Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.     

Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.     

Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.     

The Mu class glutathione transferase is abundant in striated muscle and is an isoform-specific regulator of ryanodine receptor calcium channels

Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle. An hGSTM2-2-like protein was isolated from rabbit skeletal muscle and sheep heart, at concentrations of approximately 17-93 microM. When added to the cytoplasmic side of RyRs, hGSTM2-2 and GST isolated from skeletal or cardiac muscle, modified channel activity in an RyR isoform-specific manner. High activity skeletal RyR1 channels were inactivated at positive potentials or activated at negative potentials by hGSTM2-2 (8-30 microM). Inactivation became faster as the positive voltage was increased. Channels recovered from inactivation when the voltage was reversed, but recovery times were significantly slowed in the presence of hGSTM2-2 and muscle GSTs. Low activity RyR1 channels were activated at both potentials. In contrast, hGSTM2-2 and GSTs isolated from muscle (1-30 microM) in the cytoplasmic solution, caused a voltage-independent inhibition of cardiac RyR2 channels. The results suggest that the major GST isoform expressed in muscle regulates Ca2+ signalling in skeletal and cardiac muscle and conserves Ca2+ stores in the sarcoplasmic reticulum.     

Response of ryanodine receptor channels to Ca2+ steps produced by rapid solution exchange.

We used a flow method for Ca2+ activation of sheep cardiac and rabbit skeletal ryanodine receptor (RyR) channels in lipid bilayers, which activated RyRs in < 20 ms and maintained a steady [Ca2+] for 5 s. [Ca2+] was rapidly altered by flowing Ca(2+)-buffered solutions containing 100 or 200 microM Ca2+ from a perfusion tube inserted in the cis, myoplasmic chamber above the bilayer. During steps from 0.1 to 100 microM, [Ca2+] reached 0.3 microM (activation threshold) and 10 microM (maximum Po) in times consistent with predictions of a solution exchange model. Immediately following rapid RyR activation, Po was 0.67 (cardiac) and 0.45 (skeletal) at a holding voltage of +40 mV (cis/trans). Po then declined (at constant [Ca2+]) in 70% of channels (n = 25) with time constants ranging from .5 to 15 s. The mechanism for Po decline, whether it be adaptation or inactivation, was not determined in this study. cis, 2 mM Mg2+ reduced the initial Po for skeletal RyRs to 0.21 and marginally slowed the declining phase. During very rapid falls in [Ca2+] from mM (inhibited) to sub-microM (sub-activating) levels, skeletal RyR did not open. We conclude the RyR gates responsible for Ca(2+)-dependent activation and inhibition of skeletal RyRs can gate independently.     

Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.     

Ryanodine receptors

RyRs are large homotetrameric proteins that are approximately 4/5 cytoplasmic and approximately 1/5 transmembrane and luminal in mass. Mutations in RyRs produce human disease and many of these disease-causing mutations are in the cytoplasmic domains. To elucidate the mechanisms of a disease and to develop interventions, it is crucial to determine how the alterations in the cytoplasmic domains communicate with the transmembrane pore of this channel. One of the major activators of all three RyR isoforms is Ca2+ and some of the disease-causing mutations are thought to alter the sensitivity of the channels to Ca2+ activation. This review examines the current state of structural understanding of the RyR channel activation.     

Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP

Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca(2+) release underlying Ca(2+) sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818-821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ～25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ～10% of the cases, all RyRs (2-10 channels; most frequently 3-4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg(2+) and high (50 mM) luminal Ca(2+) (Ca(lum)) or Sr(2+) solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ～300 μs. Coupled RyR1 were heterogeneous, where highly active RyR1 ("drivers") seemed open during the entire coupled event (P(o) = 1), while other RyR1s ("followers") displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca(2+) (Ca(cyt)) or caffeine, whereas followers flicker frequency was Ca(cyt) independent and more sensitive to inhibition by cytosolic Mg(2+). Coupled events were insensitive to varying lumen-to-cytosol Ca(2+) fluxes from ～1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca(2+)-induced Ca(2+) release. However, coupling requires specific Ca(lum) sites, as it was lost when Ca(lum) was replaced by luminal Ba(2+) or Mg(2+). In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg(2+), Ca(cyt), and Ca(lum,) which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca(2+) sparks.     

The cytosolic N-terminus of presenilin-1 potentiates mouse ryanodine receptor single channel activity

Ryanodine receptors (RyRs) amplify intracellular Ca(2+) signals by massively releasing Ca(2+) from intracellular stores. Exaggerated chronic Ca(2+) release can trigger cellular apoptosis underlying a variety of neurodegenerative diseases. Aberrant functioning of presenilin-1 (PS1) protein instigates Ca(2+)-dependent apoptosis, providing a basis for the "calcium hypothesis" of Alzheimer's disease (AD). To get insight into this problem, we hypothesized that the previously reported physical interaction between RyR and PS1 modulates functional properties of the RyR. We generated a soluble cytoplasmic N-terminal fragment of PS1 comprising the first 82 amino acid (PS1 NTF(1-82)), the candidate for interaction with putative cytoplasmic modulatory sites of the RyR, and studied its effect on single channel currents of mouse brain RyRs incorporated in lipid bilayers. PS1 NTF(1-82) strongly increased both mean currents (EC(50)=12nM, Hill coefficient (n(H)) approximately 1) and open probability for higher sublevels for single RyR channels (EC(50)=7nM, n(H) approximately 2). Bell-shaped Ca(2+)-activation curve remained unchanged, suggesting that PS1 NTF(1-82) allosterically potentiates RyRs, but that the channel still requires Ca(2+) for activation. Corroborating such an independent mechanism, the RyR potentiation by PS1 NTF(1-82) was overridden by receptor desensitization at high [Ca(2+)] (pCa>5). This potentiation of RyR by PS1 NTF(1-82) reveals a new mechanism of physiologically relevant PS1-regulated Ca(2+) release from intracellular stores, which could be alternative or additional to recently reported intracellular Ca(2+) leak channels formed by PS1 holoproteins.     

Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.

The single-channel activity of rabbit skeletal muscle ryanodine receptor (skeletal RyR) and dog cardiac RyR was studied as a function of cytosolic [Ca2+]. The studies reveal that for both skeletal and cardiac RyRs, heterogeneous populations of channels exist, rather than a uniform behavior. Skeletal muscle RyRs displayed two extremes of behavior: 1) low-activity RyRs (LA skeletal RyRs, approximately 35% of the channels) had very low open probability (Po < 0.1) at all [Ca2+] and remained closed in the presence of Mg2+ (2 mM) and ATP (1 mM); 2) high-activity RyRs (HA skeletal RyRs) had much higher activity and displayed further heterogeneity in their Po values at low [Ca2+] (< 50 nM), and in their patterns of activation by [Ca2+]. Hill coefficients for activation (nHa) varied from 0.8 to 5.2. Cardiac RyRs, in comparison, behaved more homogeneously. Most cardiac RyRs were closed at 100 nM [Ca2+] and activated in a cooperative manner (nHa ranged from 1.6 to 5.0), reaching a high Po (> 0.6) in the presence and absence of Mg2+ and ATP. Heart RyRs were much less sensitive (10x) to inhibition by [Ca2+] than skeletal RyRs. The differential heterogeneity of heart versus skeletal muscle RyRs may reflect the modulation required for calcium-induced calcium release versus depolarization-induced Ca2+ release.     

The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.     

Modulation of the oligomerization of isolated ryanodine receptors by their functional states

The calcium release channels/ryanodine receptors (RyRs) usually form two-dimensional regular lattices in the endoplasmic/sarcoplasmic reticulum membranes. However, the function and modulation of the interaction between neighboring RyRs are still unknown. Here, with an in vitro aqueous system, we demonstrate that the interaction between RyRs isolated from skeletal muscle (RyR1s) is modulated by their functional states by using photon correlation spectroscopy and [(3)H]ryanodine binding assay. High level of oligomerization is observed for resting closed RyR1s with nanomolar Ca(2+) in solution. Activation of RyR1s by micromolar Ca(2+) or/and millimolar AMP leads to the de-oligomerization of RyR1s. The oligomerization of RyR1s remains at high level when RyR1s are stabilized at closed state by Mg(2+). The modulation of RyR1-RyR1 interaction by the functional state is also observed under near-physiological conditions, suggesting that the interaction between arrayed RyR1s would be dynamically modulated during excitation-contraction coupling. These findings provide exciting new information to understand the function and operating mechanism of RyR arrays.     

Ryanodine receptors: structure, expression, molecular details, and function in calcium release

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca(2+) from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (> 2MDa) and exist as three mammalian isoforms (RyR 1-3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.     

Coupled calcium release channels and their regulation by luminal and cytosolic ions

Contraction in skeletal and cardiac muscle occurs when Ca²⁺ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca²⁺ release channels. Several isoforms of the RyR exist throughout the animal kingdom, which are modulated by ATP, Ca²⁺ and Mg²⁺ in the cytoplasm and by Ca²⁺ in the lumen of the SR. This review brings to light recent findings on their mechanisms of action in the mammalian isoforms RyR-1 and RyR-2 with an emphasis on RyR-1 from skeletal muscle. Cytoplasmic Mg²⁺ is a potent RyR antagonist that binds to two classes of cytoplasmic site, identified as low-affinity, non-specific inhibition sites and high-affinity Ca²⁺ activation sites (A-sites). Mg²⁺ inhibition at the A-sites is very sensitive to the cytoplasmic and luminal milieu. Cytoplasmic Ca²⁺, Mg²⁺ and monovalent cations compete for the A-sites. In isolated RyRs, luminal Ca²⁺ alters the Mg²⁺ affinity of the A-site by an allosteric mechanism mediated by luminal sites. However, in close-packed RyR arrays luminal Ca²⁺ can also compete with cytoplasmic ions for the A-site. Activation of RyRs by luminal Ca²⁺ has been attributed to either Ca²⁺ feedthrough to A-sites or to Ca²⁺ regulatory sites on the luminal side of the RyR. As yet there is no consensus on just how luminal Ca²⁺ alters RyR activation. Recent evidence indicates that both mechanisms operate and are likely to be important. Allosteric regulation of A-site Mg²⁺ affinity could trigger Ca²⁺ release, which is reinforced by Ca²⁺ feedthrough.     

Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

Ruthenium red modifies the cardiac and skeletal muscle Ca(2+) release channels (ryanodine receptors) by multiple mechanisms.

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca(2+) flux, [(3)H]ryanodine binding, and single channel measurements. In vesicle-Ca(2+) flux measurements, RR was more effective in inhibiting RyRs at 0.2 microM than 20 microM free Ca(2+). [(3)H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca(2+) regulatory sites of RyRs. In symmetric 0.25 M KCl with 10-20 microM cytosolic Ca(2+), cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 microM Ca(2+) + 2 mM ATP) and cardiac (200 microM Ca(2+)) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca(2+)-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.