Ryanodine receptor channel-dependent glutathione transport in the sarcoplasmic reticulum of skeletal muscle

We found that glutathione transport across endo/sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione influx could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 agonists. The effect of blockers was specific to glutathione, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarcoplasmic reticulum.     

Components of purified sarcolemma from porcine skeletal muscle

Sarcolemmal membranes were isolated from porcine skeletal muscle by modifications of a LiBr-extraction technique. Latency determinations of acetylcholinesterase, ouabain-sensitive p-nitrophenylphosphatase, [3H]ouabain binding, and (Na+ + K+)-ATPase activities indicated that 65-76% of the membranes were sealed inside-out vesicles. The preparations were enriched in cholesterol and phospholipid, and demonstrated adenylate cyclase activity and both cAMP and cGMP phosphodiesterase activities. An indication of the purity of this fraction was that the Ca2+-ATPase activity (0.13 mumol Pi mg-1 min-1 at 37 degrees C) was 3.8% of that of porcine skeletal muscle sarcoplasmic reticulum preparations. Pertussis toxin specifically catalyzed the ADP-ribosylation of a Mr 41,000 sarcolemmal protein, indicating the presence of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, Ni. An endogenous ADP-ribosyltransferase activity, with several membrane protein substrates, was also demonstrated. The addition of exogenous cAMP-dependent protein kinase or calmodulin promoted the phosphorylation of a number of sarcolemmal proteins. The calmodulin-dependent phosphorylation exhibited an approximate K 1/2 for Ca2+ of 0.5 microM, and an approximate K 1/2 for calmodulin of 0.1 microM. 125I-Calmodulin affinity labeling of the sarcolemma, using dithiobis(succinimidyl propionate), demonstrated the presence of Mr 160,000 and 280,000 calmodulin-binding components in these membranes. These results demonstrate that this porcine preparation will be valuable in the study of skeletal muscle sarcolemmal ion transport, protein and hormonal receptors, and protein kinase-catalyzed phosphorylation.     

Ryanodine receptor defects in muscle genetic diseases

Ryanodine receptor (RyR), a homotetrameric Ca2+ release channel, is one of the main actors in the generation of Ca2+ signals that trigger muscle contraction. Three genes encode three isoforms of RyRs, which have tissue-restricted distribution. RyR1 and RyR2 are typical of muscle cells, with RyR1 originally considered the skeletal muscle type and RyR2 the cardiac type. However, RyR1 and RyR2 have recently been found in numerous other cell types, including, for instance, peripheral B and T lymphocytes. In contrast, RyR3 is widely distributed among cells. RyR1 and RyR2 are localized in a specialized portion of the sarcoplasmic reticulum (SR), the terminal cisternae, which is the portion of the SR Ca2+ store that releases Ca2+ to control the process of muscle contraction. A specific role for RyR3 has not yet been established: probably, its co-expression with the other RyR isoforms contributes to qualitatively modulate Ca2+-dependent processes in muscle cells and in neurons. Several mutations in the genes encoding RyR1 and RyR2 have been identified in autosomal dominant diseases of skeletal and cardiac muscle, such as malignant hyperthermia (MH), central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). More recently, CCD cases with recessive inheritance have also been described. MH is a pharmacogenetic disease, but the others manifest as congenital myopathies. Even if their clinical phenotypes are well established, particularly in skeletal muscle, the molecular mechanisms that generate the conditions are not clear. A number of studies on cellular models have attempted to elucidate the molecular defects associated with the different mutations, but the problem of understanding how mutations in the same gene generate such an array of diverse pathological traits and diseases of widely different degrees of severity is still open. This review will consider the molecular and cellular effects of RyR mutations, summarizing recent data in the literature on Ca2+ dysregulation, which may lead to a better understanding of the functioning of RyRs.     

Ryanodine does not affect the potassium channel from the sarcoplasmic reticulum of skeletal muscle

Single K channels from skeletal muscle sarcoplasmic reticulum were incorporated into artificial membranes. Ryanodine applied to either side of the membrane did not affect the gating nor the conductance properties of those channels. These results suggest that the site of action of ryanodine is limited only to the calcium channels present in the membrane of sarcoplasmic reticulum (1).     

Kinetics of insulin binding and kinase activity of the partially purified insulin receptor from human skeletal muscle

The kinetics of insulin binding and kinase activity of soluble, partially purified insulin receptors from human skeletal muscle are considered. An equilibrium for insulin binding was obtained within 2 h at 37 degrees C. At lower temperatures the equilibrium for insulin binding was less clearly defined. Dissociation of 125I-labelled insulin was incomplete unless an excess amount of unlabelled insulin was added. Insulin-stimulatable autophosphorylation of the 95 kDa subunit was verified by gel electrophoresis. The kinase activity was measured with the synthetic polypeptide poly(Glu-Tyr(4:1] as a phosphoacceptor. The insulin receptor kinase activity correlated significantly (r = 0.92, P less than 0.0001) to the concentration of high-affinity insulin binding sites in the eluate. Autophosphorylation of the insulin receptor was necessary for the activation of the receptor kinase. When activated the receptor kinase activity was stable for at least 60 min at 21 degrees C with a pH optimum of approx. 7.8, similar to the pH optimum for insulin binding. The non-ionic detergent Triton X-100 inhibited the sensitivity of the receptor kinase to insulin. Insulin stimulated the Vmax of the kinase reaction about 3-fold, decreased the Km for ATP from 35 +/- 5 microM (mean +/- S.E.) to 8 +/- 1 microM (P less than 0.02) and induced a positive cooperativity to ATP with an increase in the Hill coefficient from 1.00 +/- 0.02 to 1.37 +/- 0.07 (P less than 0.05). According to the Hill plots, insulin itself showed no cooperativity with respect to receptor binding or kinase activation.     

Subunit composition of the purified dihydropyridine binding protein from skeletal muscle

The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).     

Lipid composition of purified transverse tubule membranes isolated from amphibian skeletal muscle

The level and proportion of lipids and their fatty acid composition were analyzed in highly purified transverse tubule membranes of amphibian skeletal muscle. Tubule membranes show (a) a higher content of lipids, (b) a higher phospholipid/cholesterol ratio and (c) a different phospholipid composition from other subcellular fractions, such as the light and heavy membranes from sarcoplasmic reticulum, which are similar in lipid profile. Transverse tubule membranes are characterized by a high percentage of phosphatidylserine and sphingomyelin and a low proportion of phosphatidylcholine compared with the other membranes. All three show a high proportion of ethanolamine plasmalogens (50% of the total ethanolamine glycerophospholipid). Transverse tubule membrane lipids contain a high proportion of 20- and 22-carbon polyunsaturated fatty acids, predominantly 20:4, 20:5, 22:5 and 22:6. Arachidonate predominates in phosphatidylinositol, eicosapentaenoate and docosahexaenoate in ethanolamine and serine glycerophospholipids.     

Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules

The purified calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubule membrane consists of three subunits: alpha with Mr 135 000, beta with Mr 50 000, and gamma with Mr 33 000. Purified receptor preparations were incorporated into phosphatidylcholine (PC) vesicles by addition of PC in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and removal of detergent by molecular sieve chromatography. Forty-five percent of the alpha, beta, and gamma polypeptides and the [3H]dihydropyridine/receptor complex were recovered in association with PC vesicles. The rate of dissociation of the purified and reconstituted dihydropyridine/receptor complex was identical with that in T-tubule membranes, and allosteric modulation by verapamil and diltiazem was retained. The reconstituted calcium antagonist receptor, when occupied by the calcium channel activator BAY K 8644, mediated specific 45Ca2+ and 133Ba2+ transport into the reconstituted vesicles. 45Ca2+ influx was blocked by the organic calcium antagonists PN200-110 (K0.5 = 0.2 microM), D600 (K0.5 = 1.0 microM), and verapamil (K0.5 = 1.5 microM) and by inorganic calcium channel antagonists (La3+ greater than Cd2+ greater than Ni2+ greater than Mg2+) as in intact T-tubules. A close quantitative correlation was observed between the presence of the alpha, beta, and gamma subunits of the calcium antagonist receptor and the ability to mediate 45Ca2+ or 133Ba2+ flux into reconstituted vesicles. Comparison of the number of reconstituted calcium antagonist receptors and functional channels supports the conclusion that only a few percent of the purified calcium antagonist receptor polypeptides are capable of mediating calcium transport as previously demonstrated for calcium antagonist receptors in intact T-tubules.     

Ryanodine interferes with charge movement repriming in amphibian skeletal muscle fibers.

Cut twitch muscle fibers mounted in a triple Vaseline-gap chamber were used to study the effects of ryanodine on intramembranous charge movement, and in particular on the repriming of charge 1. Charge 1 repriming was measured either under steady-state conditions or by using a pulse protocol designed to study the time course of repriming. This protocol consisted of repolarizing the fibers to -100 mV from a holding potential of 0 mV, and then measuring the reprimed charge moving in the potential range between -40 and +20 mV. Ryanodine at a high concentration (100 microM) did not affect the maximum amount of movable charge 1 and charge 2, or their voltage dependence. This indicates that the alkaloid does not interact with the voltage sensor molecules. However, ryanodine did reduce the amount of reprimed charge 1 by approximately 60% suggesting the possibility of a retrograde interaction between ryanodine receptors and voltage sensors.     

Ryanodine receptor channelopathies

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.     

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.     

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.     

Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.     

The purified Ca2+ antagonist receptor from skeletal muscle: subunit structure, photoaffinity labeling and endogenous protein kinase activity

Tritiated analogues of the Ca2+ channel blockers such as [3H] PN200-110, [3H] verapamil and [3H] diltiazem have been used to identify and isolate Ca2+ antagonist receptors. The Ca2+ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca2+ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction.     

The purified Ca2+ antagonist receptor from skeletal muscle: subunit structure,photoaffinity labeling and endogenous protein kinase activity

Tritiated analogues of the Ca²⁺ channel blockers such as [³H] PN200-110, [³H] verapamil and [³H] diltiazem have been used to identify and isolate Ca²⁺ antagonist receptors. The Ca²⁺ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca²⁺ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.