Abnormal ryanodine receptor channels in malignant hyperthermia.

Previous studies have demonstrated a defect associated with the calcium release mechanism of sarcoplasmic reticulum (SR) from individuals susceptible to malignant hyperthermia (MH). To examine whether SR calcium release channels were indeed altered in MH, SR vesicles were purified from normal and MH susceptible (MHS) porcine muscle. The Ca2+ dependence of calcium efflux rates from 45Ca2(+)-filled SR vesicles was then compared with the Ca2+ dependence of single-channel recordings of SR vesicles incorporated into planar lipid bilayers. The rate constants of 45Ca2+ efflux from MHS SR were two to threefold larger than from normal SR over a wide range of myoplasmic Ca2+. Normal and MHS single channels were progressively activated in a similar fashion by cis Ca2+ from pCa 7 to 4. However, below pCa 4, normal channels were inactivated by cis Ca2+, whereas MHS channels remained open for significantly longer times. The altered Ca2+ dependence of channel inactivation in MHS SR was also evident when Ca2+ was increased on the trans side while cis Ca2+ was held constant. We propose that a defect in a low-affinity Ca2+ binding site is responsible for the altered gating of MHS SR channels. Such a defect could logically result from a mutation in the gene encoding the calcium release channel, providing a testable hypothesis for the molecular basis of this inherited disorder.     

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.     

Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel

The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.     

Involvement of the 60 kDa phosphoprotein in the regulation of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca2+ and 1 microM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca2+ release from the passively loaded SR was induced by a Ca2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca2+ release between normal and MHS SR. Upon addition of 20 microM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca2+ release in normal SR significantly decreased from 659 +/- 102 to 361 +/- 105 nmol Ca2+/mg SR per s, whereas in MHS SR the rate decreased from 749 +/- 124 to 652 +/- 179 nmol Ca2+/mg SR per s. Addition of 20 microM adenosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320-327) is at least partly due to the reduced extent of the Ca2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with Mr = 55,000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.     

Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor.

The skeletal muscle ryanodine receptor of malignant hyperthermia-susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86-99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86-99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.     

Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin)

The effect of annexin VI (67-kDa calcimedin) on the activity of the Ca2+ release channel was studied using heavy sarcoplasmic reticulum membranes reconstituted into planar bilayers. Annexin VI, in a range of 5-40 nM, modified the gating behavior of the Ca2+ release channel by increasing the probability of opening by 2.7-fold and the mean open time by 82-fold relative to controls. Annexin VI caused no change in the slope conductance of the channel. The modulatory effect of annexin VI on the activity of Ca2+ release channels was Ca2+ dependent, and the annexin VI-modified channel was sensitive to both ruthenium red and ryanodine. The effect of annexin VI was observed when this protein was added specifically to the trans chamber, which corresponds to the luminal side of sarcoplasmic reticulum as determined by the ATP activation of the channel. In addition, differential extraction studies demonstrated that some annexin VI is localized within the lumen of the isolated heavy sarcoplasmic reticulum vesicles prepared by several different procedures. Annexin VI did not modify, from either the cis or trans chambers, the activity of K+ or Cl- channels from sarcoplasmic reticulum or the dihydropyridine sensitive Ca2+ channel from transverse tubules. In addition, the 38-kDa core proteolytic fragments of annexin VI had no effect on the Ca2+ release channel activity. Annexin VI is therefore a candidate for a physiological modulator of the Ca2+ release channel and as such, may play an important role in the excitation-contraction coupling.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Altered transverse tubule dihydropyridine receptor binding in malignant hyperthermia

Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.     

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.     

Postulated role of interdomain interaction between regions 1 and 2 within type 1 ryanodine receptor in the pathogenesis of porcine malignant hyperthermia

We have demonstrated recently that CICR (Ca2+-induced Ca2+ release) activity of RyR1 (ryanodine receptor 1) is held to a low level in mammalian skeletal muscle ('suppression' of the channel) and that this is largely caused by the interdomain interaction within RyR1 [Murayama, Oba, Kobayashi, Ikemoto and Ogawa (2005) Am. J. Physiol. Cell Physiol. 288, C1222-C1230]. To test the hypothesis that aberration of this suppression mechanism is involved in the development of channel dysfunctions in MH (malignant hyperthermia), we investigated properties of the RyR1 channels from normal and MHS (MH-susceptible) pig skeletal muscles with an Arg615-->Cys mutation using [3H]ryanodine binding, single-channel recordings and SR (sarcoplasmic reticulum) Ca2+ release. The RyR1 channels from MHS muscle (RyR1MHS) showed enhanced CICR activity compared with those from the normal muscle (RyR1N), although there was little or no difference in the sensitivity to several ligands tested (Ca2+, Mg2+ and adenine nucleotide), nor in the FKBP12 (FK506-binding protein 12) regulation. DP4, a domain peptide matching the Leu2442-Pro2477 region of RyR1 which was reported to activate the Ca2+ channel by weakening the interdomain interaction, activated the RyR1N channel in a concentration-dependent manner, and the highest activity of the affected channel reached a level comparable with that of the RyR1MHS channel with no added peptide. The addition of DP4 to the RyR1MHS channel produced virtually no further effect on the channel activity. These results suggest that stimulation of the RyR1MHS channel caused by affected inter-domain interaction between regions 1 and 2 is an underlying mechanism for dysfunction of Ca2+ homoeostasis seen in the MH phenotype.     

Abnormal sarcoplasmic reticulum ryanodine receptor in malignant hyperthermia

Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant hyperthermia (MH) has a defect associated with the mechanism of calcium release from its intracellular storage sites in the sarcoplasmic reticulum (SR). In this report we demonstrate that the [3H]ryanodine receptor of isolated MH-susceptible (MHS) porcine heavy SR exhibits an altered Ca2+ dependence of [3H]ryanodine binding at the low affinity Ca2+ site as well as a lower Kd for ryanodine (92 versus 265 nM) when compared to normal porcine SR. The Bmax of the normal and MHS [3H] ryanodine receptor (9.3-12.6 pmol/mg) was not significantly different, and analysis of MHS and normal SR proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal a significant difference in the intensity of Coomassie Blue staining of the spanning protein/ryanodine receptor region of the gels (Mr greater than 300,000). We also find that MHS porcine muscle intact fiber bundles exhibit a 5-10-fold lower ryanodine threshold for twitch and tetanus inhibition, and contracture onset when compared to normal muscle. Since the SR ryanodine receptor is a calcium release channel as well as a component intimately involved in transverse tubule-SR communication, abnormalities in the skeletal muscle ryanodine receptor may be responsible for the abnormal SR calcium release and contractile properties demonstrated by MHS muscle.     

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.     

Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticulum protein structure and calcium release.

In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Transverse tubule calcium regulation in malignant hyperthermia

Transverse tubule (TT) calcium transport and permeability were examined in the inherited skeletal muscle disorder malignant hyperthermia (MH). ATP-dependent calcium uptake by TT vesicles isolated from normal and MH-susceptible (MHS) pig muscle had a similar dependence on ionized Ca2+ concentration (K1/2 for Ca2+ of 0.21 +/- 0.04 and 0.25 +/- 0.05 microM for MHS and normal TT, respectively), as well as a similar Vmax (20.9 +/- 2.0 and 23.7 +/- 4.5 nmol Ca/mg protein/min for MHS and normal TT, respectively). Furthermore, the stimulation of calcium uptake by either calmodulin or cAMP-dependent protein kinase was similar in normal and MHS TT. Halothane concentrations greater than 2 mM inhibited calcium uptake by either normal or MHS TT to a similar extent (IC50 = 8 mM). Dantrolene (10 microM), nitrendipine (1 microM), and Bay K 8644 (1 microM) had no significant effect on either the initial rates of calcium uptake or maximal calcium accumulation of either MHS or normal TT vesicles. However, in the absence of any added agents, maximum calcium accumulation by MHS TT was significantly less than by normal TT (90 +/- 10 versus 130 +/- 9 nmol Ca/mg protein after 15 min of uptake). This difference was not due to an increased permeability of MHS TT to calcium, nor was it due to a difference in the sarcoplasmic reticulum contamination (less than 5%) of the MHS and normal preparations. Although our results indicate there is no significant defect in MHS TT calcium regulation, the diminished maximum calcium accumulation by MHS TT may contribute to the abnormal sarcoplasmic calcium homeostasis in skeletal muscle during an MH crisis.     

Heavy metal-induced Ca2+ release from sarcoplasmic reticulum

Two distinct forms of Ca2+ release from isolated sarcoplasmic reticulum vesicles in response to additions of heavy metals (silver and mercurials) are described. One form of heavy metal-induced Ca2+ release involves the ruthenium red-sensitive Ca2+ release channel localized in terminal cisternae. The other form of heavy metal-induced Ca2+ release appears to involve all portions of the sarcoplasmic reticulum and is insensitive to ruthenium red. This latter form of Ca2+ release occurs over a similar range of heavy metal concentrations as inhibition of the sarcoplasmic reticulum Ca2+ pump but does not appear to be a result solely of such pump inhibition. Both forms of Ca2+ release are inhibited by glutathione, an endogenous constituent of muscle fibers, and by dithiothreitol, agents which prevent sulfhydryl oxidation. To assess the role of any sulfhydryl oxidation in sarcoplasmic reticulum Ca2+ release physiologically, dithiothreitol and glutathione were introduced inside muscle fibers and effects on excitation-contraction coupling examined. The results strongly suggest that sulfhydryl oxidation plays no essential role in skeletal muscle excitation-contraction coupling.     

Redox regulation of calcium release in skeletal and cardiac muscle

In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 microM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 microM [Ca2+]. In 10 microM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] < or = 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 microM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed.     

Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.     

Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum

Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.