Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum

The effect of the plant alkaloid ryanodine on the skeletal muscle sarcoplasmic reticulum Ca2+ release channel was studied by determining the Ca2+ permeability of "heavy" vesicles passively loaded with 45Ca2+ in the presence or absence of ryanodine. Depending on the experimental conditions, ryanodine either stimulated or inhibited Ca2+ efflux. Vesicles were rendered permeable to 45Ca2+ at a ryanodine concentration of 0.01 microM when diluted into a medium containing the two Ca2+ release channel inhibitors Mg2+ and ruthenium red. At ryanodine concentrations greater than 10 microM, 45Ca2+ efflux was inhibited in channel-activating (5 microM Ca2+) or -inhibiting (10 mM Mg2+ plus 10 microM ruthenium red) media. An optimal stimulatory effect was observed when vesicles were incubated with ryanodine at 37 degrees C and in media that caused partial opening of the channel. Similar results to those described above were obtained using cardiac sarcoplasmic reticulum vesicles that were capable of rapid 45Ca2+ efflux. Use of the slowly permeating molecule L-[3H]glucose allowed measurement of channel-mediated efflux rates from vesicles in the presence and absence of ryanodine. At low activating concentrations, ryanodine did not appreciably change the regulation of L-glucose efflux rates by external Ca2+, Mg2+, and adenine nucleotide. These results suggested two possible modes of action of ryanodine: 1) a change in the gating mechanism of the channel which is not readily detected using the slowly permeating molecule L-glucose or 2) a change in channel structure which prevents its complete closing.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel.     

Temperature and Ca2+ dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart

Opening and closing of the cardiac ryanodine (Ry) receptor (RyR) are coordinated by the free intracellular Ca2+ concentration, thus making the Ca2+ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4 degrees C, as indicated by Ry sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca2+-dependent kinetics of [3H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [3H]ryanodine binding sites determined at 20 degrees C was 1.54 times higher in rat than burbot heart (0.401 +/- 0.039 and 0.264 +/- 0.019 pmol/mg protein, respectively) (P < 0.02), while the binding affinity (Kd) for [3H]ryanodine was similar (3.38 +/- 0.63 and 4.38 +/- 1.14 nM for rat and burbot, respectively) (P = 0.47). The high-affinity [3H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca2+ concentration at both 20 degrees C and 2 degrees C and did not differ between the two species. Half-maximal [3H]ryanodine binding occurred at 0.191 +/- 0.027 microM and 0.164 +/- 0.034 microM Ca2+ for rat and at 0.212 +/- 0.035 microM and 0.188 +/- 0.039 microM Ca2+ for burbot (P = 0.65), at 2 degrees C and 20 degrees C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca2+-binding affinity at 20 degrees C was 4.4 and 5.9 times lower, respectively, than in the burbot. At 20 degrees C, the rate of [3H]ryanodine binding to the high-affinity binding site was similar in rat and burbot but was drastically slowed in rat at 2 degrees C. At 2 degrees C, [3H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10 degrees C and 20 degrees C, the rate of dissociation was two to three times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism, where the closing of the Ca2+ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [3H]ryanodine binding in rat compared with burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.     

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.     

Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.     

Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum.

In the absence of cyclic nucleotides heart microsomes have two classes of calcium binding sites with binding constants of 0.69 and 0.071 micron-1 and capacities of 2.2 and 9.7 nmol/mg protein, respectively. Neither cyclic AMP nor monobutyryl cyclic AMP affect binding but cyclic GMP and monobutyryl cyclic GMP cause the complete loss of the high affinity calcium binding sites, Cyclic GMP (but not monobutyryl cyclic GMP) also causes a decrease in the binding constant of the low affinity binding sites. AMP, GMP and Tris-butyrate do not affect calcium binding. The effects of the cyclic nucleotides are direct and are not mediated by protein phosphorylation. Phosphorylation of microsomal proteins increases the binding constant but not the capacity of the high affinity calcium binding sites. The capacity and also, perhaps, binding constant of the low affinity sites is also increased by phosphorylation. In additon to their effects on calcium binding the cyclic nucleotides also affect the movements of calcium into and out of the microsomes. The effects are again direct and not mediated by protein phosphorylation. Cyclic GMP decreases the rate of Ca2+ efflux from preloaded cardiac microsomes and also appears to decrease the rate of uptake of Ca2+ by cardiac microsomes though this effect is less clear cut than the action on efflux. The cyclic nucleotide has a half maximal effect at a concentration of 100 microns. By contrast cyclic AMP increases the rate of influx of Ca2+ into heart microsomes and the rate of efflux of Ca2+ from preloaded preparations. The effect is, however, rather slight. It is suggested that the most obvious interpretation of these results is that cyclic GMP decreases the Ca2+ permeability of the cardiac microsomal membrane while cyclic AMP increases the permeability. In contrast to the results found with membrane preparations from certain other tissues phosphorylation of cardiac microsomal proteins does not appear to alter Ca2+ efflux or influx out of, or into, cardiac microsomal preparations. It is thus concluded that phosphorylation of cardiac microsomal proteins does not affect the Ca2+ permeability of the microsomal membrane.     

Functional expression of recombinant type 1 ryanodine receptor in insect cells

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by palmitoyl carnitine.

Studies of [3H]ryanodine binding, 45Ca2+ efflux, and single channel recordings in planar bilayers indicated that the fatty acid metabolite palmitoyl carnitine produced a direct stimulation of the Ca2+ release channel (ryanodine receptor) of rabbit and pig skeletal muscle junctional sarcoplasmic reticulum. At a concentration of 50 microM, palmitoyl carnitine (a) stimulated [3H]ryanodine binding 1.6-fold in a competitive manner at all pCa in the range 6 to 3; (b) released approximately 65% (30 nmol) of passively loaded 45Ca2+/mg protein; and (c) increased 7-fold the open probability of Ca2+ release channels incorporated into planar bilayers. Neither carnitine nor palmitic acid could reproduce the effect of palmitoyl carnitine on [3H]ryanodine binding, 45Ca2+ release, or channel open probability. 45Ca2+ release was induced by several long-chain acyl carnitines (C14, C16, C18) and acyl coenzyme A derivatives (C12, C14, C16), but not by the short-chain derivative C8 or by free saturated fatty acids of chain length C8 to C18, at room temperature or 36 degrees C. This newly identified interaction of esterified fatty acids and ryanodine receptors may represent a pathway by which metabolism of skeletal muscle could influence intracellular Ca2+ and may be responsible for the pathophysiology of disorders of beta-oxidation such as carnitine palmitoyl transferase II deficiency.     

Two novel types of calcium release from skeletal sarcoplasmic reticulum by phosphatidylinositol 4,5-biphosphate.

In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the time to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 microM PIP2 were not modified by ruthenium red. PIP2 (>0.1 microM) markedly accelerated the rate of 45Ca2+ efflux from SR vesicles in a concentration-dependent manner. The PIP(2)-induced 45Ca2+ efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced 45Ca2+ release was clearly different from that in the Ca(2+)-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [3H]ryanodine or [3H]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca(2+)-induced Ca2+ release channels (the ryanodine receptor 1).     

Stimulation and inhibition of [3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.     

Critical amino acid residues determine the binding affinity and the Ca2+ release efficacy of maurocalcine in skeletal muscle cells

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.     

Physiological differences between the alpha and beta ryanodine receptors of fish skeletal muscle.

Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction.     

Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine.

We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.     

Reactive disulfides trigger Ca2+ release from sarcoplasmic reticulum via an oxidation reaction

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to the S-S bond such as 2,2'-dithiodipyridine (2,2'-DTDP), 4,4'-dithiodipyridine, and N-succinimidyl 3(2-pyridyldithio)propionate (SPDP) trigger Ca2+ release from sarcoplasmic reticulum (SR) vesicles. They are known to specifically oxidize free SH sites via a thiol-disulfide exchange reaction with the stoichiometric production of thiopyridone. Thus, the formation of a mixed S-S bond between an accessible SH site on an SR protein and a RDS causes large increases in SR Ca2+ permeability. Reducing agents, glutathione (GSH) or dithiothreitol reverse the effect of RDSs and permit rapid re-uptake of Ca2+ by the Ca2+, Mg2+-ATPase. The RDSs, 2,2'-DTDP, 4,4'-dithiodipyridine and SPDP displaced [3H]ryanodine binding to the Ca2+-receptor complex at IC50 values of 7.5 +/- 0.2, 1.5 +/- 0.1, and 15.4 +/- 0.1 microM, respectively. RDSs did not alter the rapid initial phase of Ca2+ uptake by the pump, stimulated ATPase activity, and induced release from passively loaded vesicles with nonactivated pumps; thus they act at a Ca2+ release channel and not at the Ca2+, Mg2+-ATPase. Efflux rates increased in 0.25-1.0 mM [Mg2+]free then decreased in 2-5 mM [Mg2+]free. Adenine nucleotides inhibited the oxidation of SHs on SR protein by RDSs and thus reduced Ca2+ efflux rates. However, once RDSs oxidized these SH sites and opened the Ca2+ release pathway, subsequent additions of nucleotides stimulated Ca2+ efflux. In skinned fibers, 2,2'-dithiodipyridine elicited rapid twitches which were blocked by ruthenium red. These results indicate that RDSs trigger Ca2+ release from SR by oxidizing a critical SH group, and thus provide a method to covalently label the protein(s) involved in causing these changes in Ca2+ permeability.     

Dantrolene and azumolene inhibit [3H]PN200-110 binding to porcine skeletal muscle dihydropyridine receptors.

We tested whether the hydantoin muscle relaxants dantrolene, azumolene, or aminodantrolene could alter the binding of [3H]PN200-110 to transverse tubule dihydropyridine receptors or the binding of [3H]ryanodine to junctional sarcoplasmic reticulum Ca2+ release channels. All three drugs inhibited [3H]PN200-110 binding with azumolene (IC50 approximately 20 microM) 3-5 times more potent than dantrolene or aminodantrolene. In contrast, 100 microM azumolene and dantrolene produced a small inhibition of [3H]ryanodine binding (less than 25%) while aminodantrolene was essentially inert. Hence there was a preferential interaction of hydantoins with dihydropyridine receptors instead of ryanodine receptors. Skeletal muscle dihydropyridine receptors may participate in the mechanism of action of dantrolene and azumolene.     

The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer

The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.