Gertrude Hellmann

Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.

Aspects of the mechanism of action of local anesthetics on the sarcoplasmic reticulum of skeletal muscle

1. The effect was studied of local anesthetics (tetracaine, dibucaine, procaine and xylocaine) on the forward and the backward reactions of the calcium pump of skeletal muscle sarcoplasmic reticulum. 2. The inhibition of the rate of calcium uptake, the rate of calcium-dependent ATP splitting and the rate of calcium-dependent ATP-ADP phosphate exchange by sarcoplasmic reticulum in the presence of the above drugs is at least partially due to the inhibition of the phosphoprotein formation from ATP. 3. The rate of the ADP-induced calcium release from sarcoplasmic reticulum and the rate of ATP synthesis driven by the calcium efflux are inhibited on account of a reduction of the phosphoprotein formation by orthophosphate. 4. The phosphorylation of calcium transport ATPase by either ATP or orthophosphate is diminished by the local anesthetics owing to a reduction in the apparent calcium affinity of sarcoplasmic reticulum emmbranes on the outside and on the inside, respectively. 5. The drug-induced calcium efflux from calcium-preloaded sarcoplasmic reticulum vesicles, a reaction not requiring ADP, is probably not mediated by calcium transport ATPase.

Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings

The actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (P(o)) of the calcium release channel at activating calcium concentrations (20-100 microM Ca(2+)) by 60-100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6-2 mM) decreased P(o). Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced P(o) at activating Ca(2+) (20-100 microM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca(2+) (0.6 microM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca(2+). High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca(2+). The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.

Inhibition of calcium release from skeletal muscle sarcoplasmic reticulum by calmodulin.

The effect of calmodulin on calcium release from heavy sarcoplasmic reticulum isolated from rabbit skeletal muscle was investigated with actively and passively calcium loaded sarcoplasmic reticulum vesicles and measured either spectrophotometrically with arsenazo III or by Millipore filtration technique. The transient calcium-, caffeine- and AMP-induced calcium release from actively loaded sarcoplasmic reticulum vesicles was reduced to 29%, 51% and 59% of the respective control value by 1 microM exogenous calmodulin. Stopped-flow measurements demonstrate that calmodulin reduces the apparent rate of caffeine-induced calcium release from actively loaded sarcoplasmic reticulum. The rate of calcium uptake measured in the presence of ruthenium red, which blocks the calcium release channel, was not affected by calmodulin or calmodulin-dependent phosphorylation of sarcoplasmic reticulum vesicles with ATP[S]. The rate of the calcium-, caffeine- and AMP-induced calcium release from passively loaded sarcoplasmic reticulum vesicles was reduced 1.4-2.0-fold by 1 microM exogenous calmodulin, i.e. the half-time of release was maximally increased by a factor of two, whilst calmodulin-dependent phosphorylation of a 57 kDa protein with ATP[S] had no effect. The data indicate that calmodulin itself regulates the calcium release channel of sarcoplasmic reticulum.

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum.

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.

Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca2+ affinity of regulatory Ca2+ sites in single channel recordings and [3H]ryanodine binding

The actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid (p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20-100 microM) increased the mean open probability (Po) of the calcium channel at subactivating (20 nM), maximally activating (20-100 microM and inhibitory (1-4 mM) Ca2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 microM 4-CMPS caused a transient increase in Po, followed by a low activity state within 1 min. At inhibitory Ca2+ concentrations Po was increased to values observed with maximally activating Ca2+ or lower, inhibitory Ca2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [3H]Ryanodine binding was increased up to four-fold with 3-15 microM 4-CMPS/p-CMB (Hill coefficient 1.7-2.0) at 4 microM Ca2+ and reduced at high concentrations (50-200 microM). The increase in [3H]ryanodine binding by 10 microM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 microM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.

Calmodulin·(Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.

ATP-Pi and ITP-Pi exchange by cardiac sarcoplasmic reticulum

Abstract ATP-P i and ITP-P i exchange is demonstrated in cardiac sarcoplasmic reticulum isolated from dogs. Both reactions require calcium outside the sarcoplasmic reticulum and inside, as well as magnesium and ADP or IDP. ATP-P i or ITP-P i exchange by sarcoplasmic reticulum is maximally activated at μM concentrations of calcium outside the sarcoplasmic reticulum, considerably inhibited by mM concentrations of calcium in the medium and abolished at nM concentrations of calcium in the medium; these last concentrations do not activate phosphorylation of the calcium transport ATPase by ATP or ITP. Phospholipase A-treated sarcoplasmic reticulum vesicles do not exhibit any nucleoside triphosphate-P i exchange at calcium concentrations between 0.01 and 0.3 mM, but both reactions are partially activated by mM calcium concentrations, indicating that calcium in the mM range inside the sarcoplasmic reticulum is essential for nucleoside triphosphate-P i exchange. Drugs like prenylamine, chlorpromazine, quinidine, tetracaine and dibucaine inhibit ATP-P i exchange and calcium-dependent ATPase to a similar extent. Inhibitors of mitochondrial ATP-P i exchange do not affect ATP-P i exchange by sarcoplasmic reticulum; dicyclohexyl carbodiimide was an exception in causing increased rate of phosphate exchange in sarcoplasmic reticulum. It is suggested that nucleoside triphosphate-P i exchange occurs via an exchange of inorganic phosphate with the phosphate of the phosphoprotein formed from nucleoside triphosphate, which is dephosphorylated by nucleoside diphosphate, resulting in ATP or ITP formation.

Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding.

Abstract. The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [3H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (Po) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in Po (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [3H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (Po > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (Po= 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[125I]calmodulin binding to HSR. 1 mm MgCl2 reduced Po from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (Po > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.

The reversal of the calcium pump of cardiac sarcoplasmic reticulum

SummaryThe reversal of the calcium pump of cardiac sarcoplasmic reticulum (SR) prepared from dogs was investigated. Phosphorylation of the calcium transport ATPase by orthophosphate and ATP synthesis from ADP and orthophosphate by SR passively preloaded with calcium are demonstrated. The ADP-dependent calcium efflux from SR loaded with calcium in the presence of acetylphosphate is stoichiometrically coupled to ATP synthesis from ADP and orthophosphate.ZusammenfassungDie Umkehr der Calcium-Pumpe des myokardialen sarkoplasmatischen Retikulums (SR) wurde untersucht. SR-Vesikel, die passiv durch Präinkubation mit hohen Calciumkonzentrationen mit Calcium beladen wurden, synthetisieren ATP aus Orthophosphat und ADP. Die ATP-Synthese erfolgt über die Phosphoryllerung der Calcium-Transport-ATPase des SR durch Orthophosphat und eine Dephosphorylierung des E∼P durch ADP. Die Rate der Calcium-Freisetzung aus SR-Vesikel — die Calcium-Beladung erfolgte aktiv in Gegenwart von Acetylphosphat — wird durch ADP erhöht. Der ADP-abhängige Calcium-Efflux ist stöchiometrisch mit der ATP-Synthese aus Orthophosphat und ADP gekoppelt.