Distinct effects on Ca2+ handling caused by malignant hyperthermia and central core disease mutations in RyR1

Malignant hyperthermia (MH) and central core disease (CCD) are disorders of skeletal muscle Ca2+ homeostasis that are linked to mutations in the type 1 ryanodine receptor (RyR1). Certain RyR1 mutations result in an MH-selective phenotype (MH-only), whereas others result in a mixed phenotype (MH + CCD). We characterized effects on Ca2+ handling and excitation-contraction (EC) coupling of MH-only and MH + CCD mutations in RyR1 after expression in skeletal myotubes derived from RyR1-null (dyspedic) mice. Compared to wild-type RyR1-expressing myotubes, MH + CCD- and MH-only-expressing myotubes exhibited voltage-gated Ca2+ release (VGCR) that activated at more negative potentials and displayed a significantly higher incidence of spontaneous Ca2+ oscillations. However, maximal VGCR was reduced only for MH + CCD mutants (Y4795C, R2435L, and R2163H) in which spontaneous Ca2+ oscillations occurred with significantly longer duration (Y4795C and R2435L) or higher frequency (R2163H). Notably, myotubes expressing these MH + CCD mutations in RyR1 exhibited both increased [Ca2+]i and reduced sarcoplasmic reticulum (SR) Ca2+ content. We conclude that MH-only mutations modestly increase basal release-channel activity in a manner insufficient to alter net SR Ca2+ content ("compensated leak"), whereas the mixed MH + CCD phenotype arises from mutations that enhance basal activity to a level sufficient to promote SR Ca2+ depletion, elevate [Ca2+]i, and reduce maximal VGCR ("decompensated leak").     

Intracellular Ca2+ dynamics in malignant hyperthermia and central core disease: established concepts, new cellular mechanisms involved

Malignant hyperthermia (MH) and central core disease (CCD) are inherited human disorders of skeletal muscle Ca2+ homeostasis. Both MH and CCD are linked to mutations and/or deletions in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the intracellular Ca2+ release channel, which is essential to excitation-contraction (EC) coupling. Our knowledge on how mutations in RyR1 disrupt intracellular Ca2+ homeostasis and EC coupling, eventually leading to MH and CCD has been recently improved, thanks to multidisciplinary studies ranging from clinical, single channel recordings, patch-clamp experiments, and molecular biology. This review presents a brief historical perspective, on how pioneer studies resulted in associating MH and CCD to RyR1. The review is also focused on discussing novel results in regard to pathophysiological consequences of specific MH/CCD RyR1 mutant proteins, which are representative of the different cellular mechanisms that are linked to either phenotype.     

Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.     

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.     

Dynamic alterations in myoplasmic Ca2+ in malignant hyperthermia and central core disease

Ca2+ ions play a pivotal role in a wide array of cellular processes ranging from fertilization to cell death. In skeletal muscle, a mechanical interaction between plasma membrane dihydropyridine receptors (DHPRs, L-type Ca2+ channels) and Ca2+ release channels (ryanodine receptors, RyR1s) of the sarcoplasmic reticulum orchestrates a complex, bi-directional Ca2+ signaling process that converts electrical impulses in the sarcolemma into myoplasmic Ca2+ transients during excitation-contraction coupling. Mutations in the genes that encode the two proteins that coordinate this electrochemical conversion process (the DHPR and RyR1) result in a variety of skeletal muscle disorders including malignant hyperthermia (MH), central core disease (CCD), multiminicore disease, nemaline rod myopathy, and hypokalemic periodic paralysis. Although RyR1 and DHPR disease mutations are thought to alter excitability and Ca2+ homeostasis in skeletal muscle, only recently has research begun to probe the molecular mechanisms by which these genetic defects lead to distinct clinical and histopathological manifestations. This review focuses on recent advances in determining the impact of MH and CCD mutations in RyR1 on muscle Ca2+ signaling and how these effects contribute to disease-specific aspects of these disorders.     

Ryanodine receptor channelopathies

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.     

Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca(2)+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH(2)-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca(2)+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca(2)+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 microM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca(2)+ release was smaller and activated at more negative potentials for myotubes expressing the NH(2)-terminal CCD mutations. The shift in the voltage dependence of SR Ca(2)+ release correlated strongly with changes in resting Ca(2)+, SR Ca(2)+ store depletion, and peak voltage-gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca(2)+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca(2)+ leak. These results demonstrate that the NH(2)-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca(2)+ leak, store depletion, and a reduction in voltage-gated Ca(2)+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.     

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.     

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).     

Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors

The calcium release channels/ryanodine receptors (RyRs) are potential/putative targets of cADPR (cyclic ADP-ribose) action in many tissue systems. In striated muscles, where RyRs predominate, cADPR action on these channels is controversial. Here cADPR modulation of cardiac and skeletal muscle RyR channels was tested. We considered factors reported as necessary for cADPR action, such as the presence of calmodulin and/or FK binding proteins (FKBPs). We found: 1) The RyR channel isoforms were insensitive to cADPR (or its metabolite NAADP [nicotinic acid adenine dinucleotide phosphate]) under all conditions examined, as studied by: 1a) single channel recordings in planar lipid bilayers; 1b) macroscopic behavior of the RyRs in sarcoplasmic reticulum (SR) microsomes (including crude microsome preparations likely to retain putative cADPR cofactors) at room temperature and at 37 degrees C (net energized Ca2+ uptake or passive Ca2+ leak); 2) [32P]cADPR did not bind significantly to SR microsomes; 3) cADPR did not affect FKBP association to SR membranes. We conclude that cADPR does not interact directly with RyRs or RyR-associated SR proteins. Our results under in vitro conditions suggest that c ADPR effects on Ca2+ signaling observed in vivo in mammalian striated muscle cells may reflect indirect modulation of RyRs or RyR-independent Ca2+ release systems.     

Involvement of the cardiac ryanodine receptor/calcium release channel in catecholaminergic polymorphic ventricular tachycardia.

The cardiac ryanodine receptor (RyR2), the major calcium release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes, has recently been shown to be involved in at least two forms of sudden cardiac death (SCD): (1) Catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial polymorphic VT (FPVT); and (2) Arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Eleven RyR2 missense mutations have been linked to these diseases. All eleven RyR2 mutations cluster into 3 regions of RyR2 that are homologous to the three malignant hyperthermia (MH)/central core disease (CCD) mutation regions of the skeletal muscle ryanodine receptor/calcium release channel RyR1. MH/CCD RyR1 mutations have been shown to alter calcium-induced calcium release. Sympathetic nervous system stimulation leads to phosphorylation of RyR2 by protein kinase A (PKA). PKA phosphorylation of RyR2 activates the channel. In conditions associated with high rates of SCD such as heart failure RyR2 is PKA hyperphosphorylated resulting in "leaky" channels. SR calcium leak during diastole can generate "delayed after depolarizations" that can trigger fatal cardiac arrhythmias (e.g., VT). We propose that RyR2 mutations linked to genetic forms of catecholaminergic-induced SCD may alter the regulation of the channel resulting in increased SR calcium leak during sympathetic stimulation.     

The pore region of the skeletal muscle ryanodine receptor is a primary locus for excitation-contraction uncoupling in central core disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced approximately 90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.     

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.     

All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells

The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that is probably caused by Ca²⁺ release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca²⁺ release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to monitor changes in [Ca²⁺]_i and perfused with either cold (0°C), room temperature (RT), or RT buffer containing 40 mM caffeine. Control cells showed no significant response to cold or caffeine, whereas robust Ca²⁺ transients were recorded in response to both RC and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response and that all three isoforms respond in a similar manner. Ca²⁺ release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state. calcium release channel; sarcoplasmic reticulum; excitation-contraction coupling     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers

Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction, Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.     

Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region

The type 1 ryanodine receptor (RyR1) is a Ca²⁺ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca²⁺ release, resulting in abnormal Ca²⁺ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca²⁺ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca²⁺ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca²⁺ in the ER and an increase in resting cytoplasmic Ca²⁺. RyR1 channel activity was quantitatively evaluated by [³H]ryanodine binding and three parameters (sensitivity to activating Ca²⁺, sensitivity to inactivating Ca²⁺ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca²⁺ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca²⁺ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca²⁺ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.     

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 30 S lobster skeletal muscle Ca2+ release channel (ryanodine receptor) has functional properties distinct from the mammalian channel proteins.

The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized ryanodine receptor (RyR) of lobster skeletal muscle has been isolated by rate density centrifugation as a 30 S protein complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified 30 S receptor revealed a single high molecular weight protein band with a mobility intermediate between those of the mammalian skeletal and cardiac M(r) 565,000 RyR polypeptides. Immunoblot analysis showed no or only minimal cross-reactivity with the rabbit skeletal and canine cardiac RyR polypeptides. By immunofluorescence the lobster RyR was localized to the junctions of the A-I bands. Following planar lipid bilayer reconstitution of the purified 30 S lobster RyR, single channel K+ and Ca2+ currents were observed which were modified by ryanodine and optimally activated by millimolar concentrations of cis (cytoplasmic) Ca2+. Vesicle-45Ca2+ flux measurements also indicated an optimal activation of the lobster Ca2+ channel by millimolar Ca2+, whereas 45Ca2+ efflux from mammalian skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicles is optimally activated by micromolar Ca2+. Further, mammalian muscle SR Ca2+ release activity is modulated by Mg2+ and ATP, whereas neither ligand appreciably affected 45Ca2+ efflux from lobster SR vesicles. These results suggested that lobster and mammalian muscle express immunologically and functionally distinct SR Ca2+ release channel protein complexes.