Skeletal Sarcoplasmic Reticulum Research Articles

Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform.

Background and PurposeStatins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca2+ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors.Experimental ApproachRyR1 and RyR2 single‐channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage‐clamp conditions. LC‐MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy‐acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca2+ release.Key ResultsHydroxy acid simvastatin (active at HMG‐CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca2+ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro‐drug form of simvastatin (inactive at HMG‐CoA reductase) also activated RyR1, suggesting that the HMG‐CoA inhibitor pharmacophore was not responsible for RyR1 activation.Conclusion and ImplicationsSimvastatin interacts with RyR1 to increase SR Ca2+ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca2+‐dependent arrhythmias and sudden cardiac death.

Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors

BackgroundScorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. MethodsRecombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. Results[3H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with Kd=17.4±4.0nM. Intrepicalcin significantly augments the bell-shaped [Ca2+]-[3H]ryanodine binding curve at all [Ca2+] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca2+ release at an initial dose=45.3±2.5nM, and depletes ~50% of Ca2+ load from skeletal sarcoplasmic reticulum vesicles. ConclusionsWe conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. General significanceThis is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs.

Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

Simvastatin Activates Single Skeletal RyR1 Channels but Exerts More Complex Regulation of the Cardiac Isoform, RyR2

Statins are widely prescribed for the treatment of hypercholesterolemia. Although effective in lowering cholesterol levels, statin use has been associated with muscle weakness, myopathies and, in rare cases, rhabdomyolysis. The mechanisms underlying toxicity have not been elucidated, however, cerivastatin, a compound removed from the market due to reports of rhabdomyolysis, has been shown to trigger Ca2+ release from isolated skeletal sarcoplasmic reticulum (SR) vesicles (Inoue et al., 2003). To investigate whether statins can directly activate RyR channels, we incorporated single sheep cardiac RyR2 or mouse skeletal RyR1 channels into planar phospholipid bilayers under voltage-clamp conditions and added simvastatin, a commonly prescribed statin, to the cytosolic channel side. In the presence of 10 μM cytosolic [Ca2+], 10 μM simvastatin (carboxylate form), significantly increased RyR1 open probability (Po) from 0.020±0.013 to 0.269±0.068 (SEM, n=13, p<0.001). This effect was fully reversible after washout of simvastatin (0.033±0.029, SEM, n=6). The ability of simvastatin to activate RyR1 was further highlighted by our experiments showing that simvastatin is more effective than caffeine at stimulating [3H]ryanodine binding to skeletal SR (n=3, p<0.01). Unexpectedly, we found that 10 µM simvastatin did not activate RyR2 but that higher concentrations were required to increase Po. Low concentrations of simvastatin (1 µM) tended to reduce Po. These results may explain why the most frequently reported adverse effects of statins are of skeletal rather than cardiac origin. Confirmation that the direct modulation of RyR1 and RyR2 observed in bilayer experiments is relevant in cells is shown in permeabilised cardiac and skeletal myocytes where simvastatin increased the frequency of Ca2+ sparks in skeletal cells but decreased frequency in cardiac cells (see poster by Lotteau et al.).Funded by the British Heart Foundation.

Differential Effects of Temperature and Lipids on the Gating of RyR and SR K+ Channels

The ryanodine receptor (RyR) is the pathway for release of the sarcoplasmic reticulum (SR) Ca2+ required for cardiac and skeletal muscle contraction. The opening of SR K+ channels is thought to support this process. Movements of the RyR and SR K+ channel gates required for channel opening and closing will be dependent on the biophysical properties of the SR membrane which, in turn, will be affected by factors such as temperature and membrane composition. Cardiac RyR channel opening is hypersensitive to temperature changes (Sitsapesan et al., 1991, J Physiol., 434:469-488) although single-channel recordings are invariably performed at room temperature. We have therefore incorporated mammalian cardiac and skeletal RyR and SR K+ channels into bilayers under voltage-clamp conditions to investigate the effects of changing temperature and membrane lipid composition on single-channel behaviour.Changing bilayer lipid composition from phosphatidylethanolamine (PE) only, to a more physiological composition containing a 5:4:1 ratio of PE: phosphatidylserine (PS): phosphatidylcholine (PC) at 23oC, significantly increased the open probability (Po) and single channel conductance of both channels. For example, RyR Po increased from 0.06±0.03 to 0.26±0.08 (n=8; SEM; P<0.05). With bilayer lipid ratio of 5 PE: 4 PS: 1 PC, raising temperature from 10oC to 37oC caused a graded decrease in RyR Po. In contrast, SR K+ channels exhibited non-linear changes in Po with a minimum at approximately 23oC. Our results demonstrate the importance of investigating intracellular ion channel function at physiological temperatures and show that changes in SR membrane lipid composition markedly influence the behaviour of ion channels in the SR.Supported by the British Heart Foundation.

FRET-Based RyR-Selective Detection Reveals No Significant Competition between CaM and S100A1 Binding to RyRs

Using fluorescence resonance energy transfer (FRET), we directly tested the hypothesis that S100A1 competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. This hypothesis is largely based on competition assays using isolated sarcoplasmic reticulum (SR) membranes from skeletal muscle and evidence that S100A1 binds to peptides corresponding to one of the proposed CaM binding domains on RyR1. However, competition between S100A1 and CaM for RyR binding has not been directly detected. We targeted a donor-labeled FKBP12.6 (D-FKBP) to the cytoplasmic domain of RyR1 or RyR2 in SR vesicles isolated from pig skeletal or cardiac muscle. FRET was detected as a decrease of D-FKBP fluorescence in the presence of 100nM acceptor-labeled CaM (A-CaM) and used to index CaM binding to RyR. Upon pre-incubating SR with [S100A1] ranging from 0.01 to >100 μM, we found partial inhibition of FRET, with μM KI, for both skeletal and cardiac SR. By comparison, FRET was completely inhibited by unlabeled WT-CaM (KI ∼100nM), indicating that A-CaM and WT-CaM bind RyR with similar affinities and at the same site. Similar results were obtained using co-sedimentation assays conducted under similar experimental conditions, to detect competition between S100A1 and CaM binding to SR membranes. Taken together, these results indicate that CaM-RyR binding may not be significantly competed by S100A1 under normal physiologic conditions. Furthermore, structural analysis of FRET data suggests that S100A1 allosterically interacts with RyR-CaM binding. Initial results from a complementary FRET approach, using acceptor-labeled S100A1, further support the conclusion that S100A1 does not significantly compete with CaM-RyR binding in skeletal or cardiac muscle.

DHBP Block of Ryanodine Receptor Channels

1,1′-diheptyl-4,4′-bipyridinium dibromide (DHBP), a divalent organic cation, was reported to inhibit muscle contraction, which was attributed to inhibition of ryanodine receptor (RyR) mediated Ca2+ release from the sarcoplasmic reticulum (SR). Still, single RyR channel studies are lacking and the mechanism by which DHBP blocks RyRs is unknown. We reconstituted RyRs from skeletal muscle and heart SR microsomes into planar bilayers. We observed with DHBP addition to the cytosolic surface of RyRs (clamped at Vm = 0 mV) decreased channel conductance (IC50 ∼ 1 mM). The percentage block in channel conductance elicited by DHBP was invariable in the range −30 to +30 mV. DHBP did not affect RyR gating characteristics, which was measured under Ca2+ conditions that produced low, moderate or high Po and in the presence/absence of physiological modulators (ATP, Mg2+). DHBP added to luminal and cytosolic sides of the RyRs, resulted in additive effects on channel conductance. DHBP also induced inhibition of RyR-mediated SR Ca2+ release from skeletal SR microsomes. A similar block of RyRs conductance was observed with other bipyridinum derivatives, none of which affected SERCA ATPase activity. Previous reports in the literature indicated block of RyRs by DHBP in cells required lower drug concentrations, suggesting a possible cellular mechanism of DHBP accumulation. In summary, DHBP blocks RyRs Ca2+ conductance resembling the action of polyamines and organic cations. DHBP properties make it an attractive experimental probe for understanding the role of RyR conductance in Ca2+ release from intracellular Ca2+ stores (Supported by American Heart Association and Eskridge Foundation).

Benzodiazepines and Benzothiazepines as Modulators of the Sarcoplasmic Reticulum Calcium ATP-ase and Ryanodine Receptors in Striated Muscle

We have reported that CGP-37157, a benzothiazepine (BZT) derivative of clonazepam utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger, also activates ryanodine receptors (RyRs) and inhibits the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase (SERCA). We extended the studies to other BZT (e.g., clonazepan, diltiazem) as well as to benzodiazepines (BZD; e.g. diazepan, lorazepan). We aimed to determine if these drug classes have as a common trait the ability to modulate RyRs and/or SERCA. The effects of BZD and BZT on RyRs activity were tested in SR microsomes with a Ca2+ leak assay as well as after reconstituting RyRs into lipid bilayers. The agents tested had variable potency to increase RyR-mediated Ca2+ leak from skeletal SR microsomes. As an example, while diazepam significantly increased RyR activity, most of the others (clonazepam, lorazepam) had minor effects at high doses. In contrast, diltiazem produced moderate inhibition. Planar bilayer studies confirmed the leak observations with both cardiac and skeletal RyR. In the presence of ruthenium red, most agents decreased the rate of loading which indicates inhibitory effects on SERCA activity. The effects of BZT and BZD on loading correlated with a decrease in ATPase activity of SERCA-enriched skeletal SR fractions. In summary, most BZT and BZD utilized in therapeutics or as pharmacological tools have an inhibitory action on SERCA. In contrast, they show a variety of effects on RyRs ranging from inhibition to activation. Hence, the pharmacological action of BZT and BZD on cellular Ca2+ homeostasis reported in the literature of cardiac and skeletal muscle as well as other non-muscle systems may require taking into consideration the contributions of all drug-sensitive intracellular Ca2+ transporters. (Supported by NIH-GM078665 and AHA-MWA 12180038).

CGP37157 is an Inhibitor of the Sarcoplasmic Reticulum Calcium ATPase and an Activator of Ryanodine Receptors in Striated Muscle

CGP-37157 (CGP), a benzothiazepine derivative of clonazepam, is commonly utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger. Yet, evidence suggests that CGP could also affect other targets, such as L-type Ca2+ channels and plasmalemma Na+/Ca2+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptors (RyR) and/or sarcoplasmic reticulum (SR) Ca2+ stimulated ATPase (SERCA) by CGP. SERCA activity was measured in SR microsomes with a ATP-ase activity assay or Ca2+ loading in presence of ruthenium red (RyR blocker). The effects of CGP on RyR activity were performed in SR microsomes with a Ca2+ leak assay or after reconstitution of the channels into planar lipid bilayers. CGP inhibited SERCA-mediated Ca2+ uptake of cardiac and skeletal SR microsomes (IC50's = 6.6 and 9.9 µM, respectively). The CGP effects on SERCA activity correlated with a decreased Vmax of ATPase activity of SERCA-enriched skeletal SR fractions without an apparent change in Km. CGP (≥ 5 µM) also increased RyR-mediated Ca2+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyR are directly activated by CGP (EC50's = 9.4 and 12.0 μM, respectively). In summary, we found that CPG inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca2+ homeostasis reported in the literature of cardiac, skeletal muscle as well as other non muscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca2+ transporters. (Supported by NIH R01 GM078665).

Climate Change and Housing Prices: Hedonic Estimates for Ski Resorts in Western North America

Sarcoplasmic reticulum (SR) vesicles prepared from rat ventricle muscle are isolated, and their [3H]ryanodine-binding and calcium transport properties are studied in detail under active loading conditions in the presence of pyrophosphate. Experiments are performed in tandem with rabbit skeletal SR under identical conditions to allow direct comparisons of the mechanisms by which activators and inhibitors influence the calcium release channel. Ca(++)-induced Ca++ release is demonstrated with both preparations and the cardiac channel is about 1.5-fold more sensitive to activation by Ca++, which is in excellent quantitative agreement with the ability of Ca++ to activate [3H]ryanodine-binding sites. The cardiac and skeletal receptors show major quantitative differences with respect to sensitivity to pharmacologic modulators, cations and pH. The inhibitors ruthenium red, Mg++ and neomycin are significantly more potent in inhibiting the skeletal receptor, whereas the activators daunorubicin and caffeine are significantly more potent towards the cardiac receptor. The ATP analog, beta,gamma-methyleneadenosine 59-triphosphate, enhances the binding of [3H]ryanodine to the high-affinity site in skeletal SR by a factor of 4 but has a negligible effect on the cardiac receptor, although at suboptimal Ca++ for the binding of ryanodine, beta,gamma-methyleneadenosine 59-triphosphate activates the cardiac receptor to a greater extent. High levels of salt (1 M NaCl) enhance the rate of [3H]ryanodine association with its binding sites in both preparations, although they selectively reduce the binding-site capacity in skeletal SR due to a failure to maintain a stable equilibrium. Although high- and low-affinity binding of [3H]ryanodine have a similar response to changing pH, the skeletal receptors are significantly more sensitive to pH.(ABSTRACT TRUNCATED AT 250 WORDS)

CGP-37157 Inhibits the Sarcoplasmic Reticulum Ca2+ATPase and Activates Ryanodine Receptor Channels in Striated Muscle

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC₅₀ values of 6.6 and 9.9 μM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 μM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 μM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

Charade of the SR K+-Channel: Two Ion-Channels, TRIC-A and TRIC-B, Masquerade as a Single K+-Channel

The presence of a sarcoplasmic reticulum (SR) K+-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K+-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K+ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K+-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K+-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca2+ and inhibited by luminal Ca2+. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.

CLIC2-RyR1 Interaction and Structural Characterization by Cryo-electron Microscopy

Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [ 3H]ryanodine binding, Ca 2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [ 3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca 2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

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.

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.

Calumenin, a multiple EF-hands Ca 2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum

Calumenin is a multiple EF-hand Ca 2+-binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. AY225335 and AY225336, respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca 2+concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca 2+ release, but decreased depolarization-induced Ca 2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca 2+-dependent manner.

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

Reactions with Dye Free Radicals Reveal Weak Redox Properties of Drugs

The calcium release channel (CRC) of the skeletal sarcoplasmic reticulum is rich in thiol groups and is strongly regulated by covalent modification of these thiols. Oxidizing reagents activate the release channel, whereas reducing reagents inhibit the channel. However, most CRC regulators are not thiol reagents. Here, we propose that reversible redox interactions are involved in regulation of the CRC by nonthiol reagents. This hypothesis was tested with several CRC regulators. The local anesthetics tetracaine, procaine and QX-314, which block the CRC, behaved as electron donors in reactions with dye free radicals. In contrast, ryanodine, caffeine, doxorubicin and daunorubicin, compounds known to activate the release channel, all accepted electrons from dye anion radicals. Moreover, release of Ca2+ from SR initiated by doxorubicin (acceptor) was antagonized by local anesthetics (donors). Based on the redox characterization of CRC modulators, we propose a functional model in which channel inhibitors and activators act as weak electron donors and acceptors, respectively, and shift the thiol-disulfide balance within the release protein. The consequence of this model is that, in spite of the chemical diversity of CRC modulators, a common mechanism of channel regulation involves the transient exchange of electrons between the activator/inhibitor and the CRC.

Functional implications of modifying RyR‐activating peptides for membrane permeability

1. Our aim was to determine whether lipoamino acid conjugation of peptides that are high-affinity activators of ryanodine receptor (RyR) channels would (a) render the peptides membrane permeable, (b) alter their structure or (a) reduce their activity. The peptides correspond to the A region of the II-III loop of the skeletal dihydropyridine receptor. 2. The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to approximately 20-fold. 3. Nuclear magnetic resonance showed that the alpha-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation. 4. The conjugated peptides were more effective in enhancing resting Ca2+ release, Ca2+-induced Ca2+ release and caffeine-induced Ca2+ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca2+ release from mechanically skinned extensor digitorum longus (EDL) fibres. 5. The effect of both conjugated and unconjugated peptides on Ca2+ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca2+ release or on the Ca2+ Mg2+ ATPase. 6. A small and very low affinity effect of the peptide in slowing Ca2+ uptake by the Ca2+, Mg2+ ATPase was exacerbated by lipoamino acid conjugation in both isolated SR and in skinned EDL fibres. 7. The results show that lipoamino acid conjugation of A region peptides increases their membrane permeability without impairing their structure or efficacy in activating skeletal and cardiac RyRs.