane (BLM) and improved the method of cardiac sarcoplasmic vesicle fusion into BLM. We increased fusion gradient for the vesicles, used chloride ions for fusion up to concentration of 1.2 mol/l and fused the vesicles by adding them directly to the forming BLM. Under these conditions, increased probability of fusion of vesicles containing 2-7 ryanodine channels into BLM was observed. Interestingly about 10% of the channels did not gate into BLM independently, but their gating was coupled. At 53 mmol/l calcium solution, two coupled gating channels had double conductance (191 +/- 15 pS) in comparison with the noncoupled channels (93 +/- 10 pS). Activities of the coupled channels were decreased by 5 micromol/l ryanodine and inhibited by 10 micromol/l ruthenium red similarly as single RyR2 channels. We suppose that cardiac sarcoplasmic vesicles contain single as well as coupled RyR2 channels.     

Energetics of divalent selectivity in a calcium channel: the ryanodine receptor case study

A model of the ryanodine receptor (RyR) calcium channel is used to study the energetics of binding selectivity of Ca²⁺ versus monovalent cations. RyR is a calcium-selective channel with a DDDD locus in the selectivity filter, similar to the EEEE locus of the L-type calcium channel. While the affinity of RyR for Ca²⁺ is in the millimolar range (as opposed to the micromolar range of the L-type channel), the ease of single-channel measurements compared to L-type and its similar selectivity filter make RyR an excellent candidate for studying calcium selectivity. A Poisson-Nernst-Planck/density functional theory model of RyR is used to calculate the energetics of selectivity. Ca²⁺ versus monovalent selectivity is driven by the charge/space competition mechanism in which selectivity arises from a balance of electrostatics and the excluded volume of ions in the crowded selectivity filter. While electrostatic terms dominate the selectivity, the much smaller excluded-volume term also plays a substantial role. In the D4899N and D4938N mutations of RyR that are analyzed, substantial changes in specific components of the chemical potential profiles are found far from the mutation site. These changes result in the significant reduction of Ca²⁺ selectivity found in both theory and experiments.     

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.     

Regulation of the ryanodine receptor calcium release channel: a molecular complex system

The thermal behaviour and structural changes associated with the phase transformation of 1,2-dipalmitoyl-sn-glycerol (DPG) were studied by means of simultaneous X-ray diffraction and differential scanning calorimetry. Metastable DPG solid phases are crystallized from the melted sample by thermal quenching. The metastable phase (alpha-phase) formed initially is converted into a stable phase (beta' phase) at approximately 50 degrees C on heating. It was found that the behaviour of the alpha- to beta'-phase transformation depends on the thermal history. DPG solid samples incubated at approximately 3 degrees C for more than 10 h after cooling transformed directly into the beta'-phase with heat release. On the other hand, in the solid samples without incubation, the alpha-phase once melted and then the crystallization of the beta'-phase occurred successively from the melted state.     

Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide

We have recently reported [Mészáros L.G., Minarovic I., Zahradníková A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 1996; 380: 49–52] that nitric oxide (NO) reduces the activity of the skeletal muscle ryanodine receptor Ca²⁺ release channel (RyRC), a principal component of the excitation-contraction coupling machinery in striated muscles. Since (i) as shown here, we have obtained evidence which indicates that the NO synthase (eNOS) of cardiac muscle origin co-purified with RyRC-containing sarcoplasmic reticulum (SR) fractions; and (ii) the effects of NO donors on the release channel, as well as on cardiac function, appear somewhat contradictory, we have made an attempt to investigate the response of the cardiac RyRC to NO that is generated in situ from L-arginine in the NOS reaction. We found that L-arginine-derived NO inactivates Ca²⁺ release from cardiac SR and reduces the steady-state activity (i.e. open probability) of single RyRCs fused into a planar lipid bilayer. This reduction was prevented by NOS inhibitors and the NO quencher hemoglobin and was reversed by 2-mercaptoethanol. We thus conclude that: (i) in isolated SR preparations, it is possible to assess the effects of NO that is generated from L-arginine in the NOS reaction; and (ii) cardiac RyRc responds to NO in a manner which is identical to that we have previously found with the skeletal channel. These findings suggest that the direct modulation of the RyRC by NO is a signaling mechanism which likely participates in earlier demonstrated NO-induced myocardial contractility changes.     

Membrane topology and membrane retention of the ryanodine receptor calcium release channel

The ryanodine receptor (RyR) is a Ca²⁺ release channel located in the sarcoplasmic/endoplasmic reticulum (ER) membrane and plays a critical role in excitation-contraction coupling of skeletal and cardiac muscles. RyR normally exists in a tetrameric structure and contains two functional domains: a carboxyl-terminal hydrophobic domain that contains the conduction pore of the Ca²⁺ release channel, and a large amino-terminal domain that contains sites responsible for channel regulation. Recent studies involving mutagenesis and heterologous expression have helped unravel the structure-function relationship of RyR, including transmembrane topology and intracellular localization of the Ca²⁺-release channel. The carboxyl-terminal portion of RyR contains the putative transmembrane segments and is sufficient to form a functional Ca²⁺-release channel. The amino-terminal region of the protein contains sites responsible for regulation by endogenous modulators such as Ca²⁺ and Mg²⁺ and by exogenous ligands such as caffeine. The membrane topology of RyR appears to contain an even number (four or six) of transmembrane segments with a ion selectivity filter present within a region residing between the last two segments, similar to potassium channel, whose atomic structure was described recently. The transmembrane segments also contain sequences that are responsible for localization of RyR in the endoplasmic reticulum, and this sequence is highly conserved in IP₃ receptors, which also function as Ca²⁺-release channels.     

Intracellular calcium release mediated by two muscarinic receptor subtypes

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K⁺-specific conductance of ‘small’ single-channel amplitude similar to the SK type [1]. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.     

SH oxidation stimulates calcium release channels (ryanodine receptors) from excitable cells

The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.     

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

Cardioprotective effect of selenium via modulation of cardiac ryanodine receptor calcium release channels in diabetic rat cardiomyocytes through thioredoxin system

Increased oxidative stress contributes to heart dysfunction via impaired Ca²⁺ homeostasis in diabetes. Abnormal RyR2 function related with altered cellular redox state is an important factor in the pathogenesis of diabetic cardiomyopathy, while its underlying mechanisms remain poorly understood. In the present study, we used a streptozotocin-induced rat model of diabetic cardiomyopathy and tested a hypothesis that diabetes-related alteration in RyR2 function is related with ROS-induced posttranslational modifications. For this, we used heart preparations from either a diabetic rat or a sodium selenate (NaSe)-treated (0.3 mg/kg for 4 weeks) diabetic rat as well as either NaSe- (100 nmol/L) or thioredoxin (Trx; 5 μmol/L)-incubated (30 min) diabetic cardiomyocytes. Experimental approaches included imaging of intracellular free-Ca²⁺ ([Ca²⁺]_i) under both electrically stimulated and resting Fluo-3-loaded cardiomyocytes. RyR2-mediated SR-Ca²⁺ leak was significantly enhanced in diabetic cardiomyocytes, resulting in reduced amplitude and prolonged time courses of [Ca²⁺]_i transients compared to those of controls. Both SR-Ca²⁺ leak and [Ca²⁺]_i transients were normalized by treating diabetic rats with NaSe or by incubating diabetic myocytes with NaSe or Trx. Moreover, exposure of diabetic cardiomyocytes to antioxidants significantly improved [Ca²⁺]_i handling factors such as phosphorylation/protein levels of RyR2, amount of RyR2-bound FKBP12.6 and activities of both protein kinase A and CaMKII. NaSe treatment also normalized the oxidative stress/antioxidant defense biomarkers in plasma as well as Trx activity and nuclear factor-κB phosphorylation in the diabetic rat heart. Collectively, these findings suggest that redox modification through Trx-system besides the glutathione system contributes to abnormal function of RyR2s in hyperglycemic cardiomyocytes, presenting a potential therapeutic target for treating diabetics to preserve cardiac function.     

FK506 binding protein associated with the calcium release channel (ryanodine receptor).

The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.     

Toward a molecular understanding of the structure-function of ryanodine receptor Ca2+ release channels

Identification of the genetic basis of human diseases linked to dysfunctional free calcium (Ca2+) signaling has triggered an explosion of interest in the functional characterization of the molecular components regulating intracellular Ca2+ homeostasis. There is a growing appreciation of the central role of intracellular ryanodine-sensitive Ca2+ release channel (RyR) regulation in skeletal and cardiac muscle pathologies, including malignant hyperthermia, heart failure, and sudden cardiac death. The use of cloned RyR isoforms and recombinant expression techniques has greatly facilitated the elucidation of the molecular basis of RyR Ca2+ release functionality. This review will focus on the recombinant techniques used in the functional characterization of recombinant RyR isoforms and the insights that these approaches have yielded in unraveling the mechanistic basis of RyR channel functionality.     

Regulation of the ryanodine receptor calcium release channel of the sarcoplasmic reticulum in skeletal muscle

In striated muscle contraction is under the tight control of myoplasmic calcium concentration ([Ca2+]i): the elevation in [Ca2+]i and the consequent binding of calcium to troponin C enables, while the decrease in [Ca2+]i prevents the actin-myosin interaction. Calcium ions at rest are stored in the sarcoplasmic reticulum (SR) from which they are rapidly released upon the depolarisation of the sarcolemmal and transverse (T-) tubular membranes of the muscle cell. The protein responsible for this controlled and fast release of calcium is the calcium release channel found in the membrane of the terminal cisternae of the SR. This review focuses on the physiological and pharmacological modulators of the calcium release channel and tries to draw an up-to-date picture of the events that occur between T-tubular depolarisation and the release of calcium from the SR.     

Inhibition of dihydropyridine-sensitive calcium channels by the plant alkaloid ryanodine

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200-110 with an apparent inhibition constant of approx. 95 microM and inhibits dihydropyridine-sensitive calcium channels in the same preparation with an IC50 of approx. 45 microM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1-26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.     

Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor)

Metabolically (35)S-labeled calmodulin (CaM) was used to determine the CaM binding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [(3)H]ryanodine binding and single-channel measurements. Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca(2+); in the presence of 100 microm Ca(2+), the vesicles bound 7.5 CaM molecules per tetramer. Purified RyR2 bound approximately four [(35)S]CaM molecules per RyR tetramer, both in the presence and absence of Ca(2+). At least four CaM binding domains were identified in [(35)S]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redox state as controlled by the presence of either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced by Ca(2+) concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors.     

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.     

Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel/ryanodine receptor from skeletal muscle

The calcium release channel (CRC) from skeletal muscle is an unusually large tetrameric ion channel of the sarcoplasmic reticulum, and it is a major component of the triad junction, the site of excitation contraction coupling. The three-dimensional architecture of the CRC was determined from a random conical tilt series of images extracted from electron micrographs of isolated detergent-solubilized channels prepared in a frozen-hydrated state. Three major classes of fourfold symmetric images were identified, and three-dimensional reconstructions were determined for two of these. The two independent reconstructions were almost identical, being related to each other by a 180 degrees rotation about an axis in the plane of the specimen grid. The CRC consists of a large cytoplasmic assembly (29 x 29 x 12 nm) and a smaller transmembrane assembly that protrudes 7 nm from one of its faces. A cylindrical low-density region, 2-3 nm in apparent diameter, extends down the center of the transmembrane assembly, and possibly corresponds to the transmembrane Ca(2+)-conducting pathway. At its cytoplasmic end this channel-like feature appears to be plugged by a globular mass of density. The cytoplasmic assembly is apparently constructed from 10 or more domains that are loosely packed together such that greater than 50% of the volume enveloped by the assembly is occupied by solvent. The cytoplasmic assembly is suggestive of a scaffolding and seems well adapted to maintain the structural integrity of the triad junction while allowing ions to freely diffuse to and away from the transmembrane assembly.