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.     

Cardiac sarcoplasmic reticulum contains a low-affinity site for phenylalkylamines

The distribution of the bovine cardiac binding sites for the organic calcium-channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17-fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H](-)desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel.     

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)     

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.     

Inhibition of dihydropyridine-sensitive calcium channels by the plant alkaloid ryanodine

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200-110 with an apparent inhibition constant of approx. 95 microM and inhibits dihydropyridine-sensitive calcium channels in the same preparation with an IC50 of approx. 45 microM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1-26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.     

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.     

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

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

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.     

Ryanodine stabilizes multiple conformational states of the skeletal muscle calcium release channel.

Nanomolar to micromolar ryanodine alters the gating kinetics of the Ca2+ release channel from skeletal sarcoplasmic reticulum (SR) fused with bilayer lipid membranes (BLM). In the presence of asymmetric CsCl and 100 microM CaCl2 cis, ryanodine (RY) (5-40 nM) activates the channel, increasing the open probability (po; maximum 300% of control) without changing unitary conductance (468 picosiemens (pS)). Statistical analyses of gating kinetics reveal that open and closed dwell times exhibit biexponential distributions and are significantly modified by nanomolar RY. Altered channel gating kinetics with low nanomolar RY is fully reversible and correlates well with binding kinetics of nanomolar [3H]RY with its high affinity site (Kd1 = 0.7 nM) under identical experimental conditions. RY (20-50 nM) induces occasional 1/2 conductance fluctuations which correlate with [3H]RY binding to a second site having lower affinity (Kd2 = 23 nM). RY (5-50 nM) in the presence of 500 mM CsCl significantly enhances Ca(2+)-induced Ca2+ release from actively loaded SR vesicles. Ryanodine > or = 50 nM stabilizes the channel in a 234-pS subconductance which is not readily reversible. RY (> or = 70 microM) produces a unidirectional transition from the 1/2 to a 1/4 conductance fluctuation, whereas RY > or = 200 microM causes complete closure of the channel. The RY required for stabilizing 1/4 conductance transitions and channel closure do not quantitatively correlate with [3H]RY equilibrium binding constants and is attributed to significant reduction in association kinetics with > 200 nM [3H]RY in the presence of 500 mM CsCl. These results demonstrate that RY stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting RY effector sites on the channel protein.     

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.     

The cardiac sarcoplasmic reticulum calcium-release channel: modulation of ryanodine binding and single-channel activity

[3H]Ryanodine binding to a preparation of isolated cardiac sarcoplasmic reticulum has been investigated. A method is reported which produces a very high level of specific binding. Scatchard analysis of binding up to 50 nM ryanodine yields data which infer a single class of binding sites with a Kd of 1.4 nM and a Bmax of 9.7 pmol/mg protein. Micromolar calcium is the principal activating ligand and its effects on binding are modulated by ligands which similarly affect the activity of single calcium-release channels incorporated into artificial planar phospholipid bilayers. The benzimidazole drug, sulmazole, is able to stimulate ryanodine binding in the presence of sub-activating calcium concentrations. Ryanodine binds to the native channel only when it is in its open state and stimulation of maximal ryanodine binding is achieved by ligands which are insufficient to produce full single-channel activation. A model is proposed which relates the modulation of ryanodine binding to the behaviour of single channels.     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

3H]ryanodine as a probe of changes in the functional state of the Ca(2+)-release channel in malignant hyperthermia.

The defect in malignant hyperthermia (MH) alters the binding of [3H]ryanodine to the Ca(2+)-release channel by increasing its apparent affinity for the binding site. In sarcoplasmic reticulum (SR) membranes from both normal and mutant pigs the apparent Kd is dependent on a number of parameters. Adenosine 5'-(beta,gamma-methylene)triphosphate, ionic strength, and Ca2+ each increase the apparent affinity of the binding site for [3H]ryanodine. Equilibrium and kinetic evaluation of the binding of [3H]ryanodine to these membranes demonstrates that the MH defect in pigs increases the apparent affinity of the membranes for [3H]ryanodine by increasing the amount of high affinity relative to low affinity binding sites. Both the association and dissociation of [3H]ryanodine with all three types of membranes (normal, heterozygous MH, homozygous MH) are characterized by two or more components, with the relative ratios of these components altered by the MH defect. These findings suggest that the observed Kd is the weighted average of the binding of ryanodine to two or more interconvertible states of the channel. Dilution of [3H]ryanodine bound to normal membranes at high Ca2+ into low Ca2+ solutions enhances the rate of dissociation. This conversion occurs to a much lesser extent with MH membranes, suggesting that the MH defect may alter the rate at which the high affinity form of the protein converts to the low affinity form.     

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.     

Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.     

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.     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.     

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.     

Functional characterisation of the ryanodine receptor purified from sheep cardiac muscle sarcoplasmic reticulum

Sheep cardiac muscle sarcoplasmic reticulum ryanodine receptors have been isolated by density-gradient centrifugation following solubilisation with the zwitterionic detergent, CHAPS. The functional state of the receptor complex has been assessed by quantification of [3H]ryanodine binding and by characterisation of single-channel conductance and gating properties following reconstitution into unilamellar proteo-liposomes and incorporation into planar phospholipid bilayers. A method of solubilisation is described which yields a receptor displaying high-affinity [3H]ryanodine binding (Kd 2.8 nM, Bmax 352 pmol/mg protein) and which functions as a cation-selective, ligand-regulated channel under voltage clamp conditions. Previous reports of channel activity of purified rabbit skeletal and canine cardiac muscle ryanodine receptors describe a range of sub- or variable-conductance events. In contrast, the sheep cardiac ryanodine receptor-channels isolated using the optimal conditions described in this report consistently display a single open state conductance with either Ca2+ or K+ as the charge carrying species.