Brigitte Plank

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.

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.

pH and temperature dependence of adenosine uptake in human erythrocytes

Kinetic analysis of the saturable adenosine uptake in human erythrocytes suggests the existence of two saturable components, distinguished by different Km values (1.4 and 260 micron, respectively, at pH 7.4 and 25 degrees C). Both components were abolished by p-nitrobenzylthioguanosine or dipyridamole. Total uptake was significantly higher at pH 8 than at pH 7 at adenosine concentrations above 2 micron. The increase in uptake at the higher pH was brought about mainly by an increase in the maximum rate of transport of the low-affinity uptake system. With rising temperature the Km and the V of both uptake components increased. No transition temperature was observed between 12 and 37 degrees C.

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.

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.

Alteration of Acylphosphate Formation of Cardiac Sarcoplasmic Reticulum ATPase by Calmodulin-Dependent Phosphorylation

The calcium-dependent acylphosphate formed by the calcium transport ATPase of cardiac sarcoplasmic reticulum and the calcium-, calm odulin-dependent phosphoester(s) of sarcoplasmic reticulum fractions formed by a calcium-, calmodulin-dependent membrane-bound protein kinase can be distinguished by removal of calcium and/or magnesium by EDTA or hydroxylamine treatment of the acid denaturated membranes. Both procedures decompose the acylphosphate with little effect on the phosphoester(s). Calmodulin-dependent phosphorylation (2.44 nmol/mg SR protein) reduces the apparent K(Ca) of the acylphosphate steady state level of the calcium transport ATPase from 0.56 to 0.34 μM free calcium, without affecting the maximum phosphoenzyme level (0.93 versus 0.89 nmol/mg protein), and has little, if any, effect on the Hill-coefficient (1.32 versus 1.54)