Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum

Ca2+ channels of isolated sarcoplasmic reticulum were incorporated into a planar lipid bilayer and their pharmacological properties were studied. The results show that the channel is a Ca2+-induced Ca2+ release channel like that observed in skinned muscle fibers and isolated vesicles. (i) The open channel probability was increased by the addition of micromolar amounts of Ca2+ to the cis (myoplasmic) side and further increased by millimolar ATP. (ii) The channel was closed by millimolar Mg2+ and micromolar ruthenium red. We found that two disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), when added to the cis side open the channel and lock it irreversibly at open without changing the single channel conductance. Ca2+ efflux from SR vesicles was also enhanced by SITS and DIDS, as monitored by a tracer assay. Further, Ag+ activated the channel transiently. These results suggest that certain amino and SH residues play important roles in gating the Ca2+ channel.     

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 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.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.     

Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel.

Purified canine cardiac sarcoplasmic reticulum vesicles were passively loaded with 45CaCl2 and assayed for Ca2+ releasing activity according to a rapid quench protocol. Ca2+ release from a subpopulation of vesicles was found to be activated by micromolar Ca2+ and millimolar adenine nucleotides, and inhibited by millimolar Mg2+ and micromolar ruthenium red. 45Ca2+ release in the presence of 10 microM free Ca2+ gave a half-time for efflux of 20 ms. Addition of 5 mM ATP to 10 microM free Ca2+ increased efflux twofold (t1/2 = 10 ms). A high-conductance calcium-conducting channel was incorporated into planar lipid bilayers from the purified cardiac sarcoplasmic reticulum fractions. The channel displayed a unitary conductance of 75 +/- 3 pS in 53 mM trans Ca2+ and was selective for Ca2+ vs. Tris+ by a ratio of 8.74. The channel was dependent on cis Ca2+ for activity and was also stimulated by millimolar ATP. Micromolar ruthenium red and millimolar Mg2+ were inhibitory, and reduced open probability in single-channel recordings. These studies suggest that cardiac sarcoplasmic reticulum contains a high-conductance Ca2+ channel that releases Ca2+ with rates significant to excitation-contraction coupling.     

Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum.

Two types of divalent cation conducting channels from rabbit skeletal muscle sarcoplasmic reticulum (SR) were incorporated into planar lipid bilayers. A high conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy density SR fractions. The 100-pS channel was activated by adenine nucleotides and Ca2+ and inhibited by Mg2+ and ruthenium red. A 10-pS calcium and barium conducting channel could be incorporated into planar lipid bilayers from light, intermediate, and heavy density SR vesicles. 10-pS channel activity in bilayers was not dependent on cis Ca2+ and was only weakly dependent on adenine nucleotides. Ruthenium red at concentrations up to 1 mM had no effect and Mg2+ was only marginally effective in inhibiting macroscopic Ba2+ currents from this channel. Calcium releasing activity in intermediate and heavy density SR fractions was assayed according to a rapid quench protocol and compared with the results obtained in the bilayer. Results from this comparison indicate that the 10-pS channel is probably not involved in rapid Ca2+- and adenine nucleotide-induced Ca2+ release from isolated SR vesicles.     

Regulation of cardiac muscle Ca2+ release channel by sarcoplasmic reticulum lumenal Ca2+.

The cardiac muscle sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor) is a ligand-gated channel that is activated by micromolar cytoplasmic Ca2+ concentrations and inactivated by millimolar cytoplasmic Ca2+ concentrations. The effects of sarcoplasmic reticulum lumenal Ca2+ on the purified release channel were examined in single channel measurements using the planar lipid bilayer method. In the presence of caffeine and nanomolar cytosolic Ca2+ concentrations, lumenal-to-cytosolic Ca2+ fluxes >/=0.25 pA activated the channel. At the maximally activating cytosolic Ca2+ concentration of 4 microM, lumenal Ca2+ fluxes of 8 pA and greater caused a decline in channel activity. Lumenal Ca2+ fluxes primarily increased channel activity by increasing the duration of mean open times. Addition of the fast Ca2+-complexing buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cytosolic side of the bilayer increased lumenal Ca2+-activated channel activities, suggesting that it lowered Ca2+ concentrations at cytosolic Ca2+-inactivating sites. Regulation of channel activities by lumenal Ca2+ could be also observed in the absence of caffeine and in the presence of 5 mM MgATP. These results suggest that lumenal Ca2+ can regulate cardiac Ca2+ release channel activity by passing through the open channel and binding to the channel's cytosolic Ca2+ activation and inactivation sites.     

Fast release of calcium from sarcoplasmic reticulum vesicles monitored by chlortetracycline fluorescence

Rapid Ca2+ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free Ca2+ concentration. Ca2+ efflux was activated by extravesicular Ca2+ with an apparent dissociation constant of 25 microM and was inhibited with an inhibition constant of 120 microM in the absence of Mg2+. Caffeine enhanced the Ca2+ release rate by increasing only the affinity of Ca2+ for the activation site. Mg2+ reduced the Ca2+ release rate by competitive binding to the activation site. ATP increased the Ca2+ release rate very much without changing the affinities of Ca2+ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca2+ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca2+ release in the presence of ATP reached 80 s-1. This value is considered to be sufficient to cause muscular contraction.     

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.     

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. I. Ca2+ channel blockers

The effects of Ruthenium red and tetracaine, which inhibit Ca2+-induced Ca2+ release from the isolated sarcoplasmic reticulum (e.g., Ohnishi, S.T. (1979) J. Biochem. (Tokyo) 86, 1147-1150), on several types of Ca2+ release in vitro were investigated. Ca2+ release was triggered by several methods: (1) addition of quercetin or caffeine, (2) Ca2+ jump, and (3) replacement of potassium gluconate with choline chloride to produce membrane depolarization. The time-course of Ca2+ release was monitored using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Ruthenium red inhibited all of these types of Ca2+ release with the same concentration for half-inhibition C1/2 = 0.08-0.10 microM. Similarly, tetracaine inhibited these types of Ca2+ release with C1/2 = 0.07-0.11 mM. Procaine also inhibits both types of Ca2+ release induced by method 2 and 3 with C1/2 = 0.67-1.00 mM. These results suggest that Ruthenium red, tetracaine and procaine interfere with a common mechanism of the different types of Ca2+ release. On the basis of several pieces of evidence we propose that Ruthenium red and tetracaine block the Ca2+ channel of sarcoplasmic reticulum.     

Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation by Ca2+ and ATP and modulation by Mg2+

A high-conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy-density skeletal muscle sarcoplasmic reticulum (SR) fractions into planar lipid bilayers of the Mueller-Rudin type. cis Ca2+ in the range of 2-950 microM increased open probability (Po) in single channel records without affecting open event lifetimes. Millimolar ATP was found to be as good as or better than Ca2+ in activation; however, both Ca2+ and ATP were required to fully activate the channel, i.e., to bring Po = 1. Exponential fits to open and closed single channel lifetimes suggested that the channel may exist in many distinct states. Two open and two closed states were identified when the channel was activated by either Ca2+ or ATP alone or by Ca2+ plus nucleotide. Mg2+ was found to permeate the SR Ca channel in a trans-to-cis direction such that iMg2+/iCa2+ = 0.40. cis Mg2+ was inhibitory and in single channel recordings produced an unresolvable flickering of Ca- and nucleotide-activated channels. At nanomolar cis Ca2+, 4 microM Mg2+ completely inhibited nucleotide-activated channels. In the presence of 2 microM cis Ca2+, the nucleotide-activated macroscopic Ba conductance was inhibited by cis Mg2+ with an IC50 equal to 1.5 mM.     

The effect of trifluoroperazine on the sarcoplasmic reticulum membrane

The inhibitory effect of trifluoroperazine (25-200 microM) on the sarcoplasmic reticulum calcium pump was studied in sarcoplasmic reticulum vesicles isolated from skeletal muscle. It was found that the lowest effective concentrations of trifluoroperazine (10 microM) displaces the Ca2+ dependence of sarcoplasmic reticulum ATPase to higher Ca2+ concentrations. Higher trifluoroperazine concentrations (100 microM) inhibit the enzyme even at saturating Ca2+. If trifluoroperazine is added to vesicles filled with calcium in the presence of ATP, inhibition of the catalytic cycle is accompanied by rapid release of accumulated calcium. ATPase inhibition and calcium release are produced by identical concentrations of trifluoroperazine and, most likely, by the same enzyme perturbation. These effects are related to partition of trifluoroperazine ino the sarcoplasmic reticulum membrane, and consequent alteration of the enzyme assembly within the membrane structure, and of the bilayer surface properties. The effect of trifluoroperazine was also studied on dissociated ('chemically skinned') cardiac cells undergoing phasic contractile activity which is totally dependent on calcium uptake and release by sarcoplasmic reticulum, and is not influenced by inhibitors of slow calcium channels. It was found that trifluoroperazine interferes with calcium transport by sarcoplasmic reticulum in situ, as well as with the role of sarcoplasmic reticulum in contractile activation.     

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.     

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.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.     

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.