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.     

Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex

Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.     

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.     

Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum

The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.     

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.     

Direct binding of verapamil to the ryanodine receptor channel of sarcoplasmic reticulum.

Radioligand binding experiments and single channel recordings demonstrate that verapamil interacts with the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum of rabbit skeletal muscle. In isolated triads, verapamil decreased binding of [3H]Ryanodine with an IC50 of approximately 8 microM at an optimal pH 8.5 and pCa 4.3. Nitrendipine and d-cis-diltiazem did not interfere with binding of [3H]Ryanodine to triads, suggesting that the action of verapamil does not involve the dihydropyridine receptor. Single channel recordings showed that verapamil blocked Ca2+ release channels by decreasing open probability, duration of open events, and number of events per unit time. A direct interaction of verapamil with the ryanodine receptor peptide was demonstrated after purification of the approximately 400 kDa receptor protein from Chaps-solubilized triads. The purified receptor displayed high affinity for [3H]Ryanodine with a Kd of approximately 5 nM and a Bmax of approximately 400 pmol/mg. Verapamil and D600 decreased [3H]Ryanodine binding noncompetitively by reducing the Bmax. Thus the presence of binding sites for phenylalkylamines in the Ca2+ release channel was confirmed. Verapamil blockade of Ca2+ release channels may explain some of the paralyzing effects of phenylalkylamines observed during excitation-contraction coupling of skeletal muscle.     

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.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

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+.     

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.     

Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle

Ryanodine is a specific ligand for the calcium release channel which mediates calcium release in excitation-contraction coupling in muscle. In this study, ryanodine binding in sarcoplasmic reticulum from heart muscle and skeletal muscle is further compared and correlated with function. The new findings include the following: (1) Two types of binding, high affinity (KD1 approximately 5-10 nM) and low affinity (KD2 approximately 3 microM), can now be discerned for the skeletal muscle receptor. KD1 is approximately the same as and KD2 of similar magnitude to that previously reported for heart. (2) The dissociation rates for the high-affinity binding have been directly measured for both heart and skeletal muscle (t1/2 approximately 30-40 min). These rates are more rapid than previously reported (t1/2 approximately 14 h). (3) KD1's obtained from the ratio of the dissociation and association rate constants agree with the dissociation constant measured by equilibrium binding Scatchard analysis. (4) Ryanodine binding to the low-affinity site can be correlated with a decrease in the dissociation rate constant (k-1) of the high-affinity site, and thereby in the apparent dissociation constant (KD1). The inhibition constant (KI) for inhibiting the high-affinity off rate obtained from a double-reciprocal plot of the change in off rate vs [ryanodine] is practically the same in heart (0.66 microM) and skeletal muscle (0.64 microM) and in the range of the KD2. The binding of cold ryanodine to the low-affinity site appears to lock the bound [3H]ryanodine onto the high-affinity site rather than to exchange with it. Thus, in this sense, the ryanodine receptor exhibits "positive cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of inositol 1,4,5-trisphosphate receptor in rat liver

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from an intracellular store. We have investigated the properties of the [32P]IP3 binding sites in rat liver. Two specific [32P]IP3 receptors with KD of 2.3 and 88 nM and respective capacities of 33 fmol/mg protein and 195 fmol/mg protein have been detected in a crude membrane fraction prepared from rat liver homogenate. The pretreatment of the liver with IP3-dependent hormones increased two-fold the capacity of the high affinity site. This effect was partly reversed by dibutyryl cyclic AMP. Permeabilized hepatocytes also displayed two [32P]IP3 binding sites with KD of 1.5 and 84 nM and respective capacities of 8 and 300 fmol/10(6) cells. We have measured the [32P]IP3 binding and the IP3-induced 45Ca2+ release in the same batch of permeabilized hepatocytes. In a low Mg2+ medium, the EC50 for 45Ca2+ release was in close correlation with the KD for the low affinity site. These data suggest that an equilibrium between two states of the IP3 receptor is regulated by hormone action and the low affinity state is responsible for the intracellular Ca2+ release.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that of ruthenium red. These compounds inhibited the open channel with lower affinities (Kd = 170 microM, 4APd; Kd = 656 microM, 4APt), and had Hill coefficients of close to 1. The results suggest that ruthenium red block of the ryanodine receptor is due to binding to multiple sites located in the conduction pore of the channel.     

Effect of gadolinium on the ryanodine receptor/sarcoplasmic reticulum calcium release channel of skeletal muscle

The effect of gadolinium ions on the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptor (RyR1) was studied using heavy SR (HSR) vesicles and RyR1 isolated from rabbit fast twitch muscle. In the [(3)H]ryanodine binding assay, 5 microM Gd(3+) increased the K(d) of the [(3)H]ryanodine binding of the vesicles from 33.8 nM to 45.6 nM while B(max), referring to the binding capacity, was not affected significantly. In the presence of 18 nM[(3)H]ryanodine and 100 microM free Ca(2+), Gd(3+) inhibited the binding of the radiolabeled ryanodine with an apparent K(d) value of 14.7 microM and a Hill coefficient of 3.17. In (45)Ca(2+) experiments the time constant of (45)Ca(2+) efflux from HSR vesicles increased from 90.9 (+/- 11.1) ms to 187.7 (+/- 24.9) ms in the presence of 20 microM gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic (cis) (IC(50) = 5.65 +/- 0.33 microM, n(Hill) = 4.71) and the luminal (trans) side (IC(50) = 5.47 +/- 0.24 microM, n(Hill) = 4.31). The degree of inhibition on the cis side didn't show calcium dependency in the 100 microM to 1 mM Ca(2+) concentration range which indicates no competition with calcium on its regulatory binding sites. When Gd(3+) was applied at the trans side, EGTA was present at the cis side to prevent the binding of Gd(+3) to the cytoplasmic calcium binding regulatory sites of the RyR1 if Gd(3+) accidentally passed through the channel. The inhibition of the channel did not show any voltage dependence, which would be the case if Gd(3+) exerted its effect after getting to the cis side. Our results suggest the presence of inhibitory binding sites for Gd(3+) on both sides of the RyR1 with similar Hill coefficients and IC(50) values.     

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.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Quantitation of ryanodine receptor of rabbit skeletal muscle, heart and brain

The total number of high-affinity ryanodine receptor (RyR) binding sites present in skeletal and cardiac muscle and in brain tissue of the rabbit was determined by [3H]ryanodine binding to subfractions obtained by differential centrifugation of homogenates prepared in a low-ionic strength medium, containing 0.5% Chaps. In all three tissues at least 80% of [3H]ryanodine binding was recovered in the total membrane (TM) fraction obtained by centrifuging between 650 g for 10 min and 120,000 x g for 90 min. Skeletal muscle displayed higher contents of high-affinity RyR sites (about 49 pmol/g wet wt) than heart and brain (about 12 pmol and 3.5 pmol/g wet wt, respectively). The affinity for ryanodine, as well as the affinity for Ca2+, in the absence or presence of Ca2(+)-releasing drugs (caffeine and doxorubicin) of TM from skeletal muscle, were found to be identical to those of purified terminal cisternae. As low as 1 g of tissue was sufficient to perform several experiments.     

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.     

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.