Skeletal Muscle Ryanodine Receptor Channels Are Activated by the Fungal Metabolite, Gliotoxin

Interactions between the reactive disulfide fungal metabolite, gliotoxin (GTX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent complex with 100 μm ³⁵S-GTX, which was reversed by 1 mm dithiothreitol (DTT) or 1 mm glutathione. GTX (80–240 μm), added to either cytoplasmic (cis) or luminal (trans) solutions, increased the rate of Ca²⁺ release from SR vesicles and the frequency of opening of single RyR channels in lipid bilayers. Channel activation was reversed upon addition of 2 mm DTT to the cis solution, showing that the activation was due to an oxidation reaction (2 mm DTT added to the cis solution in the absence of GTX did not affect RyR activity). Furthermore, RyRs were not activated by trans GTX if the cis chamber contained DTT, suggesting that GTX oxidized a site in or near the membrane. In contrast to cis DTT, 2 mm DTT in the trans solution increased RyR activity when added either alone or with 200 μm trans GTX. The results suggest that (i) GTX increases RyR channel activity by oxidizing cysteine residues that are close to the membrane and located on RyR, or associated proteins, and (ii) a disulfide bridge or nitrosothiol, accessible only from the luminal solution, normally suppresses RyR channel activity. Some of the actions of GTX in altering Ca²⁺ homeostatsis might depend on its modification of RyR calcium channels. Received: 12 November 1999/Revised: 14 March 2000     

Structural Determinants of Skeletal Muscle Ryanodine Receptor Gating

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca²⁺. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912–Glu-4948) and a linker helix (Val-4830–Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.     

Role of Ryanodine Receptors in the Assembly of Calcium Release Units in Skeletal Muscle

Abstract. In muscle cells, excitation–contraction (e–c) coupling is mediated by “calcium release units,” junctions between the sarcoplasmic reticulum (SR) and exterior membranes. Two proteins, which face each other, are known to functionally interact in those structures: the ryanodine receptors (RyRs), or SR calcium release channels, and the dihydropyridine receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors, and tetrads are organized in arrays that face arrays of feet (or RyRs). Triadin is a protein of the SR located at the SR–exterior membrane junctions, whose role is not known. We have structurally characterized calcium release units in a skeletal muscle cell line (1B5) lacking Ry₁R. Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR–exterior membrane junctions lacking foot structures (dyspedic). These results suggest that components other than Ry₁Rs are responsible for targeting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 cells are not grouped into tetrads as in normal skeletal muscle cells suggesting that anchoring to Ry₁Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by finding a “restoration of tetrads” in junctional domains of surface membranes after transfection of 1B5 cells with cDNA encoding for Ry₁R.     

A Procedure for Purification of the Ryanodine Receptor from Skeletal Muscle

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636–16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [³H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.     

Characterization of the Calcium-release Channel/Ryanodine Receptor from Zebrafish Skeletal Muscle

Calcium (Ca²⁺)-mediated signaling is fueled by two sources for Ca²⁺: Ca²⁺ can enter through Ca²⁺ channels located in the plasma membrane and can also be released from intracellular stores. In the present study the intracellular Ca²⁺ release channel/ryanodine receptor (RyR) from zebrafish skeletal muscle was characterized. Two RyR isoforms could be identified using immunoblotting and single-channel recordings. Biophysical properties as well as the regulation by modulators of RyR, ryanodine, ruthenium red and caffeine, were measured. Comparison with other RyRs showed that the zebrafish RyRs have features observed with all RyRs described to date and thus, can serve as a model system in future genetic and physiological studies. However, some differences in the biophysical properties were observed. The slope conductance for both isoforms was higher than that of the mammalian RyR type 1 (RyR1) measured with divalent ions. Also, inhibition by millimolar Ca²⁺ concentrations of the RyR isoform that is inhibited by high Ca²⁺ concentrations (teleost α RyR isoform) was attenuated when compared to mammalian RyRs. Due to the widespread expression of RyR these findings have important implications for the interpretation of the role of the RyR in Ca²⁺ signaling when comparing zebrafish with mammalian physiology, especially when analyzing mutations underlying physiological changes in zebrafish. Received: 15 February 2001/Revised: 1 June 2001     

Imperatoxin A Induces Subconductance States in Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single-channel and [³H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTx_a), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca²⁺ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTx_a in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTx_a was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTx_a reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTx_a binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTx_a binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTx_a induced substates in the ryanodine-modified skeletal CRC and increased or reduced [³H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTx_a induces subconductance states in skeletal and cardiac muscle Ca²⁺ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.     

Ryanodine Receptor Luminal Ca Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca²⁺ release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca²⁺ channel and the intra-SR Ca²⁺ buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca²⁺ regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca²⁺ dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca²⁺) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca²⁺ sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca²⁺ sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca²⁺ regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca²⁺ regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca²⁺ buffer.     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.     

Action Potential-Evoked Calcium Release Is Impaired in Single Skeletal Muscle Fibers from Heart Failure Patients

Background

Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca²⁺) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca²⁺ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers.

Methods and Findings

Using state-of-the-art electrophysiological and optical techniques in single muscle fibers from biopsies of the locomotive vastus lateralis muscle, we measured the action potential (AP)-evoked Ca²⁺ release in 4 HF patients and 4 age-matched healthy controls. The mean peak Ca²⁺ release flux in fibers obtained from HF patients (10±1.2 µM/ms) was markedly (2.6-fold) and significantly (p<0.05) smaller than in fibers from healthy volunteers (28±3.3 µM/ms). This impairment in AP-evoked Ca²⁺ release was ubiquitous and was not explained by differences in the excitability mechanisms since single APs were indistinguishable between HF patients and healthy volunteers.

Conclusions

These findings prove the feasibility of performing electrophysiological experiments in single fibers from human skeletal muscle, and offer a new approach for investigations of myopathies due to HF and other diseases. Importantly, we have demonstrated that one step in the ECC process, AP-evoked Ca²⁺ release, is impaired in single muscle fibers in HF patients.     

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca²⁺ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca²⁺ release response in HEK293 cells and bound the RyR-specific ligand [³H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K⁺ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca²⁺ release in HEK293 cells, low [³H]ryanodine binding levels, and channels that were not regulated by Ca²⁺ and did not conduct Ca²⁺ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.     

Silent Calcium Channels in Skeletal Muscle Fibers of the Crustacean Atya lanipes

The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca²⁺ action potentials when depolarized and have no detectable inward Ca²⁺ current. These fibers, however, are strictly dependent on Ca²⁺ influx for contraction, suggesting that they depend on Ca²⁺-induced Ca²⁺ release for contractile activation. The nature of the communication between Ca²⁺ channels in the sarcolemmal/tubular membrane and Ca²⁺ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr²⁺ and Ba²⁺ action potentials could be elicited in Ca²⁺-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr²⁺ or Ba²⁺ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V _max , increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca²⁺ concentration the channels were silent, i.e., no inward Ca²⁺ current was detected. In Ca²⁺-free solutions, inward currents carried by 138 mm Sr²⁺ or Ba²⁺ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca²⁺ channels. Peak inward current density values were low, ca.−33 μA/cm² for Sr²⁺ and −14 μA/cm² for Ba²⁺, suggesting that Ca²⁺ channels are present at a very low density. It is concluded that Ca²⁺-induced Ca²⁺ release in this crustacean muscle operates with an unusually high gain: Ca²⁺ influx through the silent Ca²⁺ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca²⁺ in the immediate vicinity of the sarcoplasmic reticulum Ca²⁺ release channels to trigger the highly amplified release of Ca²⁺ required for tension generation. Received: 5 April 1999/Revised: 15 September 1999     

Voltage-Activated Elementary Calcium Release Events in Isolated Mouse Skeletal Muscle Fibers

The elementary Ca²⁺-release events underlying voltage-activated myoplasmic Ca²⁺ transients in mammalian muscle remain elusive. Here, we looked for such events in confocal line-scan (x,t) images of fluo-3 fluorescence taken from isolated adult mouse skeletal muscle fibers held under voltage-clamp conditions. In response to step depolarizations, spatially segregated fluorescence signals could be detected that were riding on a global increase in fluorescence. These discrete signals were separated using digital filtering in the spatial domain; mean values for their spatial half-width and amplitude were 1.99 ± 0.09 μm and 0.16 ± 0.005 ΔF/F ₀ (n = 151), respectively. Under control conditions, the duration of the events was limited by the pulse duration. In contrast, in the presence of maurocalcine, a scorpion toxin suspected to disrupt the process of repolarization-induced ryanodine receptor (RyR) closure, events uninterrupted by the end of the pulse were readily detected. Overall results establish these voltage-activated low-amplitude local Ca²⁺ signals as inherent components of the physiological Ca²⁺-release process of mammalian muscle and suggest that they result from the opening of either one RyR or a coherently operating group of RyRs, under the control of the plasma membrane polarization.     

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca²⁺, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca²⁺, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca²⁺, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca²⁺ stabilized the closed conformation by a factor of two. We conclude that the Ca²⁺-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca²⁺-dependent structural dynamics of bound CaM.     

Growth Associated Protein 43 Is Expressed in Skeletal Muscle Fibers and Is Localized in Proximity of Mitochondria and Calcium Release Units

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.     

Identification, Characterization and Partial Purification of a Thiol-protease Which Cleaves Specifically the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel

A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca²⁺ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl₂, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca²⁺ at a submicromolar concentration, the isolated thiol-protease has lost the Ca²⁺ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca²⁺-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94. Received: 10 December 1996/Revised: 11 August 1997     

Cardiac Sarcalumenin: Phosphorylation, Comparison with the Skeletal Muscle Sarcalumenin and Modulation of Ryanodine Receptor

Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca²⁺ binding protein (HCP) with respect to: (i) Ca²⁺ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg²⁺ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La³⁺; (iv) phosphorylation is obtained with [γ-³²P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La³⁺. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca²⁺- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified. Received: 15 December 1998/Revised: 25 March 1999     

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

Background

Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive.

Methodology/Principal Findings

To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s.

Conclusions/Significance

We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.     

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release

Aberrant Zn²⁺ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn²⁺ modulates cardiac ryanodine receptor gating and Ca²⁺ dynamics in isolated cardiomyocytes. We reveal that Zn²⁺ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn²⁺ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca²⁺. At concentrations of free Zn²⁺ >1 nm, Zn²⁺ is the main activating ligand, and the dependence on Ca²⁺ is removed. Zn²⁺ is therefore a higher affinity activator of RyR2 than Ca²⁺. Millimolar levels of free Zn²⁺ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn²⁺ modulates both the frequency and amplitude of Ca²⁺ waves in a concentration-dependent manner and that physiological levels of Zn²⁺ elicit Ca²⁺ release in the absence of activating levels of cytosolic Ca²⁺. This highlights a new role for intracellular Zn²⁺ in shaping Ca²⁺ dynamics in cardiomyocytes through modulation of RyR2 gating.     

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ∼16.5 nm in diameter and ∼ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.